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Abstracts and Author Index
Desinfektionsmittel (DIP01-03) .........................................................................................................................................4-5
Diagnostische Verfahren (DVP01-28)..............................................................................................................................5-15
Diagnostic methods
Eukaryontische Krankheitserreger (EKP01-07) .............................................................................................................21-23
Eukaryotic pathogens
FEMS-Satellitensymposium: „Life inside cells“ (FEMS-P01-10) .................................................................................32-36
FEMS-satellite symposium: “Life Inside Cells“
Freie Themen (FTP01-38) ..............................................................................................................................................37-51
Free topics
Gastrointestinale Infektionen (GIP01-19).......................................................................................................................51-58
Gastrointestinal infections
Allgemeine Hygiene und Krankenhaushygiene (HYP01-17).........................................................................................62-68
General and hospital hygiene
Infektionsimmunologie (IIP01-22) .................................................................................................................................73-81
Infection and immunity
Klinische Mikrobiologie, Infektiologie, Fallvorstellungen (KMP01-21) .......................................................................87-94
Clinical microbiology, infectious diseases, clinical case reports
Lebensmittelmikrobiologie und –hygiene (LMP01-08) .................................................................................................97-99
Food microbiology and food hygiene
Mikrobielle Pathogenität (MPP01-74)........................................................................................................................100-126
Microbial pathogenicity
Mikrobielle Systematik, Populationsgenetik und Infektionsepidemiologie (MSP01-13)...........................................141-146
Microbiological classification, population genetics and epidemiology of infections
Nationale Referenzzentren und Konsiliarlaboratorien (RKP01-14) ...........................................................................149-154
National reference centers and consultant laboratories
Qualitätssicherung (QSP01-02) .........................................................................................................................................157
Quality management
Oral Presentations
Diagnostische Verfahren (DVV01-14) ...........................................................................................................................15-21
Diagnostic methods
Eukaryontische Krankheitserreger (EKV01-11).............................................................................................................23-27
Eukaryotic pathogens
Eingeladene Sprecher (ESV01-17) .................................................................................................................................27-32
Invited Spreakers
FEMS-Satellitensymposium: „Life inside cells“ (FEMS-V01-04).................................................................................36-37
FEMS-satellite symposium: “Life Inside Cells“
Gastrointestinale Infektionen (GIV01-11)......................................................................................................................58-62
Gastrointestinal infections
Allgemeine Hygiene und Krankenhaushygiene (HYV01-12) ........................................................................................68-73
General and hospital hygiene
Infektionsimmunologie (IIV01-13) ................................................................................................................................81-87
Infection and immunity
Klinische Mikrobiologie, Infektiologie, Fallvorstellungen (KMV01-07) ......................................................................94-97
Clinical microbiology, infectious diseases, clinical case reports
Lebensmittelmikrobiologie und –hygiene (LMV01-02)...............................................................................................99-100
Food microbiology and food hygiene
Mikrobielle Pathogenität (MPV01-44) .......................................................................................................................126-141
Microbial pathogenicity
Mikrobielle Systematik, Populationsgenetik und Infektionsepidemiologie (MSV01-07) ..........................................146-149
Microbiological classification, population genetics and epidemiology of infections
Nationale Referenzzentren und Konsiliarlaboratorien (RKV01-07) ..........................................................................154-156
National reference centers and consultant laboratories
Qualitätssicherung (QSV01-07) .................................................................................................................................157-160
Quality management
Author Index.......................................................................................................................................................................162-172
Keine Veröffentlichung gewünscht:
No publication requested:
EKV08, GIP08, MPV23, MPV30
Keine Abstractveröffentlichung gewünscht:
No abstract publication requested:
MPP58, RKV03
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Impact of mycobacterial outer membrane structures on the
biocidal efficacy of disinfectants
K. Steinhauer1, J. Käding2, E. Frenzel3
Schuelke & Mayr GmbH, Research & Development
Norderstedt, Germany
University of Applied Sciences Luebeck, Department of Applied
Science, Luebeck, Germany
University Hamburg, Department Microbiology, Hamburg
Mycobacteria are significantly more resistant to chemical agents
including antibiotics and disinfectants compared to other Grampositive bacteria. This increased resistance is mainly due to their
unique cell wall structure, which contains large amounts of fatty
acids and mycolic acids. Due to their clinical importance and
their intrinsic resistance to many chemical agents mycobacteria
are important organisms to study in infection control.
In mycobacteria diffusion of small and hydrophilic molecules
across the outer membrane is mediated by pore proteins, called
porins. However, little is known about the uptake of biocides
and their mode of action against mycobacteria.
The aim of this study was therefore to investigate the impact of
mycobacterial outer membrane structures and porin expression
on the biocidal efficacy of disinfectants.
Mycobacterium smegmatis wt and three isogenic porin mutants
were used in quantitative suspension tests (EN 14348) to assess
whether the loss of porins influences the sensitivity of
M.smegmatis under simulated practical conditions. Porin loss
was found to result in a significantly increased resistance
towards the tested disinfectants.
Furthermore, porin expression was found to be involved in
biofilm formation and consequently to also account for
clumping of M. smegmatis, which is used as a test organism
when testing according to AFNOR. Our data indicates that
clumping of M. smegmatis significantly impacts efficacy testing.
Reduction factors were found to vary significantly (> 2 log)
when using different methods for homogenization. The same
phenomenom was also found, when using M. terrae, which is
used as a test organism when assessing the tuberculocidal
efficacy of disinfectants according to EN and DGHM-standards.
In conclusion our data shows that porins play an important role
in the efficacy of biocides against mycobacteria. Furthermore
we could demonstrate, that the methods used for
homogenization of the mycobacterial test suspensions have a
significant impact on the efficacy of the tested disinfectants.
Testing of the disinfectants-neutralize effect of Tryptic-SoyAgar with the neutralizers LTHTh in a surface-independent
B. Gerten1
Heipha Dr. Müller GmbH, Research & Developement
Eppelheim, Germany
For the inactivation of disinfectants used in pharmaceutical
processes it is mandatory to inactivate residuals of disinfectants
by neutralizing agents.
Such neutralizing agents are added to the media used in
hygiene/environmental monitoring. Although these neutralizers
are used for decades, there are only a few studies showing their
exact effect to the disinfectants.
The current study shows the neutralizing activity of Tryptic Soy
Agar (TSA) with LTHTh against different disinfectants with a
panel of EP/USP test strains. LTHTh is a commonly used
combination of neutralizers consisting of lecithin, tween,
histidin and thiosulphate.
In this study a method was developed to analyse the neutralizing
effect of LTHTh independently of the surfaces to be tested.
Therefore the disinfectants were applied evenly on the plates
using a spiral plater. After a defined period the test strains were
inoculated in the same way. The recovery rates on plates with
and without neutralizers were determined and compared to each
The results show that the neutralizer combination LTHTh is able
to inactivate disinfectants containing different active agents, like
alcohols, quaternary ammonium compounds as well as
Sanitation care with less risk for human and environment –
The Viennese WIDES-Database for disinfectants
M. Jaros1, M. Klade2
Vienna Ombuds-Office for Environmental Protection, Vienna
IFF/IFZ, Inter-University Research Center for Technology
Work and Culture, Graz, Austria
In hospitals disinfectants are routinely used to prevent
infections. As a matter of fact their application serves as a
sanitary measure. However, disinfectants themselves show
certain characteristics, which are dangerous to human beings
and environment. The continuous contact with disinfectants may
cause allergies and asthma, as shown by various studies.
Moreover, disinfectants in waste water may affect the
performance of sewage treatment plants or persist in the aquatic
environment. Appropriate measures enabling a comparative
assessment of these hazards arising from disinfectants have not
been available until now. Within the scope of the project
OEkoKauf Vienna the municipality of Vienna developed an
user-friendly database, which enables the purchasing department
and/or sanitation commissioner of hospitals to select those
disinfectants from the market supply, which pose less risk for
hospital staff, patients and environment. The database was
developed in cooperation with the Austrian Society for Hygiene,
Microbiology and Preventive Medicine (OEGHMP) and the
General Accidents Insurance Corporation (AUVA) amongst
others. The Inter-University Research Center for Technology,
Work and Culture (IFF/IFZ) in Graz designed an evaluation
scheme, which can be used to assess and compare
(eco)toxicological and human health characteristics of
antimicrobial active substances and commercial disinfectants.
Data about toxic, mutagenic, allergenic and dermal effects, as
well as effects on sewage treatment plants and surface water
were considered to be the most important indicators for hazard
potentials and compiled in a list of corresponding impact
categories. The evaluation scheme provides rules how to deduce
valuation numbers from R phrases or data sets and how to
consider data gaps.
Only disinfectants were integrated into the database, whose
efficiency was accredited by independent societies for hygiene.
Users of the database, mainly purchasers of disinfectants and
infection control teams, can retrieve the register of assessed
products, which can be sorted according to application mode
and the required anti-microbial spectra. Users can recall detailed
product information as well as toxicological data collection. The
possibility to compare potential risk to human health and
environment of different products by a mouse click facilitates
the consideration of associated risks in purchasing decisions.
The WIDES-Database will be published via Internet in July
2007. q.v.:
Real-Time RT-PCR for detection of bacterial contamination
in blood products
M. Störmer1, K. Kleesiek1, J. Dreier1
Heart- and Diabetes Center NRW, Institute for Laboratory and
Transfusion Medicine, Bad Oeynhausen, Germany
Transfusion-associated sepsis, mostly due to platelet concentrate
(PC) contamination, is recognized as the most frequent
infectious complication in transfusion therapy, surpassing by up
to 2 orders of magnitude the incidence of transfusion-associated
viral transmission. The bacterial contamination of PCs is a
major problem due to the current requirement to store PCs at
room temperature with agitation to preserve platelet function.
As a result small bacterial inocula can grow into very high
numbers within a short period of time. Therefore a screening
method allowing the early detection of very low levels of
bacteria in PCs would improve transfusion safety. Here we
present the applicability of our real-time reverse transcription
(RT)-PCR method as a routine screening system in blood banks.
We adapted a novel high-volume nucleic acid extraction
procedure based on magnetic separation technology with a
broad-range 23S rRNA real-time RT-PCR assay for fast and
sensitive detection while the BacT/Alert automated culturing
system served as the reference method. We spiked two model
bacteria, S. epidermidis and E. coli, to a single pool of
apheresis-derived single-donor platelets, and assayed the PCs by
real-time RT-PCR analysis employing an improved
primer?probe system and locked nucleic acid technology.
Coamplification of human beta2-microglobulin mRNA served as
an internal control (IC).
Studies to determine the optimal sampling-time for real-time
PCR screening showed that the sampling should not be carried
out earlier than 24 h after preparation. For automated magnetic
bead?based extraction technology with the real-time RT-PCR,
the 95%-detection limit was 29 CFU/mL for S. epidermidis and
22 CFU/mL for E. coli. No false-positive results of culturenegative tested PCs were observed, due to nucleic acid
contamination of reagents, during testing of 1500 PCs.
The high-volume nucleic acid extraction, the improved primerprobe system and the integration of an IC make the RT-PCR
assay appropriate for platelet bacteria screening. This assay has
a much shorter turnaround time than culture, facilitating the
testing of PCs before release.
Use of Bartonella adhesin A (BadA) immunoblotting in the
serodiagnosis of Bartonella henselae infections
C. Wagner1, T. Riess1, D. Linke2, C. Eberhardt1, A. Schäfer1
S. Reutter1, V. Kempf1
University Hospital of Tuebingen, Institute for Medical
Microbiology and Hygiene, Tuebingen, Germany
Max-Planck-Institute for Developmental Biology, Department
of Protein Evolution, Tuebingen, Germany
Bartonella henselae causes a variety of human diseases (e.g., cat
scratch disease, and the vasculoproliferative disorders bacillary
angiomatosis and peliosis hepatis). The laboratory diagnosis of
B. henselae infections is usually based on the detection of antiB. henselae antibodies by an indirect immunofluorescence assay
(IFA) which, unfortunately, suffers a significant amount of
cross-reactivity and hence is prone to deliver false positive
results. In this pilot study, we evaluated the use of a potential
two-step serodiagnosis of B. henselae infections by combining
IFA and anti-Bartonella adhesin A (BadA) immunoblotting.
Our data revealed that ~75% of the IFA positive sera of patients,
with a suspected B. henselae-infection, reacted specifically with
BadA and only ~20% of the IFA negative sera of healthy blood
donors. Although Yersinia adhesin A (YadA) is structurally
closely related to BadA, no cross-reactivity of sera from patients
pseudotuberculosis-infection with BadA was detected in
immunoblotting. Unfortunately, recombinantly expressed BadAdomains (head, connector, stalk fragment) were not suitable for
immunoblotting. Finally, the best resolution for full-length
BadA immunoblotting was obtained when whole cell lysates of
B. henselae were separated using continuous 4-15% sodium
dodecyl sulfate polyacrylamide gels. In summary, our results
show that BadA-antibodies are reliably detectable in the sera of
B. henselae-infected patients and, therefore, this pilot study
suggests to include BadA-immunoblotting in the laboratory
diagnosis of B. henselae infections.
Evaluation of the GenoType® MTBDRplus assay for the
direct detection of isoniazid- and rifampicin-resistant
Mycobacterium tuberculosis
K. Bögli-Stuber1, B. Léchenne1, A. Hilty1, T. Bodmer1
University of Bern, Institute for Infectious Diseases, Bern
Background: The world-wide emergence of drug-resistant
Mycobacterium tuberculosis (Mtb) strains compromises the
efficacy of modern tuberculosis (TB) drug regimens, of which
rifampicin (RMP) and isoniazid (INH) are considered the
pillars. Resistance to both drugs in Mtb is chromosomally
determined by missense mutations and/or deletions. The novel
GenoType® MTBDRplus assay (HAIN Lifescience GmbH,
Nehren, Germany) is designed to detect genetic events that are
associated with INH and RMP resistance directly from smearpositive respiratory specimens, and thus allows the rapid
identification of multidrug-resistant Mtb (MDR-TB).
Methods: Mtb patient isolates and the corresponding sediments
of smear-positive respiratory specimens were selected from our
repositories. Drug susceptibility was assessed by the radiometric
method (Bactec 460-TB instrument, PRISE, Becton-Dickinson,
Germany). The critical concentrations in mg/L were: RMP, 2.0;
INH, 0.1 and 0.4. GenoType® MTBDRplus was performed
according to the manufacturer’s instructions, in a blinded
manner, from DNA obtained from both the strains and the
Results: Ten Mtb isolates were evaluated. Of these, four had a
MDR-TB phenotype, two were high-level INH-resistant, two
were low-level INH-resistant, and two were susceptible to INH
and RMP. There was a 100% agreement, in this collection of 10
Mtb isolates, between standard drug susceptibility testing and
GenoType® MTBDRplus results; in addition, the results of the
GenoType® MTBDRplus assay obtained from the strains and
from the respective sediments were concordant. The turnaround-time of the GenoType® MTBDRplus assay was
approximately 5 hours.
Conclusions: These results indicate that GenoType®
MTBDRplus may have a role to play in the rapid detection of
MDR-TB in the respiratory specimens of patients with
suspected MDR-TB, e.g. with a relapse or coming from regions
with a known MDR-TB risk. The assay’s performance will
depend upon the nature of the genetic events leading to INH or
RMP resistance in the Mtb strains encountered in a particular
clinical setting.
Increasing sensitivity through use of a direct fluorescent
antibody test for Legionella pneumophila in
bronchoalveolar lavage samples by immunomagnetic
separation based on BioMags
S. Sethi1, M. Gore2, K. Sethi3
Institute of Medical Microbiology and Hygiene, Microbiology
Homburg/Saar, Germany
Integrated DNA Technologies Inc., DNA Technologies
Coralville, USA
Seroscan Laboratories, Serology, Heidelberg, Germany
In the present study, immunomagnetic separation of
L.pneumophila from mock bronchoalveloar lavage (BAL) fluid
samples, which were artificially spiked with L.pneumophila, and
culture positive patient BAL fluid samples, was achieved using
BioMags (superparamagnetic particles ) loaded with purified
rabbit immunoglobulin G specific for L.pneumophila. Bacteria
binding onto.
BioMag-immunomatrix were directly stained with a
L.pneumophila species-specific DFA reagent and examined
immunomagnetic separation (BIMS) followed by DFA staining
(BIMS-DFA) could correctly identify all the 20 (100 %) BAL
samples which were spiked with low numbers (2 x102 CFU) of
L.pneumophila. Cultures could be recovered from 15 (75 %) of
these 20 spiked BAL samples, 5 (25 %) of the samples failed to
yield positive cultures. Both culture and BIMS-DFA methods
showed 100 % positive results when spiked BAL samples
containing high bacterial load (104 CFU) were tested. The
findings with true patient culture positive BAL specimens which
were examined retrospectively indicated that BIMS-DFA is
significantly more sensitive for detecting L.pneumophila than
conventional cytospin method of DFA staining (cytospin-DFA).
Out of the 25 culture-positive BAL specimens tested, 7 (28 %)
proved negative by cytospin-DFA whereas BIMS-DFA
correctly detected all the 25 (100 %) specimens. It is suggested
that the BIMS-DFA procedure increases the sensitivity of DFA
testing for L.pneumophila in large volume samples such as BAL
Development of a phage typing system for Salmonella
enterica serovar infantis
T. Miller1, R. Prager1, W. Rabsch1
Robert-Koch-Institute, Wernigerode, Germany
The EU demands a decrease in diseases that are transferable
from animals to human (zoonosis) for consumers protection.
Salmonella Infantis is one zoonotic agent, causative of human
enteritis that has increased in recent years. The incidences in
humans show that S. Infantis is a new emerging pathogen. An
EU-wide Salmonella study of the prevalence of Salmonella (S.)
in hen flocks, initiated by the European Food Safety Authority
(EFSA) was carried out in all member states. The three most
frequently serovars in the EU-study were in descending order: S.
Enteritidis, S. Infantis and S. Typhimurium [1]. There are phage
typing schemes for S. Enteritidis and S. Typhimurium which are
used worldwide for outbreak investigations. We develop a
phage typing scheme for S. Infantis, necessary for
epidemiological surveillance and control of this pathogen. The
phages were isolated from 119 lysogenic strains collected from
1973 until 2006. The strains were isolated from human faecal
smears and different animal products as well. A total of 150 S.
Infantis strains were typed. The most discriminating phages (n =
22) were used for the final typing scheme and further validation.
Additionally we investigated the presence of the phage encoded
virulence gene sopE in S. Infantis strains by PCR as an
additional epidemiological marker.
Characterization and detection of extended-spectrum betalactamase (ESBL)-producing Enterobacteriaceae by use of
different updated automated susceptibility testing systems
K. - A. Moder1, J. Färber1, F. Layer1, I. Tammer1, W. König1
B. König1
Otto-von-Guericke-University Magdeburg, Institute of Medical
Microbiology, Magdeburg, Germany
Multi resistant organism cause severe infections, which are
complicated to handle including limited treatment options.
Especially ESBL producing Enterobacteriaceae have important
infection control implications and are still a major challenge to
detect in routine clinical microbiology laboratories. The
validation of the different screening tests for ESBL basically
apply to E. coli and Klebsiella spp.. Problems detecting an
ESBL production particularly occur with various Enterobacter
spp., Serratia spp. and Citrobacter spp. No CLSI
recommendations exist for ESBL detection and reporting for
organisms besides E. coli and Klebsiella spp. or for detecting
plasmid-mediated AmpC beta-lactamases.
There exist several different test methods, among them
automated identification and antimicrobial susceptibility testing
systems, e.g. the BD PhoenixTM (BD Diagnostic Systems,
Sparks, MD) and the Vitek®2 System (bioMerieux, Marcy
Etoile, France). Few data exist about the compatibility of the
various ESBL detection methods. So we tested 120 isolates of
different Enterobacteriaceae for ESBL production using the BD
PhoenixTM Automated Microbiology System (NMIC/ID-50 and
NMIC/ID-70 BD PhoenixTM GN Combo panels) and the
Vitek®2 System (Vitek®2 ID-GNB for identification; ASTN041- and AST-N062 panels for antimicrobial susceptibility
testing). We screened for the common ESBL gene families
(TEM, SHV, OXA, CMY and CTX-M) by PCR as reference
Using E-Test we detected 89 ESBL producing and 31 non ESBL
producing strains. The performance of the automated systems
was variable, in particular with organism such as Enterobacter
spp. and Citrobacter spp. Noticeable were the different results
generated with the various panels from both systems testing the
same isolate. The integration of an ESBL confirmation test into
the panels reduce time to accurate ESBL detection.
Nevertheless, concerning organism such as Enterobacter spp.,
manual test for confirmation an ESBL production is
Detection of extended-spectrum -lactamase (ESBL) producing Enterobacteriaceae with a selective chromogenic
agar medium
J. Färber1, K. - A. Moder1, F. Layer1, I. Tammer1, W. König1
B. König1
Otto-von-Guericke University of Magdeburg, Institute of
Medical Microbiology, Magdeburg, Germany
Nosocomial infections caused by extended-spectrum betalactamase (ESBL) and ampC beta-lactamase producing gramnegative bacteria complicate therapy and limit treatment
options. The CLSI has issued guidelines for phenotypic
confirmation of suspected ESBL strains among E. coli and
Kelebsiella spp. No Interpretation criteria exist for other
organisms or for detecting plasmid-mediated AmpC betalactamases.
Meanwhile there exist several different test methods, among
them automated identification and antimicrobial susceptibility
testing systems, e.g. the Vitek®2 System (bioMerieux, Marcy
Etoile, France). In addition chromogenic agar mediums, e.g.
the chrom IDTM ESBL (bioMérieux, Marcy l'
Etoile, France) is
available for ESBL screening.
Therefore we tested 120 isolates of different Enterobacteriaceae
for ESBL production. We compared the performance of the
Vitek®2 System (Vitek®2 ID-GNB for identification; AST-N020
and AST-N041 panels for antimicrobial susceptibility testing)
with the chromogenic agar medium chrom IDTM ESBL. The
detection of the common ESBL gene families (TEM, SHV,
OXA, CMY and CTX-M) by PCR was used as reference
Using E-Test we detected 89 ESBL producing and 31 non ESBL
producing strains. The main advantage of the chrom IDTM ESBL
agar is its sensitivity, which enable the recovery and
identification of most ESBL producing organism within 24
hours. The Vitek®2 system showed variable results, in particular
with organism such as Enterobacter spp. and Citrobacter spp..
In conclusion, the new agar screen plate is a convenient method
to screen for ESBL organisms, even possible out of clinical
samples, with the potential for incorporation into routine clinical
laboratory service.
DNA-microarray for genotyping antibiotic susceptibility of
multidrug resistant P. aeruginosa isolates
J. Weile1, C. Knabbe1
Robert-Bosch-Hospital, Laboratory Medicine, Stuttgart
Multidrug-resistant bacteria in both the hospital and community
environment are of great concern as it is the major cause of
failure in the treatment of infectious diseases. Regarding the
worldwide increase of multidrug resistant Pseudomonas
aeruginosa, new strategies using molecular diagnostics are
needed. P. aeruginosa is characterized by an intrinsic resistance
to various antimicrobial agents and the ability to develop
multidrug resistance during antibiotic therapy. Standard
laboratory methods based on phenotypically determined
microbiological and biochemical characteristics require usually
48 to 72 hours. One approach to cope with this problem is the
development of new, faster methods for antibiotic susceptibility
and species identification, allowing an earlier therapy onset and
a more adequate therapy, particularly for critically ill or
immunocompromised patients in intensive care units.We
developed a DNA-microarray based biochip for genotyping P.
aeruginosa in order to determine antibiotic susceptibility and
virulence. The inclusion of virulence factors like toxins or
alginate production broadens the information about the
virulence potential of P. aeruginosa at the same time. The whole
procedure can be performed in less than 5 hours and consists of
DNA isolation directly from primary clinical specimen, target
gene amplification concomitant with fluorescence labeling,
DNAse I fragmentation and array hybridization. A collective of
P. aeruginosa clinical isolates from ICU was analyzed. The
array covers mutations in regulatory genes (mexR, nfxB, mexT,
mexZ, nalC, nalD, ampD, ampR) of efflux systems and AmpClactamase, as well as gyrA and parC . A variety of
aminoglycoside modifying enzymes (aph(3’), aac(6’), aac(3),
aadA, aadB) and oxa-, imp-, vim-, ges-, per-, and gim- lactamases were included. Concerning the genotype-phenotype
correlation in the test collection, the coverage of relevant
resistance determinants for antibiotics used in a calculated
therapy of critical-ill patients was about 90 %. It was also
possible to retrospectively detect genetic determinants for
resistance in isolates which initially showed no corresponding
resistance phenotype, but developed such under therapy,
demonstrating the advantage of molecular diagnostics over the
classical phenotyping methods.
Applications for quantitative real-time immuno-PCRs (qRTiPCRs): Highly sensitive detection of staphylococcal toxins
in clinical specimens and food-derived samples
A. Fischer1, C. von Eiff1, T. Kuczius2, G. Peters1, K. Becker1
University Hospital Muenster, Institute of Medical
Microbiology, Muenster, Germany
University Hospital Muenster, Institute for Hygiene, Muenster
As immunological methods used until now are known to be
limited in sensitivity and specificity, there is an obvious need for
new highly sensitive and specific methods for the detection of
Staphylococcus aureus exotoxins. To extend the sensitivity of
nucleic acid amplification techniques to the detection of toxic
shock syndrome-mediating staphylococcal superantigen toxins
TSST-1 and staphylococcal enterotoxin B (SEB, also
responsible for staphylococcal food poisoning), two quantitative
real time immuno-PCR (qRT-iPCR) approaches with highly
increased performance have been developed.
The detection of TSST-1 and SEB, respectively, was achieved
by coating toxin-specific antibodies (ABs) to microtiter plates in
order to capture the target superantigens followed by specific
detection of the antigen-AB complex. The resulting
immunocomplex was subsequently detected using an antibody
covalently bound to a reporter-DNA molecule followed by realtime amplification of the reporter-DNA. Amplification was
carried out in an iCycler iQ real-time PCR system.
After optimization in buffered systems, qRT-iPCR protocols
have been used to detect SEB and TSST-1 in a variety of
clinical specimens and food-derived samples. For this reason,
samples were spiked with a dilution series of the respective
toxin and subsequently detected by qRT-iPCR. SEB and TSST1 were successfully detected in culture supernatants, serum,
urine, and milk at minimum concentrations as low as 100pg/ml
(approx. 4amol/µl) and 1ng/ml, respectively. Thus, the limit of
detection (LOD) was lowered up to 100-fold compared to
commercially available ELISA or RPLA.
The qRT-iPCR introduced here offers an ultra-sensitive
detection of staphylococcal superantigens in clinical specimens
and food-derived samples. Furthermore, the qRT-iPCR
approach offers high versatility for adaptation to a broad range
of toxins and other antigen targets.
Real Time PCR assay for detection of species of the genus
S. Günther1, P. Schierack1, M. Grobbel1, L. H. Wieler1
C. Ewers1
Institute of Microbiology and Epizootics, Berlin, Germany
Infections caused by species of the genus Mannheimia cause
diverse disease complexes in many wild and domestic animals
worldwide. Fast and accurate detection of single species within
the genus remains an unsolved problem till today. To solve this
diagnostic challenge, we developed a real time PCR assay for
the rapid and specific identification of five species of the genus
Mannheimia (M.haemolytica, M.varigena, M.ruminalis,
M.granulomatis and M.glucosida) from bacterial cultures and
tissue samples. The assay was validated with reference strains,
field isolates and bacteria spiked tissue samples. The sodA gene
was used as target region for species specific primer pairs. The
real time PCR assay demonstrated species specificity for all five
examined Mannheimia spp. and a rapid test completion time of
less than 5 hours. This is a considerable advantage in
comparison to the currently used traditional phenotyping
methods to distinguish between the species of the genus.
The assay was able to detect approximately 102 bacterial cells
per gram lung tissue sample, as determined with spiked tissue
samples. We assume that the assay will be useful for fast
laboratory diagnostic assessment of particularly respiratory
infections caused by Mannheimia in animals.
Low sensitivity of direct MRSA PCR-based tests due to low
colony counts from nasal swabs.
H. von Wulffen1, S. Scherpe2, T. Brodegger1, H. Feucht2
M. Äpfelbacher2
MEDILYS c/o AK Altona, Central Labor/Microbiology
Hamburg, Germany
University Hospital Hamburg-Eppendorf, Institute for
Microbiology, Hamburg, Germany
To evaluate an inhouse real-time PCR assay for direct detection
of MRSA in clinical samples results from 491 nasal swabs taken
under routine conditions for MRSA direct testing were
compared to those achieved with a commercial test system and
to those achieved by parallel culture from same specimens.
Swabs were washed out in 300 µl lysis solution, a 100 µl aliquot
of this solution was plated out on MRSA CHROM agar (MAST
Diagnostics). After the lysis reaction 5 µl each was employed in
the PCR tests. Cultures were read after 24 and 48 hours, results
expressed as colony forming units (cfu) per swab. Species
identification and oxacillin resistance were confirmed using the
VITEK 2 system (Biomérieux).Using culture as gold standard
the inhouse and the commercial PCR tests yielded sensitivities
of 64.6% and 68.8%, specificities of 98.0 and 94.6%, positive
predictive values (PPV) of 77.5% and 57.9%, and negative
predictive values (NPV) of 96.2% and 96.5% respectively. If the
gold standard was extended to include culture results from other
patient specimens from the same time period, the sensitivities
were 65.5% and 69.2%, the specificities 99.5% and 97.7%, the
PPV 95.0% and 79.4%, and the NPV 95.6% and 96.1%
respectively. For the inhouse test positive cultures yielded a
median of 550 cfu (95% CI 72-1000) for positive PCR results
(n=30) opposed to a median of only 9 cfu (95% CI 3-120) for
negative PCR results (n=17). For the commercial test these
medians were also 550 cfu (95% CI 100-1200) for positive PCR
results (n=32) and 9 cfu (95% CI 3-24) for negative PCR results
(n=15). The results suggest that PCR tests are suitable for
screening purposes (high NPV values), but they need to be
backed up by parallel cultures due to frequently low colony
counts from nasal swabs that lie below detection limits of PCR
based tests.
Comparison of commercial DNA preparation kits for the
detection of Brucellae in tissue using quantitative real-time
H. Tomaso1, H. Scholz1, S. Al Dahouk2, U. Wernery3
M. Pfeffer1, M. Eickhoff4
Bundeswehr Institute for Microbiology, Bacteriology, Munich
Bundeswehr Central Hospital Koblenz, Koblenz, Germany
Central Veterinary Research Laboratory, Dubai, United Arab
Qiagen Diagnostics GmbH, Hamburg, Germany
Brucellosis is a ‘re-emerging’ zoonosis with a worldwide
distribution. Humans are mainly infected by B. melitenis, B.
abortus, and B. suis. Brucella is the most frequent cause of
laboratory-acquired infections. The identification of Brucella
species with conventional microbiological techniques is timeconsuming and hazardous. Real-time PCR assays allow for the
rapid and specific detection of Brucella species, but the amount
of bacteria in tissues is frequently extremely low. Therefore, the
step of DNA preparation is critical to avoid false negative PCR
The aim of this study was therefore to assess the performance of
commercially available kits for the preparation of Brucella DNA
from tissues.
We evaluated the following kits: QIAampae DNA Mini Kit
(QIAGEN), peqGoldae Tissue DNA Mini Kit (PeqLab),
UltraCleanae Tissue DNA Isolation Kit (MoBio), DNA
Isolation Kit for Cells and Tissues (Roche), and NucleoSpinae
Tissue (Macherey-Nagel). Twelve tissue samples of two camels
with culture proven generalized Brucella melitensis infections
were processed. Identification of the organisms was performed
using conventional microbiological techniques including species
specific AMOS PCR. Tissue samples were inactivated in
formalin and homogenized using a Bio 101 FastPrep instrument
(Dianova, Hamburg, Germany). A 100-µL sample was further
processed with the above mentioned kits. All DNA preparations
were performed in duplicates and the amount of DNA was
determined in triplicates using a real-time PCR assay with
hybridization probes targeting the insertion sequence 711. Cycle
threshold values were used to calculate the amount of DNA that
was prepared from the tissue samples. We also assessed the
costs per sample, the time required and ease of handling for the
respective kits. We observed differences in the amount of DNA
that was harvested of up to two log steps among the kits. The
time required and the costs also differed markedly.
In summary, this study provides data that should facilitate the
choice of DNA purification kits suitable for specific clinical
samples with regard to sample volume, DNA yield, costs and
hands-on time.
Evaluation of broad-range PCR for the diagnostic of
infectious endocarditis
T. Vollmer1, J. Dreier1, C. Piper2, C. Freytag1, K. Kleesiek1
Institute for Laboratory and Transfusion Medicine, Heart- and
Diabetes Center NRW, Bad Oeynhausen, Germany
Clinic for Cardiology, Heart- and Diabetes Center NRW
Bad Oeynhausen, Germany
Infectious endocarditis (IE) is associated with a high degree of
mortality and morbidity. The crucial factor in diagnosis and
effective therapy is the identification of the etiologic agent. To
date blood cultures are the gold standard in laboratory testing
and identification of the pathogen. Pathogen identification based
on culturing methods and broad-range PCR has also been
described for heart valves after surgical excision. Due to slow
growing, fastidious bacteria or previous antibiotic treatment,
blood cultures as well as cultures of surgically removed
specimen often give a negative result. In the present study, the
performance of a broad-range 16S rDNA assay is compared to
that of an automated culture system (21 days at 37°C) with
additional attention to clinical implications. A total of 323 heart
valves of patients with definite or suspected IE were surgically
removed between September 2001 and December 2006 and
were screened for bacterial infection. In case of positive results,
isolates were identified according to standard procedures. A
total of 156 (48%) surgically removed heart valves were
detected positive of which 36 (11%) were detected in both
systems. 90 infected heart valves (28%) were detected only by
broad-range PCR, whereas in 30 cases (9%) positive results
were detected only by culture. These results showed increased
diagnosis of bacterial infection by broad-range PCR
amplification which therefore seems to be a powerful method
for the identification of the causative organism of IE. The most
frequently identified pathogens are Streptococcus spp. (PCR:
47%, culture 18%), Staphylococcus spp. (PCR: 18%, culture:
29%) and Enterococcus spp. (PCR: 7%, culture: 26%). Further
analysis of the patient files is under way with regard to patient?s
history including antibiotic treatment, diagnosis and clinical
outcome. This retrospective examination is carried out to
differentiate between acute, appropriate attended, successfully
healed or rejected cases of IE. The disease stadium at the time of
heart valve replacement is crucial for the ensured occurrence of
an etiologic agent and thus for the determination of the
diagnostic limitations of both methods.
Rapid detection of pathogenic bacteria in blood samples by
eubacterial Gram-Differentiating multiplex real-time PCR
in conjunction with pre-analytic DNA preparation kit
S. Gebert1, D. Siegel1, M. Lorenz2, C. Disqué2
N. Wellinghausen1
University Hospital, Institute for Medical Microbiology and
Hygiene, Ulm, Germany
Molzym GmbH & Co.KG, Bremen, Germany
Rapid detection of bacterial pathogens in blood from septic
patients is essential for adequate antimicrobial therapy and
prognosis of patients. Blood cultures are the diagnostic tool of
choice but usually take a few days until identification of the
Aim of this study is to facilitate detection and identification of
bacteria in blood samples by molecular methods before positive
signalling of blood cultures in an automated system.
We used a eubacterial 16S rDNA based real-time LightCycler
PCR assay that enables detection of bacterial DNA and
simultaneous differentiation of Gram-positive and Gramnegative bacteria by dual-colour multiplex FRET probe design
(Gram-diff-PCR). The assay has a sensitivity of 50-500 fg
DNA/PCR tested with several bacterial pathogens and detects
all relevant species. For investigation of spiked blood samples,
DNA preparation was performed by MolYsis (Molzym GmbH
& Co.KG, Bremen, Germany). MolYsis DNA preparation is
characterised by lysis of human cells and degradation of human
DNA prior to bacterial cell lysis which leads to minimisation of
human background DNA and selective enrichment of bacterial
DNA. In spiked citrate blood samples, the Gram-diff-PCR assay
in conjunction with MolYsis DNA preparation allowed
detection of 2-5 CFU E. coli and S. aureus per PCR-reaction,
i.e. 40-100 CFU per 200 µl blood. In addition, BACTEC blood
culture bottles spiked with bacteria were analysed continuously
with the Gram-diff-PCR assay and pre-analytic MolYsis
protocol until positive signalling in the automated BACTEC
system. This procedure allowed detection of E. coli and S.
aureus in the blood culture bottle 7,5 h and 5,5 h respectively,
previous to positive signalling of the BACTEC system.
In conclusion, the Gram-diff-PCR in conjunction with MolYsis
DNA preparation allows sensitive detection and Gramdifferentiating of bacterial DNA in native blood and blood
culture samples. Based on the results of the Gram-diff-PCR
species- or genus-specific PCR assays can be applied for
subsequent identification of the respective pathogen. In the near
future, further validation of the assay on patient blood samples
will be done.
Triple Faeces Test: A successful Dutch approach for
detection of intestinal parasites
A. Buss1, B. Kesztyues1
Laboratory for Infectious Diseases, Groningen
Introduction: The Laboratory for Infectious Diseases (LvI) is a
regional public health laboratory in Groningen, Netherlands. In
2002 we introduced the by van Gool et al1 developed “Triple
Faeces Test” (TFT) as standard tool for detection of intestinal
parasites. The TFT both combines multiple sampling, SAF
(sodium acetate acetic acid formalin) fixative and the use of
chlorazol black dye. We present this diagnostic approach for
detection of parasites and discuss the advantages and
disadvantages compared to the conventional diagnostic method,
i.e. analysis of a single fresh stool specimen. Because of the
intermittent shedding of intestinal protozoa, delivery of three
stool specimens is recommended, but often not performed in
daily practice.
Material and Methods: Patients are asked to collect stool
specimens on 3 consecutive days: on day 1 in a tube containing
SAF (TFT1), on day 2 in the empty tube (TFT2)and on day 3 in
the other tube containing SAF (TFT3). An instruction form with
detailed information on how to handle the TFT collection kit is
included in each set. After day 3, the complete TFT set is
returned to the laboratory. From tube TFT2 (without fixative)
additional diagnostic for bacteria and viruses can be performed.
Results: Between september 2006 until may 2007 3644 TFTsets were analysed in our laboratory. 992 (26%) TFT-sets were
positive for at least one protozoan species. First results revealed
the low-pathogen Blastocystis hominis (n=421) as most common
species detected. Dientamoeba fragilis (n=185), and Gardia
lamblia (n=146) were the most frequently observed pathogens.
In 183 TFT-sets more than one protozoa were found.
Discussion: The increase in the costs of material and a longer
“hands-on time” by performance of a TFT compared to the
examination of a single fresh stool sample are compensated by
the higher sensitivity of the TFT due to the detection of
vegetative stages of protozoa and a higher compliance with the
request to submit multiple stool samples. Having a nonfixed
sample in the TFT (TFT2) allows the performance of additional
molecular techniques and antigen tests.
References1. T. van Gool,·R. Weijts, E. Lommerse, T. G. Mank
Triple Faeces Test: An Effective Tool for Detection of Intestinal
Parasites in Routine Clinical Practice. Eur J Clin Microbiol
Infect Dis (2003) 22:284–290
Temperature Dependency of Detection of Fastidious and
Endocarditis-causing Organisms in Delayed-Entry Samples
in the BacT/Alert 3D Blood Culture System
I. Seegmüller1, M. Wilms2, S. Stanzel3, R. R. Reinert2
University Heidelberg, Hygiene, Heidelberg, Germany
University Aachen, Medical Microbiology, Aachen, Germany
University Aachen, Institute for Medical Statistic, Aachen
Background: This study evaluates the effect of preincubation
temperature on delayed entry samples for fastidious organisms
including the HACEK group, several Streptococci spp including
S. pneumoniae, Neisseria meningitides, Haemophilus spp. and
Corynebacterium spp. for the BacT/Alert 3D system
(bioMérieux) using the FA medium.
Method: Bottles were inoculated with two different
concentrations (0.5 McFarland and a 1:100000 dilution) of each
organism and either loaded into the system immediately or
incubated at 4 °C, room temperature (RT) or 37 °C for 24h prior
to loading. For some strains bottles were supplemented with
either human blood, Bactec Fos medium or both. Logistic
regression was applied to evaluate detection rate (DR), ANOVA
to evaluate the time to detection (TTD).
Results: DR was 92.5% for bottles loaded immediately with a
mean TTD of 26.7h (Max: 81.6h) for the low concentration and
9.21h (Max: 40.3h) for the high concentration. Preincubation at
4°C did not affect the DR. The DR at RT was 90.0% for the low
concentration and 83.6% for the high concentration. At 37°C the
DR was 76.3% and 66.3% for the low and the high
concentration respectively. Bottles inoculated with Streptococci
ssp. and Haemophilus spp. failed to signal despite showing
visible indicator alteration after preincubation. TTD was 29.8h
(Max: 91.2h) and 11.9h (Max: 48.0h) , 24.4h (Max: 86.4h) and
9.6h (Max: 40.7h), 8.3h (Max: 48.0h) and 4.9h (Max: 38.1h) for
the bottles held at 4 °C, RT and 37 °C for the low and the high
concentration respectively. Differences in DR due to
preincubation temperature were statistically significant.
Eikenella corrodens and Gemella sanguis were only detected in
the presence of blood. Kingella kingae remained undetected.
Conclusion: For delayed entry samples storage at RT seems to
be advisable. It only affected the DR in one isolate (for the low
concentration mimicking bacteremia) and offers a reasonable
TTD. An absolute incubation time of blood culture bottles of
four days seems to be sufficient, as none of the bottles signalled
positive after this period. To successfully culture Kingella
kingae alternative media should be used.
traditional culture methods. Molecular assays are known to be
more sensitive than culture or microscopic techniques.
Therefore it is very likely that the PCR-RLB positive, culture
negative samples are in fact positive.
Comparison of PCR-Reverse line blot analysis and
traditional culture of dermatophytes in clinical samples.
G. J. Wisselink1, B. Kesztyues1, E. van Zanten1
K. R. van Slochteren1, S. Coops1, A. M. C. Bergmans2
R. G. F. Wintermans2, A. M. D. Kooistra-Smid1
Laboratory for Infectious Diseases, Groningen, Netherlands
Franciscus Hospital , Department of Medical Microbiology
Roosendaal, Netherlands
Separation of bacterial DNA from human DNA in clinical
samples may have an important impact on downstream
applications, involving nucleic-acid based microbial diagnostic
systems. We evaluated two commercially available reagents
(MolYsis® and Pureprove®) for their potential to isolate and
purify bacterial DNA from human DNA. We chose oral
samples, which usually contain very high amounts of both
human and bacterial cells. Three different DNA preparations
each were made from eight caries- and eight periodontal
specimens using the two reagents above and a conventional
DNA extraction strategy as reference. Based on target-specific
RT-PCR assays we compared the reduction of human DNA
versus loss of bacterial DNA. Human DNA was monitored by
targeting the -2-microglobulin gene, while bacteria were
monitored by targeting 16S rRNA gene (total bacteria and
Porphyromonas gingivalis) or the glycosyltransferase gene
(Streptococcus mutans).
We found that in most cases at least 90% of human DNA could
successfully be removed, with complete removal in eight of 16
cases using the MolYsis® protocol, and two (of 16) cases using
Pureprove®. Conversely, detection of bacterial DNA was
possible in all cases with a recovery rate generally ranging from
35% to 50%. In conclusion, both reagents are useful for
reducing background interference from the host DNA. Bacterial
DNA can be up-concentrated, possibly resulting in enhanced
sensitivity and specificity of subsequent nucleic-acid based
microbial diagnostic systems.
Traditionally, laboratory detection and identification of
dermatophytes consists of culture on selective media and
potassium hydroxide (KOH) tests. This process yields positive
results within approximately 2-6 weeks. Using PCR followed by
Reverse Line Blot (PCR-RLB) analysis it becomes possible to
obtain positive and negative results within 2-3 days. In this
study we compared traditional culture with PCR-RLB analysis.
Two hundred and three clinical samples (187 nail, 16 skin) were
analysed retrospectively by PCR-RLB after traditional culture.
Samples were processed using QIAamp® DNA mini kit
(Qiagen, Germany) with a separate pre-lysis step. PCR targeted
the ITS region between the genes coding for 18S and 5.8S
rRNA. PCR products were analysed using RLB [Bergmans et
al., submitted]. The membrane harboured 13 different probes to
identify and discriminate between 9 different dermatophyte
species within 3 genera, namely; T. rubrum, T. mentagrophytes,
T. interdigitale, T. tonsurans, T. violaceum, T. verrucosum, M.
canis (complex), M. audouinii and E. floccosum.
Culture, KOH and PCR-RLB analysis yielded 37/203, 93/200
and 97/203 positive results respectively. Of the 37 culture
positive samples 35 scored positive in the PCR-RLB.. Sixty-two
samples scored positive in PCR-RLB but remained negative in
culture, 53 of these samples were KOH positive. Of the 97 PCRRLB positive samples 79 were identified as T. rubrum, 14 as T.
interdigitale, 3 as Trichophyton sp. Sensitivity for the PCR-RLB
compared to culture for dermatophytes is 100% (34/34),
compared to the KOH the sensitivity of PCR-RLB is 92 %
These data show PCR-RLB to be a fast and very sensitive
method to detect and identify dermatophytes compared to
Selective isolation of bacterial DNA from clinical specimens
H.-P. Horz1, S. Scheer1, F. Huenger2, M. Vianna1, G. Conrads1
University Hospital RWTH Aachen, Division of Oral
Microbiology and Immunology, Department of Operative and
Preventive Dentistry & Periodontology, and Department of
Medical Microbiology, Aachen, Germany
University Hospital RWTH Aachen, Department of Infection
Control and Infectiology, Aachen, Germany
Enhanced universality of broad-ranged (inosine-containing)
16S rDNA primers as determined by terminal restriction
fragment length polymorphism (T-RFLP) profiling
B. Brands1, G. Conrads1, H. - P. Horz1
University Hospital RWTH Aachen, Division of Oral
Microbiology and Immunology, Department of Operative and
Preventive Dentistry & Periodontology, and Department of
Medical Microbiology, Aachen, Germany
Nucleic acid-based techniques offer a rapid and highly sensitive
option for detecting pathogenic bacteria directly from clinical
specimens. In particular broad-ranged (universal) bacterial
primers are useful when the etiological agent of an infectious
disease is not known and cultivation efforts remain
unsuccessful. However, the choice of primers to be used is
nontrivial since a true “universal” PCR system does not exist
and so-called universal 16S rDNA primers differ dramatically in
their ‘universality’. This limitation, frequently ignored, can lead
to false-negative results in routine microbial diagnosis and in
case of studies on the human microbiota to a distorted picture of
the true microbial diversity. To enhance the universality of 16S
rDNA primers we modified one validated broad-ranged primer
pair by replacing the essential 3’ termini of each primer with
inosine. The advantage of this base analogue is its potential to
pair with any of the four nucleotides. Whole genomic DNA was
extracted from five periodontal samples and bacterial 16S rRNA
genes amplified either with or without inosine containing
primers and with the forward primer being labeled with a
fluorescence dye. Resulting PCR products were digested with
the endonuclease Alu I followed by electrophoresis and size
determination of labeled terminal restriction fragments (T-RFs)
using automated capillary DNA sequencer technology. The
average number of 16 T-RFs detected when the original primers
were used was exceeded in most cases with an average detection
of 17 T-RFs when the forward primer contained inosine and 21
T-RFs when the reverse primer contained inosine. Based on the
interactive web-tool MICA (Microbial community analysis, and on preliminary
sequence analysis of cloned PCR-products, three of these
additional T-RFs were identified as uncultivated Treponema sp.,
uncultivated Synergistes sp. and Methanobrevibacter oralis. In
conclusion, inosine-primers can improve molecular inventories
of human microbial ecosystems and increase the sensitivity of
nucleic-acid based diagnosis of bacterial pathogens.
Identification of clinically relevant streptococcus species by
fluorescence in situ hybridization (FISH)
A. Sigge1, B. Spellerberg1, T. Zelensky1, N. Kästner1
S. Güntner1, S. Poppert1
University Hospital, Medical Microbiology, Ulm
Objectives: The current classification of the genus
Streptococcus differentiates several heterogeneous groups. A
reliable species identification of Streptococci via conventional
biochemical and phenotypic tests is not always possible,
particularly considering the viridans streptococci. Aim of the
study was to evaluate a set of 6 newly designed and 6 previously
published FISH probes for the identification of the of the most
important pyogenic streptococci and S. pneumoniae to species
level and of the viridans streptococci to group level.
Methods: A number of 240 strains including 26 ATCC and
DSM strains were evaluated. The 214 clinical isolates gained
from blood cultures, respiratory and urogenital tracts were
identified by API systems, Lancefield antigen agglutination,
Optochin susceptibility tests and sequencing of the sodA gene as
the gold standard. The following Streptococcus species were
included: S. acidominimus, S. agalactiae, S. anginosus, S.
australis, S. constellatus, S. dysgalactiae equisimilis, S. equi, S.
infantis, S. intermedius, S. massiliensis, S. mitis, S. mutans, S.
oligofermentans, S. oralis, S. parasanguis, S. peroris, S.
pneumoniae, S. pyogenes, S. salivarius, S. sanguis, S. sobrinus,
S. suis, S. uberis, S. vestibulariis.
Results: Seven FISH probes for the identification of all
Streptococcus spp., S. pyogenes, S. agalactiae, S. pneumoniae
and S. dysgalactiae subspec. equisimilishad a sensitivity and
specificity of 100%. The probes targeting the anginosus, mitis
andsalivarius and mutans groups had a sensitivity of 87 up to
100 % and a specificity of 95 up to 100% respectively.
Conclusions: The described FISH assay allows reliable
identification of the clinically most relevant Streptococci and
may thus be a suitable alternative tool, in particular for the
identification of strains that exhibit biochemical variability
belonging to the anginosus, mutans and mitis group.
Quantification of HPV16 E6*I transcripts in cervical cancer
screening using rapid real-time RT-PCR amplification Increased levels of E6*I mRNA are indicative for severe
cervical intraepithelial neoplasia (CIN II+)
S. Kösel1, S. Burggraf1, W. Engelhardt1, B. Olgemöller1
Labor Becker, Olgemoeller und Kollegen, Munich, Germany
The integration of human papilloma virus (HPV) DNA into the
host genome and the up-regulation of viral E6 and E7 proteins
play a central role in the carcinogenesis of cervical carcinoma.
Since cervical dysplasia may regress to normal cytology or
progress to cervical carcinoma, it would be valuable to have a
diagnostic tool to help decide whether conisation should be
Cervical samples of 301 HPV16 positive women were collected
in RNAlater reagent to prevent RNA degradation. Relative
levels of HPV16 DNA and HPV16 E6*I mRNA in the samples
were determined using real time PCR. HPV16 DNA was
determined in relation to the beta-globin gene. E6/E7 mRNA
was quantitated against G6PDH mRNA. Molecular genetic
findings were correlated with histological diagnoses and
cytological follow-up.
HPV16 E6*I mRNA levels were significantly higher in women
with cytologically diagnosed severe cervical dysplasia than in
those with mild-to-moderate dysplasia, borderline or normal
cytology. Viral DNA levels were not significantly different
between severe and mild-to-moderate dysplasia. The positive
predictive value for a histological diagnosis of severe cervical
dysplasia (CIN II+) increased with the amounts of E6*I mRNA
to more than 90% whereas the sensitivity decreased. The
absence of HPV16 E6*I transcripts as well as HPV16 DNA
considerably increased the negative predictive value and the
specificity. However, low concentrations (or complete absence)
of E6*I mRNA did not preclude a CIN II+ diagnosis. High
levels of viral DNA were not indicative for CIN II+.
Although the sensitivity is limited, high levels of HPV16 E6*I
mRNA are indicative of CIN II+ in cytologically diagnosed
cervical dysplasia of individual patients. Thus, women may
benefit from early histological diagnosis and treatment without
further control samples.
Serodiagnosis of Tularemia: Clinical validation of a
combination of certified diagnostic test kits and comparison
with a new bead assay based on LuminexTM technology
S. Kopf1, A. Buckendahl1, E. Seibold1, P. Kaysser1
W. D. Splettstösser1,2
Bundeswehr Institute of Microbiology, Immunology, Munich
Institute of Medical Microbiology, Virology & Hygiene
Medical Microbiology, Rostock, Germany
Background: Detection of anti-Francisella tularensis antibodies
in sera from tularemia patients still represents a keystone in the
confirmation of the clinical diagnosis. In many European
countries, certified assays which are in accordance with the in
vitro diagnostic guideline (EC directive 98/79/EG) are not
readily available.In this study we demonstrate our clinical
experience with a newly introduced and certified combination of
classic immunological tests. Additionally, we show that the
application of a new test format might be an alternative to more
conventional test systems.
Methods: We compared a fluorescent microsphere immunoassay
(FMIA) for the detection of specific antibodies against
Francisella tularensis (LPS) with established and certified
diagnostic tools (enzyme linked immunosorbent assay (ELISA)
and immunoblot). Two additional microsphere populations were
used as internal serum and conjugate control.
Results: Employing 48 positive sera from patients and 246
negative sera from blood donors the FMIA had a sensitivity and
specificity of 100% and 99.2% respectively. From 238 samples
sent for a definite diagnosis of tularemia to our reference
laboratory, 43 specimens (18.1%) gave positive ELISA results.
But only 32 of these sera (74.4%) were also reactive in the
immunoblot which was similar in the FMIA.
Conclusion: The combination of a screening assay (ELISA) with
a confirmatory test (immunoblot) can reduce the occurrence of
false positive results by almost 30%. The FMIA proved to be a
promising tool with sensitivity and specificity comparable to the
combination of classic immunoassays but showed several
additional advantageous features: Broad dynamic range,
potential of multiplexing and automation and cost reduction of
about 90%.
Species-specific signature sequence of medically important
black yeast species
G. Haase1, G. S. de Hoog2
University Hospital RWTH Aachen, Institute of Medical
Microbiology, Aachen, Germany
Centraalbureau voor Schimmelcultures, Utrecht, Netherlands
Background: Members of the herpotrichiellaceaeous black
yeasts i.e. Cladophialophora spp., Exophiala spp., Fonsecaea
spp., Phialophora spp., Ramichloridium spp., and
Rhinocladiella spp. are of medical importance because they can
cause a variety of different mycoses whereas some of them
could be even life threatening. Therefore, a rapid and reliable
identification of respective isolates is mandatory. Unfortunately
with some rare exception classical phenotypical identification of
these pleoanamorphic fungi is hardly to achieve especially in
view of some of the recently described species like e. g. E.
calicioides, E. crusticola, E. nishimurae, E. oligosperma, and E.
Method: While assigning a secondary structure to their nuclear
ITS2 RNA molecule we came across that part of its second
domain exhibits a species-specific region of about 30
nucleotides. By screening a proprietary database including all
ITS2 sequences of the currently described type species of the
above mentioned genera we could confirm our observation with
exception of E. dermatitidis and E. phaeomuriformis both
exhibiting identical sequences at this locus. In the latter case
they could be discriminated by respective sequences differences
of the ITS1 gene.
Results: When comparing the respective sequences of further
isolates (> 5) of these species e. g. E. bergeri, E. castellanii, E.
dermatitidis, E. heteromorpha, E. jeanselmei, E. lecanii-cornu,
E. oligosperma, E. phaeomuriformis, E. pisciphila, E. salmonis,
E. spinifera, E. xenobiotica we could show that they all exhibit
this distinct barcode-like sequence thereby approving that this
signature sequence is really species-specific and showing no
intraspecific variation.
Conclusion: Since the ITS2 gene exhibits longer stretches of
identical nucleotides at the 5’ end and showed length differences
upon alignment respective BLAST searches can give rise to
ambiguous or even erroneous species identification by using the
whole sequence. In contrast to such an approach usage of the
described signature sequence is highly reliable and identification
can be performed manually by simply inspecting this
particularITS2 sequence of a given isolate
Novel strongyloides spp. real-time PCR
S. Kramme1, K. Erttmann1, N. Brattig1, B. Fleischer1
C. Drosten1, M. Panning1
Bernhard-Nocht-Institute, Hamburg, Germany
Stongyloides infection is widely distributed in tropical as well as
subtropical regions worldwide. Clinical presentation of the
infection in most cases remains asymptomatic. However, in
immunocompromised hosts disseminated infection can be
serious and often fatal. Classically diagnosis relies on the
detection of larvae in fecal samples but is rather insensitive due
to low and intermittent output. Use of up to three fecal samples
can increase sensitivity. Culture methods do exist but are rather
time consuming and labour intensive. Recently PCR based
protocols for helminth parasites have been described. We
present the first real-time PCR to detect Strongyloides spp. in
stool samples. The assay targets the 28S rRNA gene. Real-time
detection is accomplished by use of hybridization probes on a
LightCycler instrument. The limit of detection is around 10
DNA copies/reaction. To detect possible inhibitory substances
the assay is equipped with a competitive internal control
meeting the requirements of modern molecular tools.
Quantification of Strongyloides spp. DNA copies per g stool is
possible. For preclinical evaluation the assay was used to
monitor Strongyloides ratti larvae excretion in a rat model. S.
ratti was detected by real-time PCR 4 days post infection. PCR
remained positive for at least 18 days. For specificity testing a
panel of 30 stored stool specimens of patients without a travel
history to endemic regions was tested and remained negative. In
addition the test remained negative when different other
helminth parasites were tested.
This novel real-time PCR will be useful to complement
diagnosis in suspected patients with negative stool specimens.
Definite diagnosis will allow for initiation of specific chemotherapy, which can be life-saving in immunocompromised hosts.
Furthermore this assay can be used in experimental settings to
gain insights into the pathogenesis of this not well understood
Development of semi-nested and quantitative real time pool
PCR methods for the detection of Mycobacterium avium
subsp. paratuberculosis in seropositive and seronegative
dairy cows
I. Völkel1, S. Urstadt1, A. Karapetyan1, S. Thebille1
M. Niederhausen1, F. Schmelz1, C. - P. Czerny1
Georg-August-University, Institute of Veterinary Medicine
Goettingen, Germany
The etiologic association of Mycobacterium avium subsp.
paratuberculosis and Morbus Crohn in humans remains still
unclear, however, the bacteria are the infectious agents of
Johne´s disease, a chronic and degenerative wasting disorder
primarily in adult wildlife and domestic ruminants, such as
cattle, sheep, and goats. Transmission to newborn calves occurs
insidiously through the fecal-oral route by introduction of
subclinically or persistently infected cattle. These asymptomatic
carriers and undiscovered shedders are one of the driving forces
of permanently occurring herd infections and are the reason of
unsuccessful eradication efforts over many years based on
serological methods. For future certification programs we
developed and evaluated several new and highly sensitive PCR
assays detecting Mycobacterium avium subsp. paratuberculosis
DNA in feces, milk, organs, tissues of infected animals, as well
as in environmental samples. Based on a conserved and specific
region within the IS 900, a semi-nested PCR was established
amplifying 587 bp and 278 bp products. Both genome regions
were also targets for the development of two real-time PCR
types on the LightCycler. Specificity of the PCRs was
confirmed by reference strains and field isolates cultivated on
HEY-medium, IS 900 specific DNA probes, and sequencing of
the amplicons. The detection limit of the semi-nested PCR was
calculated with 1 genome equivalent. The limits of the real time
PCR variants were 10 and 2 genome equivalents, respectively.
The PCRs were able to detect one shedder within 800 fecal
samples or 40 milk samples of non-infected animals under field
conditions. For an economic screening of shedders in cattle
herds feces pools were prepared on an ELISA-based selection
strategy. In continous control surveys of herds with 6-12 months
test intervals most of the PCR positve animals shedding the
pathogen were seropositive in commercial ELISAs, too.
Intermediate shedding was observed in animals with low serum
antibody values, whereas high antibody titres and positve PCR
results were correlated with clinical disorders in most of the
cases. The unmatched value of the pool PCR methods was the
detection of serologically negative shedders which could be
traced back by individual PCRs. These animals remained
seronegative over several months and showed no signs of the
disease. Fecal PCR methods are the only alternative indicating
these silent shedders within a herd rapidly and contribute to their
immediate elimination. In pooled milk samples bacteria contents
are too low.
Realtime-like species identification of candidemia using dual
colour PNA gene probes
H. Peltroche-Llacsahuanga1, R. Lütticken1, S. von Oy1
G. Haase1
University Hospital RWTH Aachen, Institute of Medical
Microbiology, Aachen, Germany
Background: Candida albicans (Ca) and Candida glabrata (Cg)
are the two most frequently isolated fungi from blood cultures
(BC). Since almost all Ca isolates are still susceptible to
fluconazole, whereas a higher proportion of Cg is only
intermediate susceptible or resistant against this inexpensive and
relatively easy-to-handle antifungal drug, immediate and
reliable discrimination of these two species is desirable. This
can not be achieved by applying conventional identification
procedures. Therefore, we evaluated performance of the newly
developed fluorescence in situ hybridisation test (FISH)
applying differently labeled peptide nucleic acid (PNA) probes
allowing discrimination of Ca and Cg within ~2.5h.
Methods: Fluorescent labelled PNA probes targeting species
specific 26S rRNA sequences of Ca and Cg were developed and
evaluated by FISH (prototype - AdvanDX, Woburn, MA, USA).
The probe reagent containing a mixture of the two PNA probes
was applied to smears made directly from positive BC bottles of
different types (BactAlert, bioMérieux, Nuertingen, Germany –
bottle types SA, SN, FA, FN, PF). After incubation for 90 min
at 55°C, unbound probe was removed by washing at 55°C for 30
min. Smears were then examined by fluorescence microscopy.
Ca and Cg were identified as bright green or red fluorescent
yeast cells, respectively. Results were compared to identification
achieved by ID 32C (bioMérieux).
Results: When testing BC of different types, 100% agreement
between the results of PNA FISH and conventional methods
was revealed (C. albicans, n = 3; C. glabrata n = 3; C.
dubliniensis n = 2; C. guilliermondii n = 1; C. krusei n = 2; C.
lusitaniae n = 2; C. parapsilosis n = 2; C. tropicalis n = 2; S.
cerevisiae n = 2; T. asahii n = 1). Testing spiked BC (n = 17)
inoculated with a mixture of Ca and Cg with these other
commonly encountered yeast species also revealed 100%
species specific results when applying the dual color PNA FISH.
Conclusion: Dual colour PNA FISH turned out as a reliable and
rapid (~2.5h) method for identification of both Ca and Cg
directly from positive BC bottles. Preliminary data show that the
hybridization time can be shortened to 30’ without obvious loss
of sensitivity. Application of dual PNA FISH in the routine
workflow turned out to be promising. Rapid discrimination of
these two most frequently encountered fungi should lead to
improvements in antifungal therapy.
Malaria investigation by Beckman Coulter Gen.S
Y. Aniwatangkoora1, S. Toonkomdang1,2, P. Kongdoung1
Y. Changtrakun2
Khon Kaen University, Department of Clinical microscopy
Faculty of Associated Medical Sciences, Khon Kaen, Thailand
Khon Kaen University, Diagnostic Microscopy Unit
Srinagarind Hospital, Faculty of Medicine, Khon Kaen
Detection of malaria infection by means of routine laboratory
instrument is still limited. The aim of this study was to evaluate
the performance of positional parameter; volume, conductivity
and scatter (VCS) performed by Beckman Coulter Gen. S in 9
cases of malaria patients prior to treatment; positive cases and in
those of negative cases; after treatment. The discriminant value
of VCS for malaria investigation was calculated, based on
criterion of greater than 5.0568 indicated the presence of malaria
with sensitivity of 96.9% and specificity of 82.5%. The results
of the positional parameters composition between 9 positive
cases and 9 negative cases of malaria exhibited the significant
differences for neutrophil mean conductivity, p<0.01 and
standard deviation conductivity for lymphocyte, p<0.01. The
discriminant value in either all positive cases or negative cases
were greater than 5.0568, showing of response to malaria by a
large activated monocyte. No significant correlation between the
percentage of parasitemia (0.15-21.1%) and the discriminant
value was detected. The presence of a typical peak at WBC
threshold (35 fl) was observed in both positive cases and
negative cases. This typical peak was not present in normal
samples. The scatterplot demonstrated heterogeneity of the
volume of monocyte and lymphocyte. The conclusion of present
study indicated possibility of malaria detection by Beckman
Coulter Gen. S. The user must first consider on the discriminant
value of positional parameters, platelet histogram, WBC
histogram and the message then focus on blood smear. Most of
studied malaria cases were Plasmodium falciparum,
investigation of patient with other malaria species should be
performed for further study to complete the knowledge.
Detection and identification of Legionella spp. from manmade water systems and aquatic environments
H. Zinecker1, R. Gutjahr2, H. Maucher1, A. Breitenstein1
Scanbec GmbH, Halle/Saale, Germany
University of Applied Science Merseburg, Department
Engineer- and Natural Sciences, Merseburg, Germany
In this study we present a rapid and sensitive detection method
for the genus Legionella and for the clinically relevant species
Legionella pneumophila. Legionella species, the cause of
legionellosis, are predominant in artifical water systems.
Legionella infections are transmitted from these via aerosols by
aspiration to the human respiratory system. Thus, providing fast,
reliable and sensitive detection methods of Legionella bacteria
for routine water diagnostics is of high urgency.
The FastScan molecular test system is based on the detection of
target molecules (rRNA) from the microorganism of interest by
means of specific oligonucleotide capture and detection probes
in an enzyme linked sandwich hybridization assay (SHA) [1].
The most sensitive assay has a detetion limit of 5 amol target
molecules that corresponds to 3x106 16S rRNA molecules and
about 450 cells Legionella pneumophila. We were able to detect
specifically 26 Legionella strains of 8 species including 15
Legionella pneumophila serogroups.
Since there is the possibility to apply the SHA test system as a
cultivation independent analytical method not only viable but
also non-culturable Legionella cells can be detected. The
problem of non-culturable Legionella cells, the occurence of
Legionella in clumps or intracellular in amoebae and finally the
overgrowing by faster propagating microorganisms are major
difficulties of the certified culture methods following ISO
11731-2 which leads to an underestimation of the real number of
Legionella cells in a sample. As the FastScan test targets RNA
only living cells as well as non culturable organisms will be
detected. Therefore the FastScan method is perfectly applicable
for risk assessment or monitoring of biocide treatments of manmade water systems.
Furthermore we demonstrate the evaluation of the new
developed test system with real water samples from cooling
towers by comparison with the cultivation based methods (ISO
11731-2, NF-T 90431).
[1] Leskela T, Tilsala-Timisjarvi A, Kusnetsov J, Neubauer P,
Breitenstein A (2005) Sensitive genus-specific detection of
Legionella by a 16S rRNA based sandwich hybridization assay.
J Microbiol Methods 62:167-79.
Combination of a unique sample preparation and a novel
PCR-based analytical tool for the detection of microorganisms in sepsis patients
S. Sachse1, M. Lehmann2, J. Landré2, K. - H. Schmidt1
S. Russwurm2, E. Straube1
Friedrich-Schiller-University Jena, Institute for Medical
Microbiology, Jena, Germany
SIRS-Lab GmbH, Jena, Germany
Purpose: In recent years molecular diagnostic has become a well
established discipline within laboratory medicine. In contrast to
culture based methods, molecular procedures e.g. nucleic acids
techniques (NAT) enable fast detection of potential pathogens
irrespective of their growth, sample source as well as
independent from already initialised antibiotic therapy.
However, NAT has hurdles such as lower sensitivity caused by
the high human DNA background and presence of inhibitory
substances in the sample. Reduction of these problems requires
an appropriate DNA preparation that eliminates inhibitory
substances like immunoglobulins or haemin. Furthermore, to
avoid false-negative results caused by disproportionately high
interfering human DNA load in contrast to low amount of
bacterial DNA present in DNA material, conventional
preparation kits reach their limits. We developed a new preanalytical tool based on the concentration of bacterial DNA
(Looxster®), followed by a multiplex PCR-based assay allowing
the detection of sepsis-relevant pathogens.
Methods: We developed a multiplex PCR-based assay in
combination with a unique sample preparation tool. The assay
was tested using artificial DNA mixtures (pro- and eukaryotes)
as well as DNA derived from blood spiked with various microorganisms. The validation process was performed using clinical
whole blood samples from patients having a suspected systemic
infection (sepsis) and controls.
Results: It was demonstrated, that the multiplex-assay is able to
specifically detect the targeted micro-organisms in a single as
well as multiple infection model. These results were validated
with clinical samples.
Conclusion: This multiplex PCR-based assay in combination
with Looxster®, a kit for bacterial DNA concentration from
complex samples, can provide the clinician with a specific and
sensitive microbiological result within the first hours of sepsis.
Thus, the clinician can initiate an appropriate antibiotic
treatment or change as well as de-escalate already started
antibiotic therapy.
A diagnostic tool for the detection of Legionella RNA
T. Schüler1, R. Möller1, A. Breitenstein2, W. Fritzsche3, J. Popp1
Friedrich-Schiller-University Jena, Institute for physical
Chemistry, Jena, Germany
ScanBec GmbH, Halle, Germany
Institute for Photonic Technology, Nanobiophotonic, Jena
In the last decade DNA microarray technology has become of
great importance in the field of bio analytics. The need for
simple, fast and, cost effective methods for the analysis of bio
molecules was the initialization to develop various detection
units. Currently bio molecules such as DNA, proteins and others
are mainly detected on biochips by marking them with
fluorescence dyes. To overcome the disadvantages of this
technique, like photo bleaching and quenching, metal
nanoparticles are widely used. Due to the unique physical
properties of metal nanoparticles for example electrical
conductivity, their specific mass or the interesting optical
attributes many approaches have been developed[1]. The
coupling of gold nanoparticles to bio molecules allows a
specific analysis of bio molecule interactions. This work
presents an electrical method for DNA detection. It is based on
the immobilization of single stranded capture DNA in a gap
between two microstructured electrodes on a specially designed
chip. The use of an enzyme instead of gold nanoparticles offers
advantages like low background, increase of the sensitivity and
a fast reaction. After the hybridization of the target DNA to the
specific partner immobilized on the chip, an enzyme
(horseradish peroxidase) is bound to a special modification in
the target molecule. Finally, an enzyme induced deposition of
silver nanoparticles leads to a bridging of the gap between the
electrodes. An increase in the conductivity over the gap serves
as signal in the reader[2]. The aim of this work was to automate
the system and realize a fast, robust, and easy way for the
detection of bio molecules. For that purpose we developed a
special reaction camber. This allows the integration of the DNAchip in a micro fluidic system, which offers some advantages
like time saving and increase of sensitivity. Additionally the
possibility of an online detection, to control the conductivity
over the gaps simultaneously, was investigated. To show the
potential of this technology a specific detection of Legionella-
RNA, especially Legionella pneumophila, was aspirated, to
enable a fast and robust detection of Legionella outside of
specialized laboratories.
1. Fritzsche, W. and T.A. Taton, Metal Nanoparticles as Labels
for Heterogeneous, Chip-Based DNA Detection.
Nanotechnology, 2003. 14: p. R63-R73.
2. Moeller, R., et al., Enzymatic Control of Metal Deposition as
Key Step for a Low-Background Electrical Detection for DNAChips. Nano Letters, 2005.
Identification of Coxiella burnetii with a novel Low-Costand-Density (LCD)-Microarray
D. Frangoulidis1, V. Heiser2, O. Landt3, H. Meyer1
Institute for Microbiology, Munich, Germany
Chipron GmbH, Berlin, Germany
TIB-MOLBIOL GmbH, Berlin, Germany
In our study we investigated the suitability of a new and fast
“low cost and density”(LCD) DNA microarray for the detection
of Coxiella (C.). burnetii, the causative agent of Q fever. Results
were compared with a conventional and a LightCyclerHyprobePCR assay in terms of specificity and sensitivity.
The LCD-microarray has the format of a plastic slide-frame
which contains eight identical microarrays in individually
addressable hybridization fields, with oligonucleotides derived
from the IS1111-region (repetitive element, up to 20 copies) and
the adaA-gene (recently proposed as a marker for acute Q fever
disease). A non-fluorescent detection principle, based on the
formation of a clearly visible substrate precipitate, is used.
Analysis can be done with a simple transmission light scanning
device or by pure eye only.
Eleven different C. burnetii strains derived from animals and
humans with acute and chronic disease were analyzed. All
strains showed in all three assays a positive reaction with both
targets, with the exception of one chronic and one animalderived strain that were negative for adaA. The sensitivity was
determined to be 10 genomic copies/µl template for IS1111 and
100 copies for adaA. No unspecific reactions were seen with a
DNA panel of related bacteria. The results of the realtime-and
conventional PCR-method were similar to the LCD-Chip.
This new technique provides a very fast, economic, sensitive
and specific detection of Coxiella burnetii. In addition, since no
sophisticated lab equipment is needed, this method could be
introduced into the field to conduct clinical, experimental and
epidemiological studies in animals and man.
MALDI-TOF for species identification of nonfermenting
bacteria: Evaluation and Comparison to 16S rDNA
A. Mellmann1, J. Cloud2, T. Maier3, U. Keckevoet1
I. Ramminger1, P. Iwen4, J. Dunn5, G. Hall6, D. Wilson6
P. R. LaSala7, M. Kostrzewa3, D. Harmsen8
University Hospital Muenster, Institut für Hygiene, Muenster
ARUP Inst. Clin. Exper. Pathol., Salt Lake City, Utah, USA
Bruker Daltonik GmbH, Leipzig, Germany
University of Nebraska Medical Center, Omaha, Nebraska
Cook Children’s Medical Center, Fort Worth, Texas, USA
Cleveland Clinic Foundation, Cleveland, Ohio, USA
University of Texas Medical Branch, Dept. of Pathology
Clinical Microbiology, Galveston, Texas, USA
University Hospital Muenster, Polyclinic for Parodontology
Muenster, Germany
Nonfermenting bacteria (NFs) are ubiquitous environmental
opportunists causing infection in severely ill and immunocompromised patients. Due to their limited biochemical
misidentification occurs frequently. Therefore, we evaluated
matrix-assisted laser desorption/ionization time-of-flight
(MALDI-TOF) mass spectrometry for species identification.
Using 277 NF culture collection strains composed of 29 genera
most relevant for human infections, a reference database was
established for MALDI-TOF based species identification by
following manufacturer’s recommendations for microflex
testing and MALDI BioTyperTM software (Bruker Daltonik
GmbH, Germany). To evaluate the database, 93 blind-coded
clinical NFs were analyzed. As “gold-standard” for species
designation, partial 16S rDNA sequencing was used.
From the 93 isolates, all 13 Acinetobacter isolates were
excluded due to their ill-defined species designations. Using 16S
rDNA sequencing, 57 of the remaining 80 isolates produced
unique species identification ( 99% sequence similarity). 10
isolates were identified to genus level ( 97% similarity), 11
isolates gave no unique result, and 2 isolates gave a sequence
similarity <97%. MALDI-TOF identified 57 of the 67 isolates
(85.1%) to the correct species or genus within 5 minutes without
any substantial costs for consumables.
MALDI-TOF provides accurate, fast, and economical results
and represents a promising alternative for species identification
in routine diagnostics of NFs. Future expansion of the database
will enhance the utility of this methodology to identify unusual
bacterial pathogens.
Diagnostic DNA chips to detect -Lactam resistance in the
D. M. Leinberger1,2, V. Grimm2, M. Rubtsova3, A. Henn2
K. Schröppel4,5, T. A. Wichelhaus6, C. Knabbe4, J. Weile4
R. D. Schmid2, T. T. Bachmann1,2
University, Division of Pathway Medicine, Edinburgh
United Kingdom
University, Institute of Technical Biochemistry, Stuttgart
National Research Center for Antibiotics, Moscow, Russia
Robert-Bosch-Hospital, Department of Clin. Chem. and
Laboratory Medicine, Stuttgart, Germany
Eberhard-Karl-University, Institute of Medical Microbiology
and Hygiene, Tuebingen, Germany
Hospital of J. W. Goethe-University, Institute of Medical
Microbiology and Infection Control, Frankfurt am Main
Culture based antimicrobial susceptibility testing methods are
mostly too slow to allow for a rapid prospective therapy and in
practice the calculated therapy is selected according to a clinical
standard. Microbial resistance against beta-lactam antibiotics in
gram negative bacteria predominantly originates from extended
spectrum beta-lactamases (ESBL) whose activity against betalactam antibiotics and resistance against inhibitors is determined
by mutations in members of the blaTEM, blaSHV, or blaCTX-M gene
family. Additionally, plasmid-mediated AmpC lactamases have
emerged in the clinics during the past decade as a new severe
problem since they are likely to hydrolyze modern
cephalosporins. The more and more frequently detected OXAtype enzymes may be even more considered as a critical thread
because they can confer resistance against carbapenems which
are regularly used for the treatment of ESBL pathogens. Here,
we describe an ESBL-chip comprising of an integrated
diagnostic oligonucleotide microarray for the rapid
identification of ESBL in gram negative bacteria by genotyping
blaTEM, blaSHV, or blaCTX-M. The validity of the chip was
demonstrated on 60 clinical samples which where collected
during one year in a clinical routine laboratory and
phenotypically characterized as “ESBL positive”. The chip
demonstrated an excellent resolution, high phenotype genotype
correlation, and robustness towards mixed genotypes. The data
where confirmed by standard DNA sequencing. All ESBL
phenotypes could be ascribed to the presence of ESBL variants
of either blaCTX-M (78%) or blaSHV (22%), whereas no ESBL
blaTEM variant was found. Two isolates contained two ESBL
blaCTX-M variants originating from different specimens of a single
patient. The assay was performed within four hours and may be
an attractive option for rapid identification of beta lactamase
resistance in clinics with high prevalence of ESBL positive
gram negative species. Because of its full coverage of 98% of all
known variants to date, we anticipate a great value of the ESBLchip assay for epidemiologic monitoring as well. To further
increase the ESBL-chip coverage for other lactamases, we
developed new chip modules for all known plasmid-mediated
AmpC and group I, II, and V OXA-type beta lactamases which
will further enhance the clinical value of the ESBL-chip assay.
Real-time PCR-based in vitro susceptibility testing of
Borrelia burgdorferi against antimicrobial agents in the
presence of eukariotic cells.
T. Bittner1, A. Freyer1, V. Brade1, K. - P. Hunfeld1
University Hospital Frankfurt am Main, Institute for Med.
Microbiology and Hygiene, Frankfurt am Main
There is evidence that human cells may have protective effects
on B. burgdorferi isolates against antibiotic agents in vitro but
the clinical impact of such observations remains uncertain.
Nevertheless, very few isolates of Borrelia burgdorferi have
been tested for their antimicrobial susceptibility in the presence
of eukaryotic cells. As with other fastidious or intracellular
bacteria, the exact monitoring of borrelial growth in cell cultures
is laborious and no precise standardized test methods are
currently available. Here, a recA-real-time PCR-based
susceptibility assay was evaluated and used to test 5 borrelial
strains including strains obtained from patients before and after
antibiotic treatment under standardized conditions. The isolates
were tested in the presence and absence of a human acute
monocytic leukaemia cell line (THP-1) against doxycycline and
ceftriaxone, antibiotic agents that are known for their mainly
intracellular respective extracellular activity. After 72 h of
incubation, minimal inhibitory concentrations (MICs) were
determined by software-assisted calculation of bacterial growth
in samples and controls from semi-quantitative PCR results. Our
new recA-PCR-based assay system was shown to be sensitive,
convenient, and reliable. The assay is capable of testing larger
numbers of isolates and antimicrobial agents under standardized
and very precise test conditions in the presence of eukaryotic
cells. In the absence of THP-1 cells median MICs ranged
between 0,125 and 0,25 mg/l for doxycycline and between 0,016
and 0,063 mg/l for ceftriaxone. In contrast to previous reports,
however, our study did not substantiate significant differences in
the susceptibility of borrelia to both agents after co-cultivation
with THP-1 cells. Our new assay proved convenient and offers a
competent technical solution of the known difficulties
associated with in vitro testing of fastidious organisms such as
borrelia when cultivated in the presence of eukaryotic cells.
Such studies may be helpful to further analyse potential escape
mechanisms of borrelia exposed to antimicrobial agents in the
presence of other human cell lines.
Biochip-based resistance monitoring in pathogenic fungi for
clinical diagnostics
M. Mai1, S. Rupp1, N. Hauser1
Fraunhofer IGB, Molecular Biotechnology, Stuttgart, Germany
For the treatment of fungal systemic infections mostly caused by
Candida albicans, C. glabrata or Aspergillus fumigatus there
exists only a certain amount of antifungal drugs (azoles,
echinocandins, polyenes and pyrimidine analogues). With the
increasing number of immuno-compromised patients (e.g.
AIDS, organ transplants or other immunosuppressive therapies)
worldwide, antifungal treatments have increased with a
paralleled increase of the incidence of antifungal resistance.
Antifungal resistance mechanisms fall into different categories
according to their molecular principles as for instance transport
or target alterations. Target gene alterations as a cause for
antifungal resistance have been observed in several alleles of the
ERG11 gene encoding lanosterol 14a-demethylase, the target of
azoles in fungal pathogens. Mutations in TAC1 gene are
associated with azole resistance by up-regulation of the ABC
transporter genes CDR1 and CDR2. In C. albicans, many
different single point mutations and loss of heterozygosis in the
ERG11 gene as well as in the TAC1 gene have been described.
To determine new resistance mechanisms in clinical relevant
fungal pathogens a European network has been established, the
EURESFUN network (European RESistance FUNgal network).
Within this network we developed a diagnostic tool based on
minisequencing with on-chip primer extension for monitoring
resistance in clinical relevant fungal pathogens (Candida and
Aspergillus). This method enables a high parallel genotyping of
homozygote and heterozygote situations associated with
Up to now we have established a biochip with approximately
100 probes for the detection of resistance in Candida albicans.
Initially, a model-system using synthetic templateshas
beendeveloped to optimise the experimental parameters for the
chip-based enzymatic reaction. This system is further used as
control and for normalisationof signals. The optimised system
could be adapted for the detection of SNPs (single nucleotide
polymorphisms) and LOHs (loss of heterozygosis) with high
specificity and sensitivity. Using genomic DNA from clinical
isolates homozygous and heterozygous situation associated with
resistance could be detected. Furthermore we will expand our
chip by new resistance genes being identified by the network.
Rapid identification of pathogens from blood cultures by
Fluorescence in situ Hybridization (FISH)
S. Poppert1, M. Bartel1, A. Lakner1, N. Wellinghausen1
A. Essig1
University Ulm, Institute for Medical Microbiology, Ulm
Rapid identification of pathogens from blood cultures is of
major importance, because the timely initiation of an adequate
therapy greatly influences patient outcome and in addition may
lead to cost savings. As previously published rapid identification
(1-2 hours) may be achieved by Fluorescence in situ
complementary to specific sequences of the ribosomal RNA.
The method has not yet been broadly introduced into routine
diagnostics, because the number of covered pathogens is still
limited and available probes have not yet been evaluated on
sufficiently large sample collections.
In a first step we therefore evaluated 12 probes from the
literature and 17 newly designed probes, including probes for
Staphylococci, Streptococci, Enterococci, Pseudomonas,
Acinetobacter, Propionibacterium and yeasts using a collection
of more than 900 clinical isolates. Nearly all selected probes
demonstrated a sensitivity of 100% and specificities of better
than 95%.
In the next step the FISH method was assessed on 891 positive
blood cultures according to an algorithm based on the gram
stain. In 829 (93%) cases the pathogen was correctly identified
by FISH. In 56 cases (6,3 %) no result was achieved because the
respective rare pathogens were not covered by the probe set. In
just 6 cases (0,7%) FISH lead to a false result.
In conclusion FISH proved to be a highly sensitive and specific
tool for fast identification of pathogens from blood cultures. As
an easy performable and cheap method, FISH is suitable to
accelerate pathogen identification in a routine laboratory and
may thus improve patient outcome and lower therapy costs.
Rapid identification of Listeria species using MALDI-TOF
MS fingerprinting with MALDI BioTyper
S. Barbuddhe1, T. Maier2, G. Schwarz2, M. Kostrzewa2, E.
Domann1, T. Chakraborty1, T. Hain1
Institute for Medical Microbiology, Giessen, Germany
Bruker Daltonik GmbH, Leipzig/Bremen, Germany
Listeria monocytogenes is a food-borne pathogen that is the
causative agent of human listeriosis, an opportunistic infection
that primarily infects pregnant women and immunologically
compromised individuals. Rapid, accurate discrimination
between Listeria strains is essential for appropriate food quality,
therapeutic management and timely intervention for infection
control. A rapid method involving mass spectrometry (MS) is
presented that shows promise for identification, discrimination
of Listeria species and lineage typing after analysis of 111
strains. The method was compared with the PFGE analysis of 47
Listeria strains isolated from varied sources including 27 L.
monocytogenes strains associated with foodborne epidemics and
sporadic cases. Cells from a bacterial colony are applied to a
sample target plate directly or after a short extraction protocol.
After drying and addition of a chemical matrix, the samples are
analysed by matrix-assisted laser desorption ionisation time-offlight mass spectrometry (MALDI-TOF MS). This technique
examines the chemistry of the whole bacterial extracts, yielding
spectra consisting of a series of peaks mainly derived from
ribosomal proteins. Specimens can be prepared in a few minutes
from plate cultures and a spectrum can be obtained within one
minute. MS spectra for Listeria isolates showed characteristic
peaks, conserved at species level, some at lineage level. MS
may have potential for Listeria identification and lineage
subtyping of L. monocytogenes strains, and may improve food
quality and clinical infection control measures.
Multiplexed genotyping of methicillin resistant
Staphylococcus aureus isolates with padlock probes and tag
U. Nübel1, K. Kurt1, B. Strommenger1, A. Weller1, C. Cuny1
A. Alderborn2, M. Nilsson2, W. Witte1
Robert-Koch-Institute, Wernigerode, Germany
Uppsala University, Department of Genetics and Pathology
Uppsala, Sweden
We developed and tested a ligase-based assay for simultaneous
probing of core genome diversity and typing of methicillin
resistance determinants in Staphylococcus aureus isolates. This
assay uses oligonucleotide padlock probes whose two ends are
joined through ligation when they hybridize to matching target
DNA. Circularized probes are subsequently amplified by PCR
with common primers and analysed by using a microarray
equipped with universal tag probes.
Our set of padlock probes includes oligonucleotides targeting
diagnostic regions in ccrB, ccrC, and mecA genes of the
SCCmec cassette in methicillin resistant S. aureus (MRSA).
These probes determine the presence and type of SCCmec
cassettes (i. e., SCCmec types I to VI). Additional
oligonucleotides interrogate a number of highly informative
single nucleotide polymorphisms retrieved from a multilocus
sequence typing (MLST) database. These latter probes enable
the exploration of isolates'phylogenetic affiliation to major
clonal lineages of MRSA as defined by MLST, including, for
example, clonal complexes CC1, CC5, CC8, CC22, CC30.
The described assay enables multiplexed genotyping of MRSA,
based on a single-tube reaction. It usefully complements the
more common sequence-based spa typing.
Multi locus VNTR sequence typing (MLVST) for
vancomycin resistant Enterococcus faecium
B. Middendorf1, D. Harmsen2, A.W. Friedrich1, C. Georgi1
J. Top3, R. J. L. Willems3, A. Mellmann1
University Hospital Muenster, Institute for Hygiene, Muenster
University Hospital Muenster, Department of Periodontology
Muenster, Germany
University Medical Center Utrecht, Department of Medical
Microbiology, Utrecht, Netherlands
Vancomycin-resistant Enterococcus faecium (VREF) cause
nosocomial infections especially in immunocompromised and
severely ill patients. To elucidate their molecular epidemiology,
PFGE is the current “gold standard” typing method for outbreak
investigations. However, it is very laborious and difficult to
standardize for intra- and inter-laboratory comparisons.
Recently, MLST and MLVA schemes were developed to
overcome these drawbacks. However, due to their low
discriminatory power their applicability in outbreak
investigations is limited. Therefore, we established a multi locus
VNTR sequence typing (MLVST) scheme for VREF.
To identify suitable repeat regions for MLVST, we analyzed the
preliminary genome sequence of E. faecium DO with the
program ‘Tandem Repeats Finder’. From a total number of 1580
repeat regions, three were finally selected for further analysis
(VNTR0032, VNTR0047, and VNTR1398). The corresponding
regions were amplified and sequenced from a well characterized
collection of 144 VREF representing a broad spectrum of strains
isolated from environmental and clinical associated sources. To
evaluate MLVST, the discriminatory index (DI) and the
concordance to MLST and MLVA were determined for different
VNTR combinations.
The three VNTRs displayed individual DIs of 0.718, 0.794, and
0.835, whereas MLST and MLVA resulted in DIs of 0.971 and
0.935. The typeabilities for the various methods were 100 %
(VNTR0032), 91.67 % (VNTR0047), 84.03 % (VNTR1398),
100 % (MLST), and 80.56 % (MLVA). The combination of all
three VNTRs increased the DI to 0.931. Analysis between the
combination of all three VNTRs and MLST/MLVA resulted in
concordances of 93.1 % and 91.6 %. The highest DI values of
0.970 and 0.974 were accomplished by the combinations
VNTR0032/VNTR1398/atpA and VNTR1398/atpA/ddl (both
genes from the MLST scheme) with concordances to MLST of
97.3 % and 98.7 %, respectively.
In summary, MLVST could be a fast and less cost-intensive
alternative typing method for VREF. Ongoing studies
investigate the performance in comparison to PFGE.
Identification of Chlamydia pneumoniae antigens and
analysis of antibody response patterns using
S. Bunk1, I. Suznea2, J. Rupp3, M. Maass4, M. Przybylski2
A. Wendel1, C. Hermann1
University Konstanz, Biochemical Pharmacology, Konstanz
University Konstanz, Analytical Pharmacology, Konstanz
University Luebeck, Medical Microbiology and Hygiene,
Luebeck, Germany
University Hospital Salzburg, Paracelsus Private Medical
University, Salzburg, Germany
The microimmunofluorescence test (MIF) is still the gold
standard in C. pneumoniae serodiagnosis. The test is widely
used, although differences in antigen preparation and
subjectiveness in reading the results lead to intra- and
interlaboratory variances. ELISAs, using single C. pneumoniae
antigens or combinations of them for serodiagnosis, could
overcome these problems. Since there is only limited knowledge
about C. pneumoniae antigens and their diagnostic potential, our
project aimed to identify C. pneumoniae antigens by an
immunoproteomic approach to develop reliable serodiagnostics.
C. pneumonia proteins were prepared from elementary bodies
(EB), separated by 2D-gel electrophoresis and blotted to
nitrocellulose membranes. The membranes were incubated with
39 human sera deriving from 28 seropositive and 11
seronegative donors, as determined by MIF, and bound IgG
antibodies were quantitatively analysed by a LAS-3000 imaging
system. Using high resolution MALDI-FTICR-MS, we
identified 38 Chlamydia proteins originating from 33 genes
which were frequently recognized among the 39 sera. About
half of the proteins, including some with high antigenic
potential, represent Chlamydia antigens not described before.
Fifteen proteins, most of them EB-surface exposed, showed a
significantly higher frequency and intensity of recognition by
sera from donors with high titers of C. pneumoniae antibodies
compared to sera with lower antibody titers, suggesting that they
contribute to MIF reactivity. Since our proteomic approach
allowed the quantitative analysis of sera reactivity towards
particular antigens, we also investigated whether donors with
evidence for persistent C. pneumoniae infection, determined by
PCR analysis of monocytes and vascular samples, show a
different pattern of antibody response compared to donors
without any evidence for persistence. We found that twelve
proteins were differentially recognized, which was in line with
the altered protein expression of in vitro models of
C.pneumoniae persistence. Taken together, these results provide
the basis for the development of new serological testsbased on
selected antigens, which might have the potential to determine
the status of disease.
Serological proteomic analysis for identification of
immunogenic C. trachomatis proteins in severe female
genital tract infection
V. Forsbach-Birk1, U. Simnacher1, K. - I. Pfepper2
E. Soutschek2, E. Straube3, A. Essig1
University Hospital, Medical Microbiology, Ulm
Mikrogen, Neuried, Germany
University Hospital, Medical Microbiology, Jena
Chlamydia trachomatis is the most common sexually
transmitted infectious agent in Europe and in the United States.
The infection markedly enhanceegnancy. Necessity of
diagnostic laparascopies as part of infertility investigations
might be reduced by less invasive methods of serological assays
but reliable marker proteins for chronic infections like pelvic
infls the risk for reproductive tract sequelae in women including
tubal infertility and ectopic prammatory disease (PID) are not
available. In order to characterize immunoreactive proteins in
severe C. trachomatis infections we screened chlamydial
proteins on a large scale separating proteins of elementary
bodies from C. trachomatis serovar D by 2D gelelectrophoresis.
The gels were blotted onto PVDF membranes and probed with
sera of women suffering from chronic chlamydial infection of
the upper genital tract. Immunoreactive proteins were identified
by mass spectrometry based methods. This serological proteome
analysis revealed a panel of thirteen chlamydial proteins
recognized by PID sera. Seven of them have been previously
described as immunodominant antigens during human infections
(MOMP, HSP60, HSP70, pmpD, OMP2, EF-Tu and ‘ribosomal
protein S1’). Another group of six immunoreactive chlamydial
proteins could be identified as ‘Adenylate cyclase-like protein’,
‘TARP’, ‘ATP-dependent Clp protease proteolytic subunit 1’,
‘Thio-specific Antioxidant Peroxidase’, ‘50S ribosomal protein
L1’ and ‘hypothetical protein’ (CT017). The identified
chlamydial proteins may serve as new test antigens in the
serodiagnosis of severe C. trachomatis infections and might be
used in vaccine experiments.
Genotyping of chlamydia trachomatis
J. Rödel1, S. Eberhard1
University Jena, Medical Microbiology, Jena, Germany
Genital infections by Chlamydia trachomatis serovars D
through K are frequent causes of sexually transmitted diseases.
Since 2003 an ongoing epidemic of lymphogranuloma
venereum (LGV) that is caused by the C. trachomatis serovars
L1-3 is observed in men who have sex with men in several
European countries. Commercial nucleic acid amplification tests
(NAAT) commonly used in routine diagnostics do not
differentiate between LGV and non-LGV Chlamydia strains.
Genotyping methods using restriction fragment length
polymorphism (RFLP) and sequence analysis of PCR amplicons
of variable domains of the omp1 gene coding for the C.
trachomatis major outer membrane protein can be used to
determine the distribution of C. trachomatis genotypes
according to different serovars and to accurately diagnose LGV
infections. Moreover, mixed infections with different
chlamydial strains and variants within a serovar can also be
Between 1999 and 2006 a total of 294 NAAT-positive samples
were genotyped by RFLP and sequence analysis at the
Consiliary Laboratory for Chlamydial Infections in Jena.
Serotypes E (36.4%), F (19.4%), and D (9.8%) were most
prevalent. Multiple infections with different serovars were only
rarely observed (0.3%). The predominance of serovars E, F, and
D may have impact on the development of improved serological
tests for the diagnosis of C. trachomatis infections in the context
of defining immunogenic antigens of these serovars. Since 2003,
12 probable LGV cases from different federal states were
confirmed as infections with C. trachomatis L2. Since the
epidemic of LGV infection reached Germany some years ago,
we cannot be sure that these infections are restricted only to
individuals of risk. Therefore, the genotyping of C. trachomatispositive NAAT-samples should be taken into consideration in
Antibacterial Activity of K9 Yeast Killer toxin
I. Ochigava1, W. Gräme M2
Biopharm-L, LtD, Tbilis, Georgia
Abertay University of Dundee, Biotechnology, Dundee
United Kingdom
The increasing incidence of antibiotic-resistant bacterial
infections has made identification and characterization of new
antibacterial therapies major goals of the pharmaceutical
industrial sector. Our attention was drawn to the killer toxin of
Williopsis saturnus var. mrakii K 9 (W. mrakii) because of its
high theromstability (1000C, for 10 min) and activity retention at
wide pH values (pH 2~11). This work describes the antibacterial
spectrum of crude K9 killer toxin (KT) preparation against a
range of Gram positive as Gram negative bacteria.
The killer yeast, Williopsis saturnus var. mrakii - NCYC 500,
was tested against the following bacterial cultures: E.coli –
NCIMB 10000; Staphylococcus epidermidis – NCIMB 12721;
Streptomyces griseus, Streptococcus pyogenes – NCIMB 8884;
Bacillus subtilis – NCIMB 1043, NCIMB 6633. Agar diffusion
assays were employed for killer toxin antibacterial activity using
W. mrakii live cells. For more detailed assessment of
antibacterial activity, we used a flow cytometric assay of crude
K9 toxin preparation using a fluorescent viability probe,
DiSBAC2(3). Both approaches confirmed W. mrakii and its K9
toxin exhibited antibacterial activity against Gram positive
bacteria. Although the flow cytometric assay revealed high
numbers of dead cells after the treatment with killer toxin, this
activity was sensitive-strain dependent, particularly in the case
of Bacillus spp.
These results provide one of very few reports of effective killer
yeast action against bacteria and form the basis of further
development of stable yeast toxins as novel agents in the fight
against drug resistant infections.
Arthroderma benhamiae evades complement attack by
binding host proteins
S. Schindler1, P. Zipfel1, A. Brakhage2
Leibniz Institute for Natural Product Research and Infection
Biology, Department of Infection Biology, Jena, Germany
Leibniz Institute for Natural Product Research and Infection
Biology, Department of Applied und Molecular Mikrobiology
Jena, Germany
Dermatophytes mediate human cutaneous mycosis. The role of
complement in immune evasion of dermatophytes is currently
unknown. Therefore we analyze immune evasion of the human
pathogenic dermatophyte Arthroderma benhamiae, a
teleomorph form of Trichophyton mentagrophytes. We
investigate binding of human proteins to the cell surface of this
pathogen. We demonstrate that factor H and the factor H related
protein 1 (FHR-1) bind to the cell surface of Arthroderma
benhamiae as demonstrated by direct binding assays,
immunostaining and ELISA. Human factor H is an important
complement regulary protein of the alternative pathway of
complement and FHR-1 is member of the factor H protein
family. In addition we show binding of plasminogen, the key
protease in fibrinolysis. Factor H and FHR-1 bind C3b, the
central molecule of complement activation. We argue that factor
H, bound to the surface of Arthroderma benhamiae is
functionally active and mediates cofactor activity in cleavage of
C3b. This leads to an inactivation of C3b and an inhibition of
complement activation. Thus the dermatophyte Arthroderma
benhamiae protects itself against complement attack by binding
factor H, FHR-1 and plasminogen to the cell surface.
CipC – an enigmatic fungal protein.
B. Bauer1, F. Ebel1
Ludwig-Maximilians-University, Max-von-Pettenkofer-Institute
Munich, Germany
Systemic fungal infections are a serious threat to severely
immunocompromised patients, and their increasing number is an
undesirable consequence of modern medicine. Invasive
aspergillosis is a life-threatening mycosis and its high mortality
is due to sub-optimal diagnostic and therapeutic options. The
development of novel anti-fungal strategies and drugs is
therefore an urgent need. Novel target structures should ideally
fulfil two requirements: (i) they should be specific for fungi and
(ii) they should be essentially required during infection.
The cipC gene encodes for a small 15 kDa polypeptide that was
originally described in Aspergillus nidulans as a concanamycin
induced protein. The availability of genomic information for
several fungi led to the identification of highly homologous
proteins. Corresponding mRNAs were identified in several
screens as strongly up regulated transcripts during symbiotic or
pathogenic interactions between the respective fungi and their
hosts. From these data we speculated that CipC could be a
potential target for novel anti-fungal drugs.
Analysis of the polypeptide sequence of CipC revealed no
functional protein motifs, homologous domains or homologies
to proteins of known function. However, all members of the
CipC family share a substantial homology and a conserved
molecular structure. Using a proteomic approach we originally
identified CipC as a major hyphal protein of Aspergillus
fumigatus, a finding that we could later on confirm using a
specific monoclonal antibody. Gel filtration and cross-linking
experiments suggest that CipC is a monomeric cytoplasmic
protein. To analyse the function of CipC we recently generated
an Aspergillus fumigatus cipC null mutant that is currently
under investigation.
Clinical Candida species fail to produce the secondary
metabolite gliotoxin in vitro
C. Kupfahl1, T. Ruppert2, A. Dietz1, G. Geginat1, H. Hof1
University Heidelberg, Medical Microbiology and Hygiene
Mannheim, Heidelberg, Germany
University Heidelberg, Zentrum für Molekulare Biologie
Heidelberg, Germany
Background: The secondary fungal metabolite gliotoxin exerts a
board spectrum of immunosuppressive effects in vitro and a role
of gliotoxin in the pathogenesis of invasive mycosis is
discussed. As it was previously suggested that Candida spp. are
also able to produce gliotoxin, we tested the frequency and
species distribution of gliotoxin production among clinical
relevant Candida spp..
Methods: A total of 100 clinical Candida isolates (40 C.
albicans, 15 C. glabrata, 15 C. krusei, 15 C. tropicalis, 15 C.
parapsilosis) were grown in liquid media and on agar plates for
7 days at 35°C. Yeast cells and culture supernatants were
extracted for gliotoxin and analysed with HPLC and
representative samples were further analysed with tandem mass
Results: Gliotoxin was not detectable in any of the extracts
analysed, despite the fact that various culture conditions were
tested. The detection limit for gliotoxin of the HPLC was less
than 10 ng/ml and with tandem mass spectrometry even 2 ng/ml
were unequivocally identified.
Conclusions: In contrast to previous reports, our data strongly
suggest that clinical relevant Candida spp. are not able to
produce the secondary fungal metabolite gliotoxin.
Production of authentic prostaglandins and thromboxane by
Aspergillus fumigatus in vitro
C. Kupfahl1, D. Tsikas2, G. Geginat1, H. Hof1
University Heidelberg, Medical Microbiology and Hygiene
Mannheim, Mannheim, Germany
HanoverMedical School, Institute of Clinical Pharmacology
Hanover, Germany
Background: It has been described previously that some fungi,
including Aspergillus spp., are able to produce prostaglandinlike compounds. We here investigated whether A. fumigatus,
one of the most notorious fungal pathogens, produces authentic
prostaglandins (PG) and thromboxane (Tx) and if PG and Tx
biosynthesis can be inhibited by cyclooxygenase (COX)
inhibitors such as acetylsalicylic acid (aspirin) or diclofenac.
Methods: Four clinical A. fumigatus isolates were cultured in
RPMI 1640 medium (fatty acid free) for 7 days. Subsequently
the medium was supplemented with 10 µM arachidonic acid
ethyl ester (AAE) and after an additional incubation period of
two hours, culture supernatants were sterile-filtered and
analysed for PGE2, PGD2, PGF2 , 6-keto-PGF1 , the stable
analogue of prostacyclin (PGI2), and thromboxane B2 (TxB2) the
stable analogue of TxA2, using a screening ELISA (covering
PGE1, PGE2, PGF1 , PGF2 and showing cross reactivity to
some other prostaglandins) and GC-MS/MS.
Results: All four strains tested produced prostaglandins and
thromboxane after 7 days of incubation and after feeding with
AAE as verified by GC-MS/MS. The main prostaglandin
produced was PGE2 (428±115 pg/ml). PGF2 , 6-keto-PGF1 , and
TxB2 were also detected but at lower concentrations (42±30
pg/ml, 20±7 pg/ml, and 6±4 pg/ml, respectively). In contrast,
PGD2 was not detected in any strain. Without feeding with AAE
only PGF2 and 6-keto-PGF1 were detected at concentrations
near the detection limit of the GC-MS/MS method (1 pg/ml).
Prostaglandin production of AAE-feeded strains was not
inhibited in a significant extent with relevant concentrations of
acetylsalicylic acid (1 µM to 100 µM) or diclofenac (1 µM to 10
µM) as measured by ELISA.
Conclusions: A. fumigatus is able to synthesize authentic
prostaglandins and thromboxane from arachidonic acid in vitro.
Synthesis of these potent mediators might play a role in the
pathogenesis of clinical fungal disease. Fungal prostaglandin
synthesis was not significantly inhibited by the COX inhibitors
acetylsalicylic acid or diclofenac at concentrations inhibitory for
the human COX isoforms 1 and 2. The possible involvement of
fungal prostaglandins in fungal disease warrants the search for
alternative inhibitors of the fungal prostaglandin and
thromboxane synthesis pathway. Such a COX-independent
inhibition may represent a possible new target for
supplementary antifungal therapy.
Systematic investigation of cell wall modulations in clinical
Candida glabrata isolates
O. Bader1, A. Schwarz1, M. Borg-von Zepelin1, U. Gross1
M. Weig1
Georg-August-University Goettingen, Medizinische
Microbiology, Goettingen, Germany
The cell wall of pathogenic fungi is the interface to the host
during colonization and infection, mediating a multitude of
interactive processes. Although it is known that bacterial cell
walls can mediate drug resistance, this has not yet been
investigated in fungi. To systematically identify resistance
mechanisms in pathogenic Candida species, we established a
collection (MycoLabNet-EU) containing over 400 fully or
intermediate drug resistant clinical yeast isolates within the
EURESFUN consortium. C. glabrata isolates were examined for
drug efflux with Rhodamine 6G by FACS analysis as well as
tolerance to several cell wall and other stresses. Azole crossresistant isolates generally displayed high efficiency drug efflux.
Isolates with intermediate azole MIC90 values showed only low
to medium drug efflux, but were more tolerant to cell wall
stresses. These isolates also were less susceptible to the
echinocandin Caspofungin. This may point towards
compensational modulations of the cell wall in response to azole
treatment when increased drug efflux is not available.
Toxoplasma gondii in skeletal muscle cells in vitro:
spontaneous differentiation from the tachyzoite to the
bradyzoite stage
M. Ferreira da Silva1,2, H. Santos Barbosa1, U. Gross2
C. Lüder2
Oswaldo Cruz Institute - FIOCRUZ, Department of
Ultrastructure and Cell Biology, Rio de janeiro, Brazil
Georg-August-University Goettingen , Medical Microbiology
Institute, Goettingen, Germany
Lifelong persistence of the protozoan T. gondii may rely on the
conversion from the proliferative tachyzoite stage into quiescent
encysted bradyzoites. Although persistence within muscle tissue
is critical for parasite transmission to humans via raw or
undercooked meat, to date, no attention has been paid to skeletal
muscle cells (SMC) as a cell type to study Toxoplasma stage
conversion. To investigate in vitro differentiation of
Toxoplasma, primary murine SMC and L6C10, a rat myoblast
cell line, were differentiated in vitro to mature myotubes as
judged by fusion to multinucleated syncytial cells, expression of
muscle cell-specific markers by immunofluorescent test,
expression of specific genes by RT-PCR and regular
contractions in vitro. When examined by immunofluorescence
microscopy, at 4h of infection with T. gondii tachyzoites of the
type II strain NTE, muscle cells harboured single parasites
without any evidence of stage conversion. Stage differentiation
started spontaneously at 1 day post infection (dpi), when
parasites displayed proteins specific to the bradyzoite stage (14,
2 ± 0, 8 % in primary SMC, and 35, 2 ± 4, 6 % in L6C10). At 2
and 4 dpi, vacuoles containing bradyzoites and rosettes of
Toxoplasma in asynchronous stage conversion were detected.
Toxoplasma differentiation further increased until 6 dpi, when
muscle cells harboured high percentages of bradyzoitecontaining vacuoles (38, 6 ± 1, 1 % in primary SMC, and 64 %
in L6C10). In addition, when analysed by real time RT-PCR,
total RNA from SMC primary cultures contained increasing
amounts of bradyzoite-specific ENO1 transcripts until 6 dpi.
Together, these data suggest that, without the need of an
exogenous stress factor, T. gondii parasites readily convert from
tachyzoites to bradyzoites in skeletal muscle cells.
This work was supported by DAAD and CAPES.
Characterisation of plasmodial threonine-peptidasecomplexes
B. Mordmüller1,2, A. Kreidenweiss1,2, P. Kremsner1,2
Albert-Schweitzer-Hospital, Medical Research Unit
Lambarene, Gabun
Eberhard-Karls-University, Parasitology, Tuebingen
Protein quality control mechanisms are particularly challenged
in Plasmodia because: i) they synthesize proteins with a higher
average size compared to other organisms, ii) essential proteins
contain large unstructured sequences, and iii) proteins have to
withstand temperature oscillations within their mammalian host
as well as during their life-cycle. Interference with those
mechanisms is therefore an attractive target for
chemotherapeutic agents. Destruction of misfolded proteins is a
key-determinant for the presence of a full set of functional
proteins and is central for the maintenance of protein quality.
Threonine-peptidases are the major enzyme-class responsible
for protein degradation in eukaryotic cells. In almost all
eukaryotic organisms threonine-peptidases are represented by
the proteasome with its enzymatically active subunits 1, 2,
and 5. In addition to the proteasome, plasmodia express
another threonine-peptidase (PFL1465c or PfhslV) similar to
prokaryotic proteasome progenitors. Our aim was to characterise
plasmodial threonine-peptidase complexes functionally and
structurally as well as to test existing and newly developed
inhibitors against this class of enzymes. We cloned and
expressed genes of the proteasome and PfhslV and analyzed
their localisation as well as enzymatic activities. Subsequently, a
naturally occurring epoxide-containing peptide (epoxomicin)
and its synthetic derivatives were tested in laboratory and field
isolates together with an array of other peptidase-inhibitors and
antimalarial drugs. Most inhibitors showed moderate to high
antiplasmodial activities and some were selected for further
development of Plasmodium-specific candidate compounds.
Toxoplasma gondii inhibits activation and oligomerisation of
proapoptotic Bax during Bims-induced apoptosis
D. Hippe1, A. Weber2, U. Gross1, G. Häcker2, C. Lüder1
Georg-August-University Goettingen, Medical Microbiology
Goettingen, Germany
Technical University Munich, Medizinische Microbiology
Immunologie und Hygiene, Munich, Germany
The intrinsic pathway of apoptosis is an important mechanism
of the innate immune response to eliminate pathogen-infected
cells and to prevent propagation of obligate intracellular
microorganisms. Although it has been firmly established that T
.gondii inhibits the release of cytochrome c from host cell
mitochondria into the cytosol, thereby downregulating
activation of the caspase cascade, the detailed mechanisms of
this interference have not yet been resolved. Herein, we
investigated the effect of parasitic infection on apoptosis which
had been induced in HeLa cells by the tetracycline-inducible
expression of the BH3-only protein BimS. Inducible BimSexpression led to apoptosis in non-infected cells within 3 hours.
However, concomitant infection with T. gondii protected these
cells from undergoing apoptosis. The activity of caspase 3 and
also of initiator caspase 9 was clearly reduced after parasitic
infection. Furthermore, T. gondii led to an inhibition of
cytochrome c release in BimS-expressing cells, indicating
parasite interference at a step between Bim expression and
cytochrome c release. Remarkably, the levels of cytosolic
cytochrome c were also decreased after parasitic infection of
host cells in which RNA synthesis was inhibited, indicating that
host cell transcription is not required. Infection with T. gondii
did not alter the protein levels of proapoptotic Bax and Bak.
However, T. gondii prevented Bax activation and consequently
oligomerisation within the mitochondrial membrane. In
conclusion, T. gondii blocks the mitochondrial apoptotic
pathway via interference with the activation of the proapoptotic
Bcl-2 family member Bax. This may enable the parasite to
effectively evade the innate immune response and further its
propagation in the host.
Induction of ERK kinase signalling triggers morphotype
specific killing of Candida albicans filaments by human
I. Wozniok1, A. Hornbach2,1, C. Schmitt2, J. Loeffler1
O. Kurzai2
University of Wuerzburg, Medical Clinic II, Wuerzburg
University of Wuerzburg, Institute of Hygiene and
Microbiology, Wuerzburg, Germany
Candida albicans is one of the most important fungal pathogens
in humans. The morphological plasticity of this yeast has been
linked to its pathogenic potential and filamentous forms of C.
albicans are associated with tissue invasion and infection.
Whereas a Th-1 based specific immune response has been
shown to contribute to a protective immune response against C.
albicans it is a well established concept, that neutrophils (PMN)
are a major player in the anti-Candida immune response.
Here we show that human neutrophils are capable of
discriminating between yeast forms and filaments of a single C.
albicans strain. Whereas filaments induced targeted motility,
resulting in the establishment of close contact between
neutrophils and fungal elements, yeast forms were largely
ignored throughout the observation. In transwell chemotaxis
assays, C. albicans filaments but not yeast forms induced
migratory activity in human neutrophils. In addition filamentous
forms triggered a higher oxidative burst than yeast forms of C.
albicans. PMN motility based on actin rearrangement could be
shown to be essential for inactivation of C. albicans filamentous
forms. In contrast, the inhibition of actin reorganization did not
impair the ability of human PMN to kill yeast cells. Using
inhibitors for different MAP-kinase cascades, it could be shown,
that recognition of C. albicans filaments by PMN is mediated
via the MEK/ERK MAP-kinase cascade and independent of
JNK or p38 MAPK activation. Inhibition of the ERK signalling
pathway abolished not only neutrophil chemotaxis induced by
C. albicans filaments but also the ability of human PMN to kill
C. albicans filaments. In contrast, it did not affect PMN activity
against yeast forms.
Taken together, these data show that invasive filamentous forms
of C. albicans trigger a morphotype specific activation of human
neutrophils. Therefore these cells are capable of sensing C.
albicans invasion and initiating an early immune response.
Fungal carbohydrates and their role in the immune response
to the pathogenic mould Aspergillus fumigatus – Pieces of an
emerging puzzle
F. Ebel1
Ludwig-Maximilians-University, Max-von-PettenkoferInstitute, Munich, Germany
Cells of the innate immune system constitute the first line of
defence that is responsible for the elimination of invading
microbes. Detection of microbial challengers is mediated by a
set of germ line encoded pattern recognition receptors (PRRs)
which specifically recognize so-called pathogen-associated
molecular patterns (PAMPs), structures that are essential for and
highly-conserved within large groups of microorganisms. Due to
their dynamic alteration, proteins are generally not well suited to
function as targets for PRRs, which instead preferentially
recognize lipids, carbohydrates or DNA-/RNA-molecules.
In recent time it became increasingly apparent that cell-wall
carbohydrates comprise major fungal PAMPs. The best
characterized fungal PAMP so far is ß1-3 glucan which is
recognized by the C-type lectin dectin-1. Signalling via this
PRR triggers diverse cellular responses, e.g. phagocytosis,
production of pro-inflammatory cytokines and reactive oxygen
The morphological transition from resting conidia to swollen
conidia, germlings and finally hyphae is a characteristic feature
of infections by filamentous fungi and a challenge for the
immune system. Aspergillus fumigatus is a prototypic example
for this group of pathogens and we found that the surface
exposure of ß1-3 glucan and galactomannan, two major
constituents of the Aspergillus cell wall, shows striking
variations not only between different morphotypes, but also
between hyphae grown under different conditions. This
differential display of proven or potential PAMPs is likely to
have a strong impact on the anti-Aspergillus immune response.
A novel iron source used by Candida albicans
R. Almeida1, B. Hube1
Hans-Knoell-Institute, Microbial Pathogenicity Mechanisms
Jena, Germany
The iron sequestration by host iron-binding proteins provides a
natural resistance to infections, known as “nutritional
immunity”. Hence, the majority of iron in host niches such as
the oral cavity is tighly bound to host proteins. Inside oral
epithelial cellsI iron is stored in the iron-binding protein ferritin.
Extracellular iron found in saliva is mostly bound to lactoferrin.
Therefore, iron availability is extremely limited in the oral
cavity. In this study, we aim to determine the natural iron
sources for the human pathogenic fungus Candida albicans
during oral infection. We demonstrated that this pathogen can
grow on agar at physiological pH (pH 7.4) with ferritin as the
sole source of iron while the non-pathogenic fungus
Saccharomyces cerevisiae does not. To obtain iron from ferritin
in vitro, the fungus needs the iron reductive pathway and must
acidify the medium. Additionally, we were able to show that
hyphae, but not yeast cells of C. albicans can bind ferritin and
that this binding is necessary for the usage of iron from this
protein. Immunocytochemical localization of ferritin in
epithelial cells infected with C. albicans showed ferritin on the
fungal surface during infection. To our knowledge, this is the
first observation which suggests that ferritin can be used as an
iron source by a pathogenic fungus during infection.
Figure 1: EM picture showing ferritin (dark particles)
localized on the cell wall of C. albicans.
Figure 2: C. albicans hyphae can bind ferritin
Complement degradation as immune evasion mechanism in
cerebral aspergillosis
C. Speth1, G. Rambach1, I. Mohsenipour2, M. P. Dierich1
Medical University Innsbruck, Hygiene, Microbiology and
Social Medicine, Innsbruck, Austria
Medical University Innsbruck, Department of Neurosurgery
Innsbruck, Austria
The high lethality of cerebral aspergillosis of more than 90%
indicates a profound failure of the local immune defence in the
course of pathogenesis. In order to develop immune-supportive
therapeutic strategies we investigated putative evasion strategies
of Aspergillus against the cerebral immune system. Since the
central nervous system is separated from the periphery by the
blood-brain-barrier the complement system is a central part of
the local innate immunity and therefore a main target of these
fungal evasion mechanisms.
Aspergillus sp was grown in cerebrospinal fluid (CSF) to
simulate the conditions in the brain. Western Blot analysis
showed that the fungus secreted proteolytic factors into the CSF
which degraded various complement proteins. The extent of the
proteolysis was dependent of the time period of fungal growth
and of the Aspergillus species: whereas A. fumigatus, the
predominant cause of cerebral aspergillosis, showed a rather
quick and strong degradation, the proteolysis by A. terreus was
weaker and rather slow. Fungal growth in Sabouraud medium
instead of CSF abolished the production of complementdegrading proteases, indicating the influence of exact growth
conditions. The addition of various nitrogen sources to CSF
prevented the secretion of the proteases.
The degradation of complement might represent an immune
evasion mechanism of pathogenic Aspergillus species and thus
contribute significantly to the pathogenesis of cerebral
aspergillosis. In addition this newly defined escape mechanism
of the fungus provides a new optional therapeutical target to
support the established antifungal treatments. Defined nitrogen
sources might prevent the secretion of proteolytic factors and
specific protease inhibitors could support the attack of
complement against the fungal pathogens.
CaCRASP-1/Gpm1: a multifunctional protein of Candida
albicans acting in complement escape, tissue invasion and
S. Poltermann1, M. von der Heide1, A. Kunert1, P. F. Zipfel1
Leibniz-Institute for Natural Product Research and Infection
Biology, Department of Infection Biology, Jena, Germany
Candida albicans is the major fungal pathogen of humans
causing life-threatening infections in immunocompromised
patients as well as mucosal infections in healthy individuals.
Immediately after infection the yeast is attacked by the human
immune system and activates both the alternative and classical
pathway of the complement system. In order to survive C.
albicans controls complement activation at its surface and
inactivates toxic complement activation products by binding the
human fluid-phase complement regulators factor H, factor H
like protein 1 (FHL-1) and C4BP. We have identified C.
albicans phosphoglycerate mutase (CaGpm1) as a factor H and
FHL-1 binding protein. The protein was cloned and
recombinantly expressed. Purified CaGpm1 binds the host
complement regulators factor H and FHL-1 but not C4BP.
Based on these characteristics we propose to term this protein
Candida albicans complement regulator-acquiring surface
protein 1 CaCRASP-1. CaCRASP-1/Gpm1 binds also the
human serum protein plasminogen. With a novel antiserum,
CaCRASP-1/Gpm1 was localized in the cytoplasm and in the
cell wall of C. albicans as demonstrated by flow cytometry,
immuno fluorescence microscopy and Western blotting. In vitro
analyses show that CaCRASP-1/Gpm1 bound host regulators
factor H, FHL-1 and plasminogen are functionally active, and
mediate fungal complement escape and degradation of
extracellular matrix. To demonstrate in vivo relevance of
CaCRASP-1/Gpm1 as a factor H/FHL-1 and plasminogen
binding protein, a C. albicans CaCRASP-1/Gpm1 knock out
strain (CAP3) was generated using the Ura-blaster method. The
capability of the mutant strains to bind factor H/FHL-1 and
plasminogen was assayed in Candida-ELISAs and confirmed a
role of CaCRASP-1/Gpm1 in factor H/FHL-1 and plasminogen
binding. Furthermore CAP3 displays growth defects on various
carbon sources highlighting the role of CaCRASP-1/Gpm1 in
glycolysis/gluconeogenesis. In summary we identified
CaCRASP-1/Gpm1 as a multifunctional protein that is
expressed both on the cell surface and in the cytoplasm of C.
albicans and that mediates complement escape, tissue invasion
and glycolysis/gluconeogenesis.
PbICP, a Plasmodium berghei inhibitor of cysteine
A. Rennenberg1, T. Witt1, K. Nagarajan2, T. Hogg2
C. L. Schmidt2, R. Hilgenfeld2, V. Heussler1
Bernhard-Nocht-Institute for Tropical Medicine, Department of
Molecular Parasitology, Hamburg, Germany
University of Luebeck, Institute of Biochemistry, Center for
Structural and Cell Biology in Medicine, Luebeck, Germany
Cysteine proteases fulfill many essential functions throughout
the life cycle of the malaria parasite Plasmodium. Apart from
haemoglobin degradation, Plasmodium proteases are involved in
parasite liberation and the ordered host cell death observed in
late infected hepatocytes. Regulation of the activity of these
cysteine proteases is essential for parasite survival.
We identified a potent cysteine protease inhibitor from the
rodent malaria parasite Plasmodium berghei termed PbICP
(Plasmodium berghei inhibitor of cysteine proteases). PbICP
has the potential to regulate the activity of the parasite as well as
host cell cysteine proteases. PbICP consists of a chagasin-like
C-terminal part (PbICP-C) and a non-homologous N-terminal
Western blot and immunofluorescence analysis suggested
proteolytic processing of endogenous PbICP during blood and
liver stages. Transgenic P. berghei parasites expressing a
PbICP-GFP fusion protein confirmed processing of PbICP.
Since PbICP-C but not the entire protein acts as a very potent
inhibitor of falcipain-2 and other cysteine proteases, we
hypothesize that the N-terminal extension interacts with the Cterminus to prevent protease binding and thus processing of
PbICP is a prerequisite to yield an active inhibitor.
Localisation studies in infected hepatocytes indicated that
PbICP expression is restricted to the parasite during schizont
development. However, upon merozoite formation PbICP was
also found in the host cell cytoplasm where it may act as a
regulator of parasite-dependent host cell death.
Compared to other chagasin family members PbICP-C has
additional features, which might be crucial for protease binding.
The crystal structure determination of PbICP-C:falcipain-2
complex is in progress. The elucidation of this structure will
allow the design of inhibitors specific for falcipain-2 and -3.
This work was supported by the DFG program Life inside cells”
grant numbers HE 4497/1-2 and Hi 611/5-1.
The transcription factor Tec1p is a sequence-specific DNAbinding protein in Candida albicans
M. Sehnal1, K. Schröppel2
Dr. Margarete Fischer-Bosch-Institute for Clinical
Pharmacology, Stuttgart
Eberhard-Karls-University, Medical Microbiology and
Hygiene, Tuebingen
Tec1p is a key regulator of the morphogenetic development of
C. albicans. A tec1/tec1 mutant is avirulent and does no longer
form hyphae under inducing conditions in vitro. Tec1p contains
the highly conserved TEA-DNA-binding domain (TEAD) and is
a member of the TEA/ATTS-transcription factor family.
Previous studies showed that different TEA/ATTS-transcription
factors recognize and bind to the conserved DNA sequence
`CATTCY`. This motif has been termed TEA/ATTS consensus
sequence, TCS. It was detected by in silico analysis in several
promoters of hyphae regulated genes. However, other studies
suggest that Tec1p does not directly bind to TCS DNA to exert
its regulatory function, but rather induces developmentally
regulated genes indirectly via the activation of downstream
proteins including Bcr1p. We addressed this question by
electromobility shift analysis (EMSA) of the interaction of
recombinant rTec1p with the wildtype TCS probe and several
modified DNA probes. We found a specific retardation of an
EMSA-complex of a promoter fragment derived from a
developmentally regulated proteinase gene. The DNA binding
of rTec1p was specific, because addition of excess amounts of
unlabeled promoter fragments completely inhibited the proteinDNA interaction. Furthermore, when we used double-stranded
oligonucleotide containing a putative TCS derived from the
same promoter, we observed specific binding of rTec1p. This
suggests that rTec1p is a sequence-specific DNA binding
protein. Surprisingly, this interaction was not highly stringent;
the sequence of the competing unlabeled oligonucleotides could
be modified to some extend without loosing its inhibitory
activity on the EMSA-complex. This suggests that the
individual nucleotides of the TCS have a differential impact on
the affinity of the protein to the promoter. This is supported by
data from the human TEA homolog TEF-1, where basepreference analysis of the protein-DNA complex supports the
idea of variable and perhaps species-specific TCS sequences.
DNA footprint analysis will resolve the actual interaction
between rTec1p and its optimized TCS. In conclusion, rTec1p
directly interacts with a phase-specific promoter, emphasizing
its role as a trans-acting activator during hyphal development of
C. albicans. Since the rTec1 binding motif seems to be relaxed,
a computer-based search for Tec1p-responsive promoters might
result in an incomplete set of potential target promoters.
Characterization of Pga29p, a cell wall protein of Candida
A. de Bör1, P. W. de Groot2, M. Schaller3, G. Weindl3
J. Wagener3, F. Klis2, U. Gross1f, M. Weig1
University of Goettingen, Medical Micribiology, Goettingen
University of Amsterdam, Microbiology, Amsterdam
Eberhard-Karls-University, Dermatology, Tuebingen
Covalently linked cell wall proteins (CWPs) of the dimorphic
human pathogenic fungus Candida albicans play an important
role in host-pathogen interactions that may lead to fungal
infections. Previously, we identified Pga29p/Rhd3p/orf19.5305,
a small GPI-modified glycoprotein of about 30 kDa as one of
the most abundant CWPs in the C. albicans yeast cell wall.
However, Pga29p has no obvious homologs in other fungi,
which urged us to further characterize this protein.
In order to explore the function of Pga29p, we generated
Dpga29/Dpga29 mutants and the respective reconstituted
strains. Among these strains, no altered sensitivity to several cell
wall stress conditions was observed, suggesting that Pga29p
does not play a role in cell wall rigidity.
However, in an in vitro model of oral candidosis,
Dpga29/Dpga29 mutants showed a clear reduction in virulence.
Additionally, we studied the immune response of the epithelial
cells against our strains by quantifying mRNA levels for
cytokines. Epithelial cells that were infected with the mutant
strain showed significantly reduced expression levels of GMCSF, TNF-alpha and IL-8.
Deletion of PGA29 resulted in an increased glucose/mannose
ratio in the cell wall, which prompted us to investigate the
expression of receptors that recognize pathogen associated
molecular patterns (PAMPs). We stimulated a monolayer of the
TR146 oral epithelial cell line with CWP extracts of the
Dpga29/Dpga29 mutant, revertant and wild type. Stimulation
with the Dpga29/Dpga29 CWP extract resulted in a reduced
expression of the mannose receptor.
Conclusively, our data suggest that Pga29p has an important
role in mediating virulence.
Subversion of vascular endothelial cell functions by
translocated effector proteins of the vascular tumorinducing pathogen Bartonella henselae
C. Dehio1
University of Basel, Focal area Infection Biology, Biology
Center, Basel, Switzerland
Bartonella henselae is an arthropod-borne zoonotic pathogen
which causes a broad range of clinical manifestations in
humans. Remarkably, B. henselae can chronically infect the
human vascular endothelium, resulting in the formation of
vasoproliferative lesions known as bacillary angiomatosis
peliosis. Human umbilical vein endothelial cells (HUVEC)
provide an in vitro system to study various aspects of this
intimate interaction of B. henselae with the vascular
endothelium. We have shown that the type IV secretion system
(T4SS) VirB/VirD4 is a major virulence factor of bartonellae.
T4SS are multicomponent transporters that allow bacteria to
transfer protein or DNA substrates to a wide array of target cell
types. The VirB/VirD4 T4SS mediates most virulence attributes
associated with the interaction of B. henselae with HUVEC.
These include (i) massive rearrangements of the actin
cytoskeleton, resulting in the internalization of large bacterial
factor kappa B-dependent
proinflammatory activation, leading to cell adhesion molecule
expression and chemokine secretion, and (iii) inhibition of
apoptotic cell death, resulting in enhanced endothelial cell
survival. These diverse cellular phenotypes were found to
depend on the VirB/VirD4-mediated translocation of seven
bacterial effector proteins termed BepA-BepG. BepA is
sufficient to suppress apoptotic cell death of HUVEC, e.g. as
triggered by activated cytotoxic T lymphocytes. BepG interferes
with phagocytic uptake of individual bacteria resulting in
bacterial invasion by an alternative route termed invasomemediated uptake. Further dissection of the molecular
mechanisms by which Bep proteins subvert endothelial cell
function should shed light on the process of chronic vascular
C. elegans as a model host for the study of host-pathogen
C. - L. Kurz1
C. I. M. L, Marseille, Spain
Studies using the genetically tractable organism Caenorhabditis
elegans have greatly contributed to advances in our
understanding of biological processes such as development, cell
death and RNA interference. Over the past ten years, this animal
has been increasingly used as an alternative model to dissect
host-pathogen interactions. Virulence mechanisms used by
fungi, Gram-negative and Gram-postive bacteria, including
important human pathogens, have been studied using this
nematode. Significantly, many of the identified pathogenicity
traits identified using C. elegans as a host are required for
infection in mammals. Innovative in vivo approaches aiming at
the discovery of molecules with antimicrobial activity using C.
elegans have also been developed. In addition, the establishment
of these infection systems has permitted the characterization of a
complex host innate immune response that shows some
interesting similarities with mammalian innate defense
High throughput analysis of pathogenic activities of
herpesvirus-8 encoded genes
Hochdurchsatz-Analyse pathogener Eigenschaften
Herpesvirus-8-kodierter Gene in Endothelzellen
M. Stürzl1
Division of Molecular & Experimental Surgery, Department of
Surgery, Erlangen, Germany
HHV-8 is the etiological agent of Kaposi’s sarcoma (KS) an
endothelial cell-derived tumor with a high inflammatory
component. NF- B is a key regulatory molecule in
inflammation. Using reverse transfection array analysis as an
unbiased systems biology approach the effects on NF- Bactivation of all HHV-8-encoded genes individually and of
pairwise combinations of all K- and latent genes were
investigated. More than 7,000 transfections were performed.
The strongest activation of NF- B was observed in
combinations of vFLIP, a known activator of NF- B, with the
latent genes vCyc or LNA-1. Combined activities of these genes
were clearly synergistic, could be abrogated by co-expression of
a constitutively active I B molecule and could be confirmed in
classical transfection experiments. In addition, in KS tissues a
positive relation of NF- B p65 staining and latent HHV-8infection was detected by immunohistochemical staining. Our
results indicate that co-operative effects of vFLIP, vCyc and
LNA-1, which are all encoded within a tricistronic latencyassociated gene locus in the viral genome, are key to the robust
NF- B-activation observed in HHV-8-infected cells.
Anion, cation and osmolyte channels in malaria-infected
S. Huber
University of Tuebingen, Department of Physiology, Tuebingen
The intraerythrocytic amplification of the malaria parasite
Plasmodium falciparum induces new pathways of solute
permeability in the host cell membrane. These pathways play a
pivotal role for parasite development by supplying the parasite
with nutrients, disposing of metabolic waste products, adapting
the host’s electrolyte composition to the parasite’s needs, and
preventing premature hemolysis of the host erythrocyte by
exporting inorganic and organic osmolytes. The New
Permeability Pathways allow the fast electrogenic diffusion of
ions and thus can be analyzed by patch-clamp single channel
and whole-cell recording. By employing these techniques,
several ion channel types with different electrophysiological
profiles have been identified in Plasmodium-infected but also in
uninfected erythrocytes. Among those are ClC-2 Cl- channels,
CFTR-dependent anion channels, organic osmolyte and anion
channels, and Ca2 -permeable nonselective cation channels. The
activation of these channels in uninfected cells involves PGE2
formation, Ca2 entry, ATP release, autocrine purinoceptor
signaling, and activation of several kinases. Since erythrocytic
PGE2 formation triggers the suicidal death program and
autocrine purinoceptor signaling the ATP release function of
human erythrocytes it is suggested that the intraerythrocytic
parasites accomplish the increase in host cell membrane
permeability by interfering with these two erythrocyte functions.
Neisseria porin: Role in infection and host cell survival
T. Rudel1, V. Koszjak-Pavlovic1, I. Herrmann1, O. Kepp1
Max-Planck-Institute for Infection Biology, Molecular Biology
Berlin, Germany
Obligate human pathogenic Neisseria gonorrhoeae (Ngo) cause
the venereal disease gonorrhea. Gonococci attach to cells with
the help of pili and outer membrane proteins. Attachment of
bacteria results in the induction of host cell apoptosis which is
mediated by the PorB porin, an ATP-binding -barrel protein of
the outer bacterial membrane. PorB translocates to the host cells
mitochondria during infection causing loss of membrane
potential ( m) across the inner mitochondrial membrane and
release of cytochrome c, which is required for activation of
caspases and induction of apoptosis. When expressed in host
cells, PorB translocates to mitochondria and efficiently causes
breakdown of
m, but fails to induce the release of cytochrome
c. This suggested that PorB is required but not sufficient to
induce apoptosis, and that a second signal is needed to induce
cytochrome c release during infection. To understand the role of
PorB for the dissipation of the
m, and the induction of
apoptosis, we analyzed the mitochondrial import of PorB in
detail. A genetic system based on the inducible depletion of
individual mitochondrial import factors by RNA interference
has been established in host cells for gonococcal infection.
Using this system we have delineated the import route of PorB
into host cell mitochondria. Our current view of PorB functions
in controlling host cell survival will be discussed.
The type-III secretion injectisome
G. R. Cornelis1
University Basel, Infection Biology, Biology Center, Basel
The type III secretion injectisome is a nanosyringe that injects
bacterial effector proteins into the cytosol of eukaryotic cells. It
is related to the flagellum, with which it shares structural and
functional similarities. It consists of a basal body made of
several rings spanning the bacterial membranes, connected by a
central tube. On top of the basal body, comes a short stiff
needle. The basal body contains the export apparatus, which
serves first for the export of the needle subunits and later for
effectors. This structure is sufficient for exporting effector
proteins across the two bacterial membranes but not to inject
them into the cytosol of target cells. In Yersinia, this
translocation step requires three more proteins, LcrV, YopB and
YopD, in the case of Yersinia. LcrV forms a structure at the tip
of the needle and this structure is believed to act as a scaffold
for the insertion of a pore made of YopB and YopD into the
target cell membrane. LcrV is known since the mid nineteen
fifties to be a protective antigen against plague. The length of
the needle is controlled by a mechanism involving a protein
thought to act both as a molecular ruler and a substrate
specificity switch (YscP in Yersinia)). When assembly of the
needle is complete, the molecular ruler changes the substrate
specificity of the export apparatus, which becomes ready to
export pore formers and the effectors. One protein from the
export apparatus (YscU in Yersinia) seems to specifically
recognize the various classes of export substrates. Export of the
latter will only occur upon contact with a target cell.
Hypoxia responsive mechanisms in mucosal inflammation:
implications for barrier integrity and host defense
J. Karhausen1, K. Jäckel1, I. Vollmer1, M. Faigle1, J. Kuhlicke1
S. P. Colgan2, H. E. Eltzschig1
University Hospital, Clinic for Anesthesiology and Intensive
Medicine, Tuebingen, Germany
University of Colorado, Mucosal Inflammation Program
Denver, CO, USA
Recent work has demonstrated that the colonic mucosa is
particularly challenged by low oxygen concentrations even
under physiological conditions. More specifically in the colonic
mucosa, the direct contact to the anoxic lumen of the gut and the
dependence on a complex vascular bed, provide an anatomical
setting in which further complicating factors invariably lead to
significant disturbances in oxygen supply. Our previous work
has revealed that the functional epithelial response to hypoxic
conditions manifests as diminished epithelial barrier integrity
via inflammatory pathways. However, tolerance to commensual
bacteria and immunity against pathogenic bacteria require intact
antigen uptake, recognition, processing and response
mechanisms. As a consequence loss of selectivity of dynamic
barrier function is associated with a variety of pathophysiologies
including inflammatory bowel diseases (IBD) and critical
illness. In this context, the role of bacteria has been recognized
as a more and more complex one. To exemplify this complexity,
in IBD, both eradication of bacteria (e.g.antibiotic treatment)
and introduction of certain strains of bacteria (e.g. E.coli Nissle
1918) are current therapeutic options.
Data presented will focus on the relevance of adaptive responses
that perpetuate barrier control1-3. A pivotal factor in this hypoxia
sensitive, barrier-protective gene-expression is the transcription
factor hypoxia-inducible factor 1 (HIF-1). Based on experiments
that had shown protection of barrier function by conditional HIF
overexpression in a murine model of intestinal inflammation2,
our ongoing efforts are aimed to characterize epithelial bacterial
interactions and the significance of both hypoxia and hypoxiaregulated gene expression in controlling bacterial translocation.
As such we have established models for bacterial translocation
both in vitro and in vivo and have characterized mechanisms of
bacterial translocation following epithelial hypoxic exposure.
Aim of such work is to recognize exact mechanisms involved in
the bacterial epithelial interaction leading to bacterial
translocation and to identify possible modulating factors.
1. Synnestvedt K, Furuta GT, Comerford KM, Louis N,
Karhausen J, Eltzschig HK, Hansen
KR, Thompson LF, Colgan SP. J. Clin. Invest. 2002;110:9931002.
2. Karhausen J, Furuta GT, Tomaszewski JE, Johnson RS,
Colgan CT, Haase VH. J. Clin.
Invest. 2004;114:1098-1106.
3. Furuta GT, Turner JR, Taylor CT, Hershberg RM, Comerford
K, Narravula S, Podolsky
DK, Colgan SP. J. Exp. Med. 2001;193:1027-1034.
Mycobacterium tuberculosis: Life and death in the
D. Russell1
Cornell University, Ithaca, NY, USA
Once across the barrier of the epithelium, macrophages
constitute the primary defense against microbial invasion. For
most microbes the acidic, hydrolytically-competent environment
of the phagolysosome is sufficient to kill them. Despite our
understanding of the trafficking events that regulate phagosome
maturation our appreciation of the lumenal environment within
the phagosome is only now becoming elucidated through realtime functional assays. The assays quantify pH change,
phagosome/lysosome fusion, proteolysis, lipolysis and galactosidase activity. This information is particularly important
for understanding pathogens that successfully parasitize the
endosomal/lysosomal continuum. Mycobacterium tuberculosis
infects macrophages through arresting the normal maturation
process of the phagosome, retaining its vacuole at pH 6.4 with
many of the characteristics of an early endosome. The success
of the bacterium is dependent on its ability to modulate this
compartment. Mutants defective in this behavior are avirulent,
and macrophages activated by cytokines are able to overcome
the phagosome maturation block and kill the infecting microbes.
Current studies are focusing on the transcriptional response of
the bacterium to the changing environment in the macrophage
phagosome. Manipulation of these environmental cues, such as
preventing the pH drop to pH 6.4 with Concanamycin A,
abrogates the majority of the transcriptional response in the
bacterium demonstrating that pH is the dominant signal that the
bacterium senses and responds to. Temporal analysis of the
changing transcriptional profile from 5 minutes post-infection to
14 days post-infection reveal distinct phases in the infection
process as the bacterium establishes infection and transitions
into growth phase. Our analyses focus on the changing
metabolism of the bacterium particularly with respect to carbon
acquisition and utilization and how the availability of nutrients
are influenced by the intra-phagosomal environment. These
approaches represent our ongoing attempts to unravel the
discourse that takes place between the pathogen and its host cell.
Host adaptation of Pseudomonas aeruginosa during chronic
infection of the cystic fibrosis (CF) lung.
M. Hogardt
Max-von-Pettenkofer-Institute for Hygiene and Medical
Microbiology, Ludwig-Maximilians-University Munich
Munich, Germany
Chronic Pseudomonas aeruginosa (PA) pneumonia is still the
major cause of morbidity and mortality among patients suffering
from cystic fibrosis (CF). During the switch of P. aeruginosa
from the ubiquitous free-living bacterium to a pulmonary
pathogen, numerous cell-associated and secreted components
are required to establish CF lung colonization. Once established
PA colonization leads to initially intermittent and then chronic
pulmonary infection. During this life cycle, PA is subjected to
strong selective pressure resulting in the emergence of PA
variants with multi-resistent, mucoid and typically virulenceattenuated phenotypes. This apaptive process seems to be
accelerated by the emergence of mutator strains with high
mutation frequencies and subsequent co-selection of mutations
that are beneficial for long-term persistence of PA in the CF
lung but unfavorable for its capacity to reconquer environmental
habitats and transmissibility.
Several studies provide insights into the lifestyle of PA during
chronic CF lung infection, however, the exact mechanisms
allowing PA to persist are still unknown. Strikingly, the
selection against invasive factors of PA relevant during early
infection phase seems to be linked with the positive selection for
persistence factors. Chronic infection of the CF lung by PA is
thought to be a biofilm-like infection where most bacterial cells
have no contact to host cells. Thus, it is reasonable that the
expression of the type three secretion system is reduced in PA
isolated from CF secretions. Defects of the quorum sensing
regulator RhlR result in reduced elastase production but have
been linked with a better fitness of PA to grow with
phenylalanine. We found that during persistence in the CF lung,
PA mutator strains remarkably dissimilar from their isogenic
non-mutator ancestors, while end-stage PA mutator isolates
exhibit profound alterations in metabolic pathways and redox
active proteins with evidence for an adaptation to hypoxic and
nutritional rich conditions of CF secretions that contain high
concentrations of mucin, DNA, amino acids and lipids. These
metabolic factors found to be hyperproduced in lung-adapted
mutator isolates may be key participants in the biofilm-like
survival, growth and redox homeostasis of P. aeruginosa during
chronic infection of the CF lung.
Genotypic characterisation of Staphylococcus aureus
S. Monecke1, P. Slickers1, R. Ehricht1
Technical University Dresden, Institute for Med. Microbiology
and Hygiene, Medical Fakulty C. G. Carus, Dresden
DNA microarray technology allows the simultaneous detection
of a high number of targets. Staphylococcus aureus was selected
as target organism, and a diagnostic microarray with 166 probes
covering species-specific controls, agr allelic markers,
virulence-associated genes and resistance determinants was
constructed and evaluated testing several hundred reference
strains and clinical isolates. It was shown to allow a rapid
genotype-based assessment of the virulence of a given isolate
and of its resistance properties. Presently, an expanded version
of the array is under evaluation which carries additional 239
probes allowing to recognise SCCmec and capsule types as well
as the presence of allelic variants of “MSCRAMMs”, i.e.,
microbial surface components recognising adhesive matrix
The overall hybridisation profile of a given isolate leads to some
kind of fingerprint, or dataset allowing to elucidate the
relatedness between different isolates. Hybridisation profiles
facilitate assignment of isolates to clonal groups, and there was
a good correlation of hybridisation patterns and MLST/spa types
resulting in stable hybridisation profiles for all isolates of a
given type.
These data allow to trace phylogenetic relations between clonal
groups and evolution by acquisition of mobile genetic elements.
Since the assay can be performed with high speed and
throughput, it has great potential for the analysis of resistance
and virulence genotypes, surveillance, and for typing purposes.
Furthermore, an adaptation to different technological platforms,
such as fully automated systems is underway.
The Type III secretion system of phytopathogenic bacteria
T. Nürnberger1
University of Tuebingen, Center for Plant Molecular Biology
Tuebingen, Germany
Many Gram-negative pathogens of plants and animals use type
III protein secretion systems to infect eukaryotic hosts. Type III
protein secretion systems (TTSS) are molecular syringes that
inject bacterial effectors into eukaryotic host cells to modulate
host physiology. TTSS from phytopathogenic and animal
pathogenic bacteria are composed of similar components.
However, plant pathogenic bacteria have to bridge the host cell
wall before contacting the host membrane. Hollow pilus-like
supramolecular structures are supposed to provide a conduit for
effector translocation across this barrier and into the host cell.
Evidence is accumulating that many type III effectors from plant
pathogens target elements of host immunity in order to suppress
antimicrobial responses during infection. This report
summarizes our current knowledge on the unique architecture of
phytopathogenic bacteria-derived TTSS and the molecular mode
of action of bacterial effectors inside the plant cell.
Adaptive strategies of C. pneumoniae to cope with hypoxia
J. Rupp1
University of Luebeck, Institute of Medical Microbiology and
Hygiene, Germany
It has long been recognized that sites of inflammation present a
hostile environment. Sites of bacterial infections in diseased
tissues are often characterised by low levels of nutrients, high
levels of reductive metabolites and distinct levels of hypoxia.
Thus, it is known that oxygen tension is in the range of 3-6% in
different human tissues even under physiological conditions. So
far, properties of C. pneumoniae replication, growth and
pathogenicity have invariably been investigated in atmospheric
air containing 5% CO2 with an estimated oxygen concentration
of 20%. Intracellular survival of chlamydiae strongly depends
on energy supply and metabolic turnover of the infected host
cell. However, in hypoxia host cell metabolism is inseparably
coupled to oxygen availability in the environment and is
regulated by the transcription factor “hypoxia-inducible factor1” (HIF-1). Recent studies could show that C. pneumoniae is
able to efficiently replicate in oxygen concentrations between 25%, showing enlarged intracellular inclusions (IFT) and an
intact developmental cycle (electron microscopy). Moreover,
comparing replication efficiency by recovering infectious
elementary bodies (EB) from epithelial cells cultured in
normoxia (20% O2) and hypoxia (2% O2), a more than 3- fold
increase in C. pneumoniae infectivity (IFUs/ml) in hypoxia was
observed. To adapt to low oxygen availability, host-cell
metabolism is directly targeted by chlamydiae in the early phase
of infection. C. pneumoniae induces eukaryotic HIF-1
stabilization and subsequently increases glucose uptake to
maintain infectivity in hypoxia.
Hypoxia is a common condition in healthy and diseased tissue
and even more pronounced at sites of bacterial infections with
an influx of inflammatory cells. Functional mechanisms of
obligate intracellular bacteria to survive and to interfere with
host cell metabolism in an environment of reduced oxygenation
have not been studied in detail. The impact of hypoxia on
persistence, antibiotic susceptibility and pathogenicity of C.
pneumoniae is currently investigated.
Microarray genotyping of Pseudomonas aeruginosa
L. Wiehlmann1, N. Cramer1, B. Tümmler1
Medical University Hanover, Clinical Research Group
Hanover, Germany
A novel approach for the genotyping of Pseudomonas
aeruginosa strains was developed in collaboration with clondiag
chip technologies, Jena. The probe is generated directly from the
bacterial colony by linear asymmetric multiplex primer
extension and then hybridized onto a microarray to yield an
electronically portable 58-binary marker genotype. The lowresolution array tube types P. aeruginosa strains in SNPs of the
core genome and variable elements of the accessory genome.
Clones are defined by the 15-marker genotype of the core
genome. Clonal variants are identical in SNP-genotype, but
differ in the repertoire of the accessory genome. Typing of
marker loci of the accessory genome allows to differentiate
between nosocomial spread of the same strain and the casual
presence of the same clone.
All procedures are performed with basic laboratory equipment.
The optimized protocol is easy, rapid and robust and can be
carried out by non-experienced personnel. The protocol requires
only five hours between colony picking and Web-based
electronic evaluation of genotype so that for example the source
of a nosocomial infection can be promptly identified to initiate
appropriate hygienic measures.
So far we typed more than 1500 P. aeruginosa isolates from
diverse habitats and geographic origin to describe the global
population structure of P. aeruginosa. The data indicates that
the majority of P. aeruginosa strains belong to few dominant
clones widespread in disease and environmental habitats. Most
studied loci of the genome are freely recombining with each
other, but some physically distant loci exist in fixed
combinations of genotypes suggesting that the free flow of
genes in the P. aeruginosa population is tolerated for most, but
not all loci of the genome. The non-random associations
between segments of the core and accessory genome indicate
that genome evolution and speciation of lineages is driven by
concerted diversifying selection at multiple unlinked loci.
Wiehlmann, L., Wagner, G., Cramer, N., Siebert, B., Gudowius,
P., Morales, G., Köhler, T., van Delden, C., Weinel, C.,
Slickers, P., Tümmler, B. (2007): Population structure of
Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U.S.A. 104:
The scaling laws of human travel – the spread of epidemics
in a globalized world
D. Brockmann1
Max-Planck-Institute for Dynamics and Self-Organization
Goettingen, Germany
The dynamic spatial redistribution of individuals is a key
driving force of various spatiotemporal phenomena on
geographical scales. It can synchronize populations of
interacting species, stabilize them, and diversify gene pools.
Human travel, for example, is responsible for the geographical
spread of human infectious disease. In the light of increasing
international trade, intensified human mobility and the imminent
threat of an influenza A epidemic, the knowledge of dynamical
and statistical properties of human travel is of fundamental
importance. Despite its crucial role, a quantitative assessment of
these properties on geographical scales remains elusive, and the
assumption that humans disperse diffusively still prevails in
models. I will report on the first global, quantitative assessment
of human travelling statistics (Brockmann et al., Nature 2006)
by analysing the circulation of bank notes in the United States.
Using a comprehensive data set of over a million individual
displacements, I will show that universal scaling laws underly
global human movement patterns and present a theory which
mathematically accounts for human travel.
Vaccines in the 21st century: Perspectives for new
developments and strategies
J. Vollmar1
GlaxoSmithKline GmbH, Munich, Germany
Vaccines have contributed substantially to the fight against
infectious diseases in the last century. Vaccination has
succeeded in the eradication of smallpox and is likely to
eliminate polio and measles from this planet in the foreseeable
future. This success was achieved with vaccines which were
based on randomly attenuated live viruses. At the end of the last
century additional insight in immunology initiated the
development of innovative technologies. These technologies
allow tailoring vaccines to certain infections, enhancing and
directing the immune response and thus also targeting diseases
which have been traditionally difficult for vaccine development
such as HIV, TB or Malaria. Furthermore it has been shown for
various cancers to be related to infectious diseases, e.g. Human
Papilloma Virus was identified as causative agent for the
development of cervical cancer and Helicobacter Pylori for
stomach cancer. Vaccines for those diseases are either under
development or have the just become available. New
developments even raise hope that in the future vaccination
cannot just prevent but treat infectious diseases, cancer or other
conditions such as allergies. In addition preventive measures
against diseases which are currently neither existing nor relevant
have become an important public health topic given the impact
and extremely grave threat of bioterrorism or pandemic
influenza. Vaccines play a pivotal role in the preparedness plans
for such public health emergencies.
In summary a better understanding of immunology,
pathogenesis and etiology of diseases together with new
technologies enable vaccine researchers to focus on new
indications and targets. Specific examples of technology and
immunological advances will be given for selected vaccine
Conflict of interests: Employee of GlaxoSmithKline and former
employee of Bavarian Nordic; both are vaccine manufacturers.
Diagnostics using the SmartCycler – drawbacks and
C. Wendt1
Hygiene-Institute, Heidelberg, Germany
The SmartCycler® System is a real-time thermal cycler used for
identifying DNA/RNA from prepared samples. It was developed
as a transportable system that can be used in the field, i.e. for
detection of Bacillus anthracis or poxvirus. The System enables
the detection of up to four targets within a single sample
(multiplex assays) by using multiple fluorescent dyes and
detecting the fluorescent signal in four channels.
Up to 96 reaction sites are independently programmable, thus
allowing the simultaneous run of multiple experiments with
different protocols and at different starting times. The device is
capable to perform extremely rapid heating and cooling cycles,
by using specially designed sealable reaction tubes that optimize
rapid thermal transfer and optical sensitivity. Thus results can be
delivered in as little as 20 minutes, but mostly in an hour.
In our laboratory we have used the SmartCycler to detect
quantitatively Pneumocystis jerovecii, and qualitatively
Mycoplasma pneumoniae and Chlamydophila pneumoniae from
respiratory specimens. vanA and vanB as well as mecA, femB
and PVL are detected from positive blood cultures, from
enrichment broths and from colonies. All of these are in-house
The BD GeneOhm MRSA test is a customized test to detect
MRSA directly from nasal swabs that is performed on the
SmartCycler once or twice daily including the weekends. The
different tests are run simultaneously without causing problems.
However, pipetting of samples in the reaction tubes is more
labour intensive than pipetting in plates or in conical tubes. The
device is predisposed for analysis of specimens in small
quantities or specimens for which results are needed promptly
possibly several times a day.
SeptiFast: A new real-time PCR-based assay for rapid
molecular detection of pathogens in patients with clinical
K. - P: Hunfeld1
University Hospital of Frankfurt, Institute of Medical
Microbiology & Infection Control, Frankfurt am Main
Blood culture (BC), which currently remains the gold standard
in the microbiological diagnosis of bacterial or fungal
bloodstream infections (BSI), typically becomes positive 8-36
hours after sampling, and therapy can then be adapted based on
presumptive bacterial identification suggested by Gram-stain
characteristics. A more precise pathogen identification and
susceptibility profile, however, is not available until up to 24 to
48 hours later. For a large proportion of patients with clinically
apparent sepsis, the BC is negative, making the optimum
antimicrobial therapy empiric. As revealed by clinical studies,
early detection and adequate treatment of causative pathogens
within the first 6-12 hours, however, is critical for a favorable
outcome in patients with BSI. The development of rapid
diagnostic methods for BSI, therefore, has been identified as an
important medical need to supplement conventional BC
diagnostics and molecular techniques have potential to fulfill
this need. Nucleic acid based diagnostic systems, including
polymerase chain reaction (PCR) methods as well as the
application of DNA and RNA probes are well known sensitive
techniques prone for a more rapid detection and the specific
identification of pathogens involved in BSI.To date, however,
mainly PCR-based methods designed to specifically detect
single pathogens have been widely adopted in the routine
clinical laboratory. Here, potentials and limitations of a recently
introduced multiplex real-time PCR-based assay designed for
rapid detection of 25 clinically important pathogens including
fungal agents directly from whole blood are presented. The test
specifically targets the ITS-region of bacteria and fungi and can
be completed in less than 6 hours from K-EDTA blood. Upon
clinical evaluation the test holds promise for a much shorter
average time-to-report compared to conventional BC while PCR
detection rate for various microorganisms appears to be
significantly higher compared to BC. Reflecting on the currently
available study data, this new technology will be discussed and
commented on from the perspective of a clinical microbiologist
in the context of additional clinical and microbiological
Bacterial in Paramecium
H. - D. Görtz1
University of Stuttgart, Department of Zoology, Stuttgart,
Paramecia like other protozoa are hosts of various intracellular
bacteria in the cytoplasm or even in the nuclei. The bacteria are
belonging to different higher taxa of Eubacteria. Bacteria of the
genus Holospora are highly infectious and specifically invade
the nuclei - either micro- or macronucleus. During invasion the
bacteria release a number of proteins from their periplasm.
Holospora-bacteria multiply in the nuclei exclusively. For their
transportwithin the host cell the bacteria interact with the host
cytoskeleton.The infectious forms of the bacteria can leave the
cell without destroying it. Bacteria of the genus Caedibacter and
others are known as killer-symbionts. The bacteria produce
toxins of unknown nature that are toxic for paramecia that do
not host killer-symbionts. The toxicity is related to so-called Rbodies in the bacteria. The proteins of R-bodies are encoded on
plasmid or phage genom. Other intracellular bacteria in
paramecia are neither toxic do they seem to be favorable for the
host cell. At the time being about 2 to 5 new bacteria per year
are found in paramecia from natural water samples. The
presentation is thought to give a short overview about the
diversity of intracellular bacteria in Paramecium.
Identification of a novel adhesion-promoting virulence
factor from Legionella pneumophila
S. Klar1, A. Flieger1, R. Schade2, S. Volkmar1, M. Broich1
Robert-Koch-Institute, Research Group 5, Berlin, Germany
CCM/CBF Charité, Insitute for Pharmavology, Berlin
Legionella pneumophila infects humans by entering, surviving
and even replicating in lung macrophages and epithelial cells
and thereby causes a severe pneumonia, Legionnaires`disease.
In order to screen for L. pneumophila hydrolases, we identified a
novel secreted protein, designated p50, in the culture
supernatant by N-terminal sequencing. The amino acid sequence
of p50 showed a minor homology to nucleotidases and
suggested a potential phosphodiesterase activity. But assaying a
L. pneumophila p50 knock-out mutant did not confirm this
For further characterization of p50, we expressed the protein in
E.coli and generated a polyclonal p50-antibody. Western Blot
analysis confirmed the localization of p50 in the culture
supernatants as well as in the cell lysates. Interestingly, the
molecular size of the endogenous p50 is shifted from 50 kDa to
a smaller secreted 45 kDa form suggesting processing during or
after secretion. Analysis of a L. pneumophila proA mutant,
deficient in the major secreted protease, also showed the
processed extracellular p50 form (45 kDa) and therefore
indicated restriction by another yet unknown protease.
To uncover the export mechanism for p50, we tested several
mutants defective in the known type I, II or IVB protein
secretion systems for presence of p50 in their culture
supernatants. Western Blot analysis showed that none of the
mentioned systems was responsible for p50 secretion and
therefore the p50 export mechanism still remains unknown.
For determination of p50 functions, we examined the mutant in
infection assays. Interestingly, the p50 mutant did not replicate
in human U937 macrophages and Acanthamoeba castelanii
amoebae. We furthermore found that compared to the wild type
adhesion and invasion of the mutant was strongly reduced in
human lung macrophages and epithelial cells.
In summary, our experiments showed that p50 is a novel
secreted protein, which is processed during or after export into
the culture supernatant, and is involved in L. pneumophila
adhesion to and invasion of mammalian cells.
Analysis of SseF- an effector protein translocated by the
SPI2-encoded T3SS of Salmonella enterica Typhimurium
P. Müller1, M. Hensel1
University Hospital Erlangen, Institute of Microbiology
Erlangen, Germany
Salmonella enterica is a facultative intracellular pathogen that
resides in a membrane-bound compartment referred to as
Salmonella-containing vacuole (SCV) following invasion of the
host cell. The intracellular survival and replication is dependent
on the activity of the type three secretion system encoded by the
Salmonella pathogenicity island 2 (SPI2). Effector proteins
translocated by this system have a crucial role in the
modification of host cell processes, such as intracellular vesicle
transport and the integrity of the host cell microtubule
cytoskeleton. The contribution of the various effector proteins to
these host cell modifications is only partially understood.
SseF is a SPI2 encoded effector protein that is required for
replication in HeLa cells and associates with endosomal/
lysosomal membranes and microtubules. Salmonella strains
deficient in sseF showed reduced endosome aggregation and
decreased microtubule bundling (1,2). Thus SseF seems to play
a critical role in Salmonella pathogenicity.
In this study we investigated the molecular function of SseF in
more detail. Topological properties were studied by the
detection of epitope-tagged SseF after selective permeabilization
in immunfluorescence assays as well as by Western blot
analyses of subcellular fractionated infected HeLa cells with
subsequent alkaline or high salt precipitation. In addition,
deletional analyses were performed. The data indicate that the
region contributing to the phenotype of SseF can be restricted to
a short amino acid motif.
1 Abrahams et al, Cellular Microbiology 2006; 8(5): 728-37
2 Abrahams et al, Traffic 2006; 7(8):950-65
Genomics of the intracellular endosymbiont
Blattabacterium from cockroaches
G. Tokuda1, N. Lo2, A. Yamada3, H. Watanabe4
University of the Ryukyus, Center of Molecular Biosciences,
Nishihara, Japan
University of Sydney, School of Biological Sciences
Sydney, Australia
Kyoto University, Graduate School of Agriculture, Kyoto
National Institute of Agrobiological Sciences, Tsukuba, Japan
Blattabacterium cuenoti is an endocytosymbiotic bacterium
inhabiting the mycetocytes present in the fat bodies of almost all
cockroachs. Due to the difficulties associated with culturing and
purifying this bacterium, its exact role remains to be clarified. In
the present study, B. cuenoti was collected from fat bodies of
the wood-feeding cockroach Panesthia angustipennis, and
successfully purified for the first time. Pulse-field gel
electrophoresis was performed on the purified bacterial cells,
and the size of the B. cuenoti genome was estimated to be 650
kb. This represents the first time the genome size of an
endosymbiont outside the proteobacteria has been determined,
and is in agreement with the pattern of endosymbiont genome
reduction across members of that phylum. We are currently
performing genomic sequence analyses to help reveal the nature
of the symbiosis between this bacterium and its cockroach hosts,
and will report on recent progress.
“Host-pathogen interactions”: the difficult way of chlamydia
and mollicutes into culture collections
S. Gronow1, C. Uphoff2, H.G. Drexler2
DSMZ; Deutsche Sammlung von Mikroorganismen und
Zellkulturen, Microbiology, Brunswich, Germany
DSMZ; Deutsche Sammlung von Mikroorganismen und
Zellkulturen, Human and Animal Cell Lines, Brunswich,
It often is a necessity for researchers to obtain reference material
for the evaluation of results. Such material can be a closely
related species, a type strain or a strain with known
characteristics. In general, culture collections provide this
reference material and make it accessible to the scientific
community. However, in case of Chlamydia and Mollicutes the
supply is rather limited. Another aspect is that researchers
isolating new Chlamydia and Mollicute species have difficulties
to deposit their cultures in a collection, a step that is necessary
for publication. Only very few culture collections are willing to
accept Chlamydia and Mollicutes. Therefore, the German
Culture Collection of Microorganims and Cell Cultures (DSMZ)
decided to start collecting and distributing strains of both groups
(up to biosafety level 2) in 2007.
Mollicutes are cultivated using liquid or solid media or – when
necessary – co-cultured with appropriate cell-cultures. Identity
is confirmed by sequence analysis. The control of human and
animal cell cultures regarding their infection with Mycoplasma
is another important aspect. Research results may be falsified or
misinterpreted due to the presence of mycoplasmas. At the
DSMZ, a modified fluorescence in situ hybridization (FISH)
technique is used for monitoring each cell culture deposited.
Chlamydia are cultivated in appropriate cell cultures and
infections are determined by immunofluorescence methods.
Identity is confirmed by sequence analysis. All preparations are
mycoplasma-free. A modified FISH-technique will be
established now for Chlamydia with the aim to differentiate
serovars directly in cell culture and first results will be
Low-molecular-weight inhibitors of falcipain-2 from
plasmodium falciparum
H. Li1, L. Chen1, J. Tan2, K. Nagarajan2, K. Pumpor2, T. Hogg2
C. L. Schmidt2, H. Jiang1, X. Shen1, R. Hilgenfeld2
Shanghai Institute of Materia Medica, Drug Discovery and
Design Center, Shanghai, China
University of Luebeck, Institute of Biochemistry, Luebeck
The erythrocyte life cycle of the malarial plasmodium parasites
depends on a number of proteases, especially cysteine proteases.
These proteases are involved in various cellular processes:
During the merozoite stage, they facilitate escape from the
erythrocyte and subsequent reinvasion. In the trophozoite stage,
these enzymes are critical for the degradation of hemoglobin.
Regulation of the activity of these cysteine proteases is essential
for parasite survival.
We recently determined the crystal structure of falcipain-2
(Hogg et al., 2006) and have used this structure in virtual
screening of chemical libraries, including a library of
compounds from Traditional Chinese Medicine. Several dozens
of hit molecules and derivatives thereof have been synthesized
and tested for binding to the target by applying surface plasmon
resonance. Some of them showed good inhibitory activity
against falcipain-2, as far as inhibition of the hydrolysis of ZPhe-Arg-pNA or related substrates was concerned. This is
remarkable because it is far from trivial to discover lowmolecular-weight inhibitors that block cysteine proteases
through non-covalent, competitive binding. Currently, we are
optimizing these compounds so that they will also strongly
block the degradation of hemoglobin by falcipain-2.
T. Hogg, K. Nagarajan, S. Herzberg, L. Chen, X. Shen, H.
Jiang, M. Wecke, C.J. Blohmke, R. Hilgenfeld & C.L. Schmidt
(2006): Structural and functional characterization of falcipain-2,
a hemoglobinase from the malarial parasite Plasmodium
falciparum. J. Biol. Chem.. 281, 25425-25437.
This work was supported by the DFG program Intracellular
Forms of Life under grant no. Hi 611/5-1.
Intracellular feeding: Investigation of hemoglobin and
myoglobin degradation by Plasmodium falciparum
proteases - key to antimalarial drug design
K. Nagarajan1, V. Alterio1, T. Hogg1, C. L. Schmidt1
R. Hilgenfeld1
University of Luebeck, Institute of Biochemistry, Luebeck
Plasmodium falciparum is the most dangerous of all human
malaria parasites, accounting for 80% of malaria-related deaths.
During the parasite’s intraerythrocyte life cycle, it degrades host
cell hemoglobin within an acidic organelle termed the food
vacuole, in a process which is essential for parasite growth and
development. This process involves malarial aspartic-, cysteine-,
serine- and metallo-proteases present during the various stages
of the intraerythrocyte life cycle. The aspartic proteases
(plasmepsins) are believed to be responsible for the initial
cleavage of native hemoglobin, and cysteine proteases
(falcipains) are thought to further process cleaved hemoglobin
into smaller fragments. Interference with hemoglobin
degradation holds great promise as a new mode of
Falcipain-2 belongs to the classical papain-like cysteine
proteases (C1A). It is multifunctional, cleaving hemoglobin at
acidic pH within the food vacuole and also the cytoskeletal
proteins ankyrin and band 4.1 at neutral pH, and it can also selfprocess its pro-domain at neutral pH. We recently determined
the crystal structure of falcipain-2 (Hogg et al., 2006). The
structure revealed the presence of a unique hairpin motif located
between the active-site residues His174 and Asn204. It is
believed to be responsible for the binding of hemoglobin. To
study the importance of this loop, we constructed a number of
mutants and carried out various biochemical analyses.
Interestingly, we found that falcipain-2 is also active against
myoglobin, the oxygen-carrier protein for muscle tissue.
Moreover, myoglobin, which is structurally related to the betachain of hemoglobin, is cleaved in an ordered and specific
pattern similar to hemoglobin. Surface plasmon resonance
studies were carried out for both the substrates with an inactive
mutant of falcipain-2, revealing a low micromolar binding
affinity. Interaction between the enzyme and its substrates, its
binding sites, the cleavage products and additional experimental
data on substrate binding and specificity will be presented.
Mapping of this degradation pathway will pave the way for
understanding enzyme-substrate interactions and will support
the design of antimalarial drugs targeted at falcipain-2.
T. Hogg, K. Nagarajan, S. Herzberg, L. Chen, X. Shen, H.
Jiang, M. Wecke, C.J. Blohmke, R. Hilgenfeld & C.L. Schmidt
(2006): J. Biol. Chem.. 281, 25425-25437.
This work was supported by the DFG program Intracellular
Forms of Life under grant no. Hi 611/5-1.
Coxiella burnetii inside its host cell
E. Schröpfer1, H. Meyer1, D. Frangoulidis1
Institute for Microbiology, Munich, Germany
Coxiella burnetii (C.b.) the pathogen of Q fever is an obligate
intracellular g - proteobacterium. Because of its high infectious
potential, its stability to environmental conditions and its
infectivity as an aerosol C.b. is to be handled in BSL3
laboratories and is considered to be a potential bio terror agent.
C.b. is also the only known bacterial pathogen that can survive
permanently in host cell phagolysosomes. This feature results in
permanent vacuoles within infected cells. Rather big vacuoles
appear in cell lines derived from the kidney of African Green
monkeys, like Vero E6 or BGM cells. Vero E6 cells show a
similar course of infection as BGM cells do, but in contrast to
BGM cells they tend to detach upon prolonged infection. Maybe
this effect can be explained by differences in structure of the
actin cytoskeleton.
The Hoffman modulation contrast and the Phase contrast
microscopy were used to examine unstained C. b.-infected cell
preparationsp while filamentous actin was visualised with
fluorescence labelled phalloidine and DNA with either DAPI or
propidium jodide. To locate C.b. within the cells a monoclonal
antibody either conjugated with Oregon green or a secondary
antibody with FITC was used. Promising results were discussed.
In addition these different techniques and methods are suitable
to study structural and functional features of different pathogens
in cell-based systems.
Disassembling membrane traffic - phagolysosome formation
in a test tube
U. Becken1, A. Haas1
University of Bonn, Cell Biology, Bonn, Germany
Microbes that invade the sterile part of the human body can be
internalized by professional phagocytic cells like macrophages.
The newly formed organelle, the phagosome, matures by
sequential fusion with early endosomes and subsequently late
endosomes and lysosomes. Some pathogenic microorganisms
reprogramme the development of a phagolysosome which, in
many cases, increases their virulence.
We established a novel microscopic assay to reconstitute fusion
between phagocytic and endocytic compartments in a cell-free
system. As a first model, we analysed fusion of phagosomes
containing non-pathogenic, IgG-opsonized E.coli with
lysosomes. To prepare organelles for the fusion experiment, a
green fluorescent dye is covalently coupled to the bacterial
surface and a red fluorescent, membrane-impermeant dye is
pulse-chased into lysosomes. Fusion of the purified organelles
presents itself as a colocalisation of the two fluorophors in one
In our assay phagosome-lysosome-fusion showed known
characteristics of organelle fusion in vitro, like dependency on
time, physiological temperature, ATP or cytosolic proteins.
Furthermore, we started to analyse the influence of agents that
chelate calcium or alter organelle pH and the involvement of
different proteins like SNARE- or Rab-proteins in organelle
The described assay is in principle applicable to any phagocytic
particle and to endocytic-/phagocytic compartments of any
maturation stage and will therefore be a useful tool to analyse
phagosome maturation in general and traffic alterations by
No detour required — trafficking of the horse pathogen
Rhodococcus equi within activated macrophages
K. von Bargen1, U. Becken1, T. Dykstra1, A. Haas1
University of Bonn, Institute for Cell Biology, Bonn, Germany
Rhodococcus equi is a gram-positive soil bacterium closely
related to Mycobacterium tuberculosis. Being an facultative
intracellular pathogen it can cause severe bronchopneumonia in
young horses and AIDS patients. When ingested by resting
macrophages, it arrests phagosome maturation in-between the
stages of an early and a late compartment. The bacteria multiply
within their host cell which is finally destroyed. However,
immunologically activated macrophages control rhodococcal
multiplication and the bacteria are killed by the microbicidal
effects of peroxynitrite.
Since it has been shown that the activation of macrophages
reroutes the vacuole of M. tuberculosis into the degradative
pathway, we analyzed the compartmentation of R. equi within
the activated macrophage. We find that the trafficking of R. equi
does not differ from that in resting macrophages with respect to
inhibition of fusion with lysosomes or accessibility to
fluorescent liquid phase markers up to 5 hours post infection.
The pH of R. equi containing phagosomes in resting
macrophages is characterized by an initial drop to about 6,
followed by a complete neutralization of pH. This unusual
development of phagosome pH is barely affected by activation
of macrophages prior to infection. At the same time, activation
of macrophages does not seem to change the ultrastructural
appearance of R. equi phagosomes compared to those in resting
macrophages. However, at 24 hours post infection rhodococci in
activated host cells show a loss of structural integrity.
Multiplication within macrophages is most efficiently inhibited
by stimulation with interferon- and lipopolysaccharide before
infection, but can still be controlled significantly by addition of
activating compounds when the infection has already been
These data indicate that infection control by activated
macrophages does not necessarily depend on a change of the
trafficking of intracellular pathogens.
The mitochondrion-invading bacterium Midichloria
T. Beninati1, D. Sassera2, C. Bandi2, S. Epis2, L. Sacchi3, N. Lo4
University of Sydney, Faculty of Veterinary Science
Sydney, Australia
University of Milan, Veterinary Parasitology, Milan, Italy
University of Pavia, Biology, Pavia, Italy
University of Sydney, Biological Sciences, Sydney, Australia
We have characterized and named Midichloria mitochondrii, the
only known bacterium to invade the mitochondria of any
animal. M. mitochondrii is found in the ovarian cells of the
European tick Ixodes ricinus, and represents a novel lineage
within the Rickettsiales (an order of the alpha-proteobacteria).
Within ovarian cells, M. mitochondrii penetrates the outer
mitochondrial membrane and colonizes the periplasmic space
between the two membranes. As the mitochondrial matrix is
degraded, the bacterium multiplies within the empty shell of the
organelle. Over 20 bacteria have been observed within a single
mitochondrion. PCR screening studies show that the bacterium
is found in 100% of I. ricinus females across its distribution, and
also within a number of other tick species. The functional
significance of the M. mitochondrii-tick association is unclear.
Even though the bacterium seems to behave as a ?predator?
towards the host mitochondria, this does not interfere with egg
development, thus ensuring its vertical transmission to the
progeny. We are currently using quantitative PCR to examine
how numbers of M. midichlorii and mitochondria change
throughout the life cycle of the tick.
Protein transport across the parasitophorous vacuolar
membrane in malaria parasite infected erythrocytes
K. Lingelbach1, S. Charpian1, N. Gehde1, J. Przyborski1
Philipps-University, Department of Biology, Marburg
The human malaria parasite Plasmodium falciparum invades red
blood cells where it develops within a parasitophorous vacuole,
the vacuolar membrane thereby acting as a barrier and an
interface between the parasite and the host cell. During parasite
development, a number of parasite encoded proteins are
transported to specific destinations within the host cell, where
they play roles as pathogenicity factors and/or are involved in
physiological alterations of the erythrocyte required for parasite
growth. These proteins are synthesized within the parasite,
secreted into the parasitophorous vacuole, and subsequently
translocated across the vacuolar membrane, by a yet unknown
mechanism To address this, we have begun to define the
molecular requirements for the transit of parasite proteins
through the parasitophorous vacuole, and for their subsequent
translocation across the vacuolar membrane. Using parasites
transfected with various reporter constructs we show that protein
unfolding within the vacuole is required for translocation across
the vacuolar membrane. Furthermore, to investigate the
requirements on the erythrocytic site of the vacuolar membrane,
infected cells were permeabilized with streptolysin O, which
allows the extraction of the erythrocyte cytosol whilst
maintaining the integrity of the vacuole, thus allowing access of
externally added proteins to the outer face of the vacuolar
membrane. This experimental approach enables reconstitution
of protein translocation across this membrane. Treatment of
permeabilized cells with low concentrations of trypsin blocked
protein translocation, and re-addition of erythrocyte cytosol
restored translocation activity. Harsher protease treatment
resulted in an irreversible loss of translocation activity,
indicating that two proteinaceous components, differing in their
protease sensitivities, are involved in the translocation process.
Further work identified human Hsp70 as a possible mediator in
this process, as this protein was found to be present on the
erythrocytic face of the vacuolar membrane, was trypsin
sensitive and was replenishable by the addition of erythrocyte
cytosol. Taken together, our data is consistent with a protein
pore within the membrane of the parasitophorous vacuole being
involved in the translocation of parasite proteins into the cytosol
of the host erythrocyte.
Live and let die - rhodococcus equi infection of macrophages
A. Haas1, T. Sydor1, K. von Bargen1, M. Polidori1, U. Becken1
Cell Biology Institute, Bonn, Germany
Rhodococcus equi is a Gram-positive bacterium that causes
severe bronchopneumonia in AIDS patients and in very young
horses where bacteria mostly localize to alveolar macrophages
in which they multiply and finally produce necrosis of lung
tissue. Virulence depends on the presence of a virulence
plasmid. We have previously shown that we can reproduce in a
mouse macrophage model some of the pathogenic events seen in
foals: Only plasmid-containing, virulent bacteria multiply in
murine macrophages, they are cytotoxic and establish an
unusual, arrested early-to-late endocytic compartment while
avirulent bacteria localize to phagolysosomes and are killed.
How do rhodococci establish their unusual compartment, how
can they multiply and, eventually, lyse the infected host cell?
Data are presented on the pH profiles of phagosomes containing
wild type or mutant bacteria, on interaction of phagosomes with
the endocytic system, their permissiveness for bacterial
multiplication and on the cytotoxic effects of bacterial mutants.
The parasitophorous vacuole membrane of the
microsporidian pathogen Encephalitozoon cuniculi possesses
pores for metabolite exchange
K. Rönnebäumer1, U. Gross1, W. Bohne1
University of Goettingen, Medical Microbiology, Goettingen
Microsporidia are obligate intracellular organisms of increasing
importance as pathogens in immunocompromised patients. The
model organism Encephalitozoon cuniculi is extremely well
adapted on intracellular parasitism and possesses one of the
most compact eukaryotic genomes (3x106 bp), which predicts
the loss of certain biosynthetic pathways. To characterize the
nutrient requirements of this organism, we cultivated E. cuniculi
in tissue culture medium lacking individual amino acids and
determined the growth rate by a newly developed cell ELISA. E.
cuniculi was found to be auxotroph for most of the investigated
amino acids, which emphasizes the extensive participation of
this parasite on the host cell metabolite pool. Since E. cuniculi
resides inside a parasitophorous vacuole, it is separated from the
host cell cytoplasm by a membrane and nutrients have to cross
this barrier to become available. To investigate the presence of
pores in the parasitophorous vacuole (PV) membrane, we
microinjected fluorescent dyes into infected host cells. A 0,5
kDa molecule could rapidly enter the PV while a 10 kDa
molecule was stably excluded from the PV lumen. These
experiments indicate that the PV membrane possesses pores
with an exclusion size of 10 kDa or less and functions like a
molecular sieve, which should allow exchange of smaller
molecules like amino acids. Along with our recent finding, that
the PV membrane of E. cuniculi lacks host cell membrane
proteins, this observation raises the interesting question on the
origin and biogenesis of the PV membrane as an important host
cell-parasite interface.
A review in health economics. Is psychological stress the
cause or the consequence of a mental disorder?
Inflammations and infections could be possible causes.
Psychosomatic diagnoses are depending on the actual
accuracy in diagnostics.An urgent plea for basic research in
microbiology and diagnostics, encouraging a billion
investment in cause study instead of billion spending for
combating symptoms.
E. Feldmeier1
HAW Hamburg, Health, Hamburg, Germany
Mental disorders are on a rise - worldwide. The WHO expects,
that depression will be the second most disease in 2020 - and
one of the most expensive.
The core statements of the BMBF-documentation „Like being ill
in one'
s soul“ (2007) are:
1. „Despite intensive [neuro]research the causes of depression
are still not clear“
2. Moreover, as in any report about mental disorder, „anyone
can get a depression“, from '
welfare case'to professor.
There are innumerable descriptions, like:„All of a sudden,
people loose pleasure in life, falling in deep melancholy. Some
people are getting ill „apparently without any reason“, others
survive a war staying healthy...“, supporting the view that
unknown parasites could likely be the reason, too.
Maybe there are 2 different types of depression:
a)a disorder of neurotransmitter, harming the vegetative nervous
b)psychosomatic cause: actual or childhood traumas cause a
Is the so called mental disorder the cause or the consequence?
The confusion of cause and impact has an important
consequence. Psycho-therapy is a common, nearly inevitable
treatment for mental sick patients (long lasting & expensive).
MO are persistently inert to psychological therapy.
Bacteria are changing their surface. For that, diagnostics as well
as therapy are unsure.
Diagnostics: MOs cheat the immune system, e.g. in biofilms and
by linking factor H. The common used CRP-parameter could be
a non-reliable indicator (wrong negative).
Therapy: Wrong presuppositions lead to non-effective, nonsystematical antibiotics treatment, leading to resistances or
pseudo-resistances and chronic manifestation.
Scientific history shows that '
still-unknown' parasites were
common for centuries.
MOs could also be responsible for other widespread diseases,
i.e. colitis ulcerosa, asthma, rheumatism and '
rhythm disorders (in customer language: appearing sometimes,
without any known reason).
Abundant literature available.
Antimicrobial susceptibility of coagulase-positive and
coagulase-variable Staphylococci from various indications of
swine, dogs and cats as determined in the BfT-GermVet
monitoring program 2004-2006
S. Schwarz1, C. Werckenthin2, E. Aleík, M. Grobbel3
A. Lübke-Becker3, L. H. Wieler3, J. Wallmann4
Institute forAnimal Breed (FAL), Molekulare Microbiology and
Diagnostic, Neustadt-Mariensee, Germany
Ludwig-Maximilians-University Munich, Institute for Medical
Microbiology, Infektions- and Epidemic Medicine, Munich
Free University Berlin, Institute for Microbiology and Animal
Epidemic, Department Veterinary Medicine, Berlin, Germany
Bundesamt für Verbraucherschutz und Lebensmittelsicherheit
(BVL), Berlin, Germany
The BfT-GermVet monitoring program is a complementary
program to the national resistance monitoring program GERMVet. In the BfT-GermVet program, bacteria from selected
indications of cats, dogs, swine, horses and cattle, were
investigated for their susceptibility to 24 antimicrobial agents by
broth microdilution according to CLSI standards.
A total of 248 coagulase-positive and coagulase-variable
staphylococci from two indications of swine (infections of the
skin and infections of the urinary/genital tract including strains
from the mastitis metritis agalactia syndrome) as well as two
indications of dogs/cats (respiratory tract infections and
infections of skin/ear/mouth) were analysed. The staphylococcal
isolates investigated in the BfT-GermVet program revealed a
rather uniform susceptibility status. Independently of the animal
origin and indication, the most frequently detected resistance
properties were resistances against penicillin G (53-77%) and
ampicillin (42-75%), tetracyclines (33-52%), as well as
erythromycin (13-27%). Oxacillin-resistant or gentamicinresistant staphylococci were detected in 1-9% or 2-9% of the
isolates, respectively. Evenlower prevalences of resistance of 02% were detected for amoxicillin/clavulanic acid, cephalothin,
and cefazolin. Animal-specific differences were observed with
regard to sulfonamide resistance (2% of the porcine
staphylococci). In addition, 22% of the canine/feline
staphylococci from infections of skin/ear/mouth were
chloramphenicol-resistant, whereas chloramphenicol resistance
was detectedin only 4-7% of the strains from the remaining
three indications.
The results of this study provide for the first time a
representative overview of the susceptibility status of
staphylococci from selected indications of swine, dogs and cats
in Germany.
Molecular basis of resistance to macrolides and lincosamides
among staphylococci and streptococci from various animal
sources collected in the resistance monitoring program BfTGermVet
P. Lüthje1, S. Schwarz1
Institute forAnimal Breed (FAL), Molekulare Microbiology and
Diagnostic, Neustadt-Mariensee, Germany
In this study, the erythromycin- and/or clindamycin-resistant
isolates among 248 coagulase-positive and coagulase-variable
staphylococci and 500 streptococci, collected all over Germany
during 2004–2006 in the resistance monitoring program BfTGermVet, were investigated for the genetic basis of macrolide
and/or lincosamide resistance.
The staphylococci were sampled from different disease
conditions of dogs/cats (respiratory tract infections, infections of
skin/ear/mouth) or pigs (infections of the skin, infections of the
urinary/genital tract including strains from the mastitis metritis
agalactia syndrome). The streptococci tested were -haemolytic
streptococci from respiratory tract infections, infections of
skin/ear/mouth, and infections of the urinary/genital tract of
dogs/cats as well as from urinary-genital tract/MMA of pigs or
horses. Moreover, Streptococcus suis from infections of the
central nervous system and from cases of arthritis in pigs as well
as Streptococcus equi from infections of the respiratory tract of
horses were investigated. A total of 57 resistant staphylococci
and 65 resistant streptococci were identified, differentiated
biochemically to the species/subspecies level, and tested by
PCR for the resistance genes erm(A), erm(B), erm(C), erm(TR),
msr(A), msr(D), mef(A), mph(C), lnu(A), lnu(B), and lnu(C).
The methylase genes erm(A), erm(B), and erm(C) were detected
in staphylococci, alone or in different combinations. The erm(B)
gene was the predominant gene in Staphylococcus intermedius
and streptococci. The efflux gene msr(A) as well as the genes
mph(C) and lnu(A) coding for inactivating enzymes were
detected in single staphylococcal isolates. The efflux genes
mef(A) and msr(D) were detected in three streptococci, in one of
them together with the gene erm(B). The gene lnu(B) was
detected for the first time in streptococci of animal origin,
namely in seven porcine S. dysgalactiae subsp. equisimilis
isolates with clindamycin MICs of 4 µg/ml.
In this study, a large number of staphylococci and streptococci
from different animal sources, including pets and companion
animals, has been investigated for their macrolide-lincosamide
resistance genotype. The data obtained confirm that high-level
resistance to erythromycin and clindamycin in staphylococci
and streptococci was most frequently detected and mainly due to
rRNA methylases, whereas inactivating enzymes and exporters
were prevalent at distinctly lower frequencies.
Antimicrobial susceptibility of streptococci from various
indications of swine, horses, dogs and cats as determined in
the BfT-GermVet monitoring program 2004-2006
S. Schwarz1, E. Aleík2, M. Grobbel3, A. Luebke-Becker3
C. Werckenthin2, L.H. Wieler3, J. Wallmann4
Institute forAnimal Breed (FAL), Molekulare Microbiology and
Diagnostic, Neustadt-Mariensee, Germany
Ludwig-Maximilians-University Munich, Institute for Medical
Microbiology, Infektions- and Epidemic Medicine, Munich
Free University Berlin, Institute for Microbiology and Animal
Epidemic, Department Veterinary Medicine, Berlin, Germany
Bundesamt für Verbraucherschutz und Lebensmittelsicherheit
(BVL), Berlin, Germany
The BfT-GermVet monitoring program is a complementary
program to the national resistance monitoring program GERMVet. In the BfT-GermVet program, bacteria from selected
indications of cats, dogs, swine, horses and cattle were
investigated for their susceptibility to 24 antimicrobial agents by
broth microdilution according to CLSI standards.
A total of 500 streptococci from two indications of swine ( haemolytic streptococci from infections of the urinary/genital
tract including isolates from the mastitis metritis agalactia
syndrome as well as S. suis from infections of the central
nervous system and the musculoskeletal system), two
indications of horses (S. equi from respiratory tract infections
and -haemolytic streptococci from infections of the genital
tract), as well as three indications of dogs and cats ( haemolytic streptococci from infections of the respiratory tract,
the urinary/genital tract, and skin/ear/mouth) were analysed.
Independently of their origin (animal species and disease
condition), the streptococcal isolates investigated in the BfTGermVet program revealed an overall similar susceptibility
status. The most frequently detected resistance properties were
resistances against sulfamethoxazole (20-78%), tetracycline (1793%), gentamicin (14-79%) as well as erythromycin (0-33%).
All isolates, except a single penicillin-resistant isolate (MIC of
0.25 µg/ml) and two ceftiofur-resistant isolates (MIC of 0.5
g/ml), were susceptible to penicillins and cephalosporins.
Animal-specific differences were observed for erythromycin
resistance (0-1% of the equine isolates, 10-14% of the
canine/feline isolates, 26-33% of the porcine isolates) and
tetracycline resistance (17-26% of the equine isolates, 27-43%
of the canine/feline isolates, 70-93% of the porcine isolates). In
contrast, low-level gentamicin resistance (MICs of 16-32 µg/ml)
was seen most frequently among equine streptococci (62-79%),
while porcine and canine/feline streptococci exhibited this
resistance property at 17-35% and 14-48%, respectively.
The results of this study provide for the first time a
representative overview of the susceptibility status of
streptococci from selected indications of horses, swine, dogs and
cats in Germany.
multocida and B. bronchisepticaisolates from selected
indications of dogs and cats in Germany.
Antimicrobial susceptibility of Pasteurella multocida and
Bordetella bronchiseptica from dogs and cats as determined
in the BfT-GermVet monitoring program 2004-2006
S. Schwarz1, E. Aleík2, M. Grobbel3, A. Lübke-Becker3
C. Werckenthin2, L. H. Wieler3, J. Wallmann4
Institute forAnimal Breed (FAL), Molekulare Microbiology and
Diagnostic, Neustadt-Mariensee, Germany
Ludwig-Maximilians-University Munich, Institute for Medical
Microbiology, Infektions- and Epidemic Medicine, Munich
Free University Berlin, Institute for Microbiology and Animal
Epidemic, Department Veterinary Medicine, Berlin, Germany
Bundesamt für Verbraucherschutz und Lebensmittelsicherheit
(BVL), Berlin, Germany
Introduction: Antimicrobial resistance in Escherichia coli
isolates has emerged as a major problem of health care in
Germany. For instance, according to data from the
Antimicrobial Resistance Surveillance Programme of the PaulEhrlich-Society for Chemotherapy, which monitors the spread
of resistance in frequently encountered nosocomial pathogens,
resistance to fluoroquinolones (ciprofloxacin) in E. coli
increased from 5% in 1995 to 22% in 2004. In addition, the
proportion of strains producing extended-spectrum betalactamases (ESBL) among E. coli isolates raised from <1% to
5%. Alternative options for treatment are needed to control
antimicrobial resistance. Fosfomycin (FOS) is a bactericidal
broad-spectrum antibiotic that is not structurally related to other
classes of antimicrobial agents. The objective of this study was
to determine the in vitro activity of FOS against CIP-resistant
andESBL-producing E. coli strains.
Methods: Among the 33 isolates tested, 11 were resistant to
CIP, 16 were ESBL producers, and six had both resistance
determinants. Eighteen strains were clinical isolates randomly
collected from a recent multi-Center study in Germany. The
remaining 15 strains producing known ESBLs were taken from
the stock culture collection. The in vitro activity of FOS was
compared with that of ceftazidime (CAZ), meropenem (MEM)
and piperacillin/tazobactam (P/T). MICs were determined using
the broth microdilution procedure according to the DIN
(Deutsches Institut für Normung) 58940 guidelines. Breakpoints
(bp) were those approved by DIN. However, as DINbp for FOS
do not exist, those recommended by the French Society of
Microbiology (SFM) were applied: susceptible 32 mg/l,
resistant >32 mg/l.
Results: Based on DIN breakpoints, all isolates were susceptible
to MEM, whereas 48.5% and 18.2% were intermediate or
resistant to CAZ and P/T, respectively. FOS inhibited all
isolates at 32 mg/l and 94% at 4 mg/l, with MIC50 and MIC90
values of 1 and 4 mg/l, respectively. Using CASFM bp, all
isolates weresusceptible to FOS.
Conclusion: FOS showed excellent in vitro activity against
fluoroquinolone-resistant and ESBL-producing E. coli isolates.
It is, therefore, potentiallyuseful for the treatment ofinfections
caused by E. coli strains resistant to first-line antibiotics.
The BfT-GermVet monitoring program is a complementary
program to the national resistance monitoring program GERMVet. In the BfT-GermVet program, bacteria from selected
indications of cats, dogs, swine, horses and cattle were
investigated for their susceptibility to 24 antimicrobial agents by
broth microdilution according to CLSI standards.
In dogs and cats, Pasteurella multocida is a commensal in the
oropharynx, but may also be involved in respiratory tract
infections. Canine and feline P. multocida isolates, however,
play an important role in infections of humans following a cat or
dog bite. Bordetella bronchiseptica is frequently associated with
canine infectious tracheobronchitis, also known as kennel
cough, and feline infectious upper respiratory tract disease. A
total of 92 canine/feline P. multocidaisolates from respiratory
tract infections or infections of skin/ear/mouth as well as 42
canine/feline B. bronchisepticaisolates from respiratory tract
infections were analysed. The P. multocida isolates were
susceptible to all antimicrobial agents tested, except
sulfamethoxazole to which prevalences of resistance of 43-45%
were recorded. In contrast, the B. bronchiseptica isolates were
resistant at high frequenciesagainsta number of antimicrobial
agents, including cefazolin (100%), sulfamethoxazole (81%),
and trimethoprim/sulfamethoxazole (81%). Moreover,all
isolatesexhibited high MIC values against a number of
antimicrobial agents for which no approved breakpoints
applicable to B. bronchiseptica are currently available: penicillin
G ( 32 µg/ml), oxacillin ( 32 µg/ml), ceftiofur ( 32 µg/ml),
cefquinome (8 32 µg/ml), cephalothin (8 - 64 µg/ml),
spectinomycin ( 1024 µg/ml), clindamycin ( 32 µg/ml), and
spiramycin (16 - 128 µg/ml).
The results of this study provide for the first time a
representative overview of the susceptibility status of P.
In-vitro-activity of fosfomycin against fluoroquinoloneresistant and extended-spectrum beta-lactamse-producing
isolates of Escherichia coli
M. Kresken1, J. Brauers1, B. Körber-Irrgang1
Antiinfectives-Intelligence GmbH, Rheinbach, Germany
Antimicrobial susceptibility of Pseudomonas aeruginosa
from dogs and cats and Arcanobacterium pyogenes from
cattle and swine as determined in the BfT-GermVet
monitoring program 2004-2006
C. Werckenthin1, E. Aleík1, M. Grobbel2, A. Lübke-Becker2
S. Schwarz3, L. H. Wieler2, J. Wallmann4
Ludwig-Maximilians-University Munich, Institute for Medical
Microbiology, Infection- and Epidemic Medicine, Veterinary
Faculty, Munich, Germany
Free University Berlin, Institute for Microbiology and Animal
Epidemic, Department Veterinary Medicine, Berlin, Germany
Institute for Animal Breed, Bundesforschungsanstalt für
Landwirtschaft, Neustadt-Mariensee, Germany
Bundesamt für Verbraucherschutz und Lebensmittelsicherheit
(BVL), Berlin, Germany
The BfT-GermVet monitoring program is a complementary
program to the national veterinary resistance monitoring
program GERM-Vet. In the BfT-GermVet program, bacteria
from selected indications of cats, dogs, swine, horses and cattle
were investigated for their susceptibility to 24 antimicrobial
agents by broth microdilution according to CLSI standards.
In small animal medicine, Pseudomonas aeruginosa is mainly
involved in otitis externa, infections of the respiratory and
urogenital tract as well as nosocomial infections. As many of the
antimicrobial agents commonly used in veterinary medicine are
not active against P. aeruginosa and most modern antipseudomonal agents are not approved for veterinary use, the
options for an antibiotic therapeutic intervention are very
limited. In the BfT-GermVet program, a total of 99 P.
aeruginosa isolates from infections of the skin, ear and mouth as
well as the urinary/genital tract of dogs and cats have been
breakpoints for canine isolates, susceptible 2 µg/ml, resistant
8 µg/ml) was observed in 27 % of the isolates from skin, ear and
mouth and 11 % of isolates from the urinary/genital tract
(intermediate isolates 29 % and 39 %, respectively). For the
fluoroquinolone enrofloxacin (specific breakpoints for
canine/feline isolates, susceptible 0.5 µg/ml, resistant 4
µg/ml), resistance was detected in 24 % (skin/ear/mouth) and 11
% (urinary/genital tract) with intermediate isolates in 49 % and
61 %, respectively.
Arcanobacterium pyogenes is an important pathogen of purulent
infections in veterinary medicine, especially in food-producing
animals. However, the species is, in contrast to A.
haemolyticum, seldom involved in human infections. In the BfTGermVet program, 90 strains from cattle and swine were
investigated, using a slightly modified method for susceptibility
testing, as published standardized methods are not applicable to
this species. Animal-specific breakpoints are not available.
Resistance to penicillins was not observed, as was expected
from previous data. The most prevalent resistance traits were
tetracycline resistance (33-56 %, bovine and porcine isolates)
and sulfonamide resistance (26-40 %, bovine isolates).
The results of this study provide for the first time minimum
inhibitory concentrations of representative P. aeruginosa and A.
pyogenes strains from selected indications of animals in
Environmental sampling of particulate matter and fungal
spores during demolition of a building on a hospital area
D. Hansen1, B. Blahout1, D. Benner1, W. Popp1
University Hospital Essen, Hospital Hygiene, Essen, Germany
Demolition or renovation works adjacent to hospitals pose risks
of airborne infections especially aspergillosis. During November
2005 and March 2006 an old building with 3 floors was
demolished on the area of university hospital of Essen. To
determine if there were any infectious risks for patients from
emissions from the demolition work we monitored particle and
fungal concentration of the air before and during demolition.
During the extensive demolition activity air sampling was
carried out biweekly, otherwise weekly or every two weeks. Air
sampling was conducted at 7 positions around the building. The
weather conditions were monitored at the time of sampling, too.
Concentrations of ultra fine particles, particles
0.3 µm,
0.5 µm and particles
1 µm were significantly
higher during demolition than before. Concentration of moulds,
which could be cultured at 37° C, did not differ between the two
periods. Concentration of moulds which grew at 22° C
correlated significantly with temperature and humidity and was
significantly higher before than during demolition period.
We conclude that infectious risks for patients during demolition
work in hospital areas may be lower than generally assumed.
Identification and characterization of class 1 and class 2
integrons among Escherichia coli isolates from farm animals
and companion animals collected in the BfT-GermVet
monitoring study
K. Kadlec1, S. Schwarz1
Institute for Animal Breed (FAL), Neustadt-Mariensee
In the BfT-GermVet monitoring study, 417 Escherichia coli
isolates collected in 2004-2006 all over Germany from various
disease conditions of pigs (n=87), horses (n=102) or cats/dogs
(n=228) were investigated for their susceptibility to 24 different
antimicrobial agents or combinations of agents. This study dealt
with the identification of integron-associated resistance genes
among multiresistant E. coli isolates.
Class 1 and class 2 integrons were detected by previously
described PCR assays. Amplicons of the variable parts of the
integrons were compared by restriction analysis and at least one
representative of each type of amplicon was cloned and
sequenced. Transformation and conjugation experiments were
conducted to confirm a plasmid location of the integrons.
Class 1 and class 2 integrons were detected in 74 isolates. Nine
isolates harboured both types of integrons, one isolate harboured
two different class 1 integrons, 45 a single class 1 integron, and
19 isolates a single class 2 integron. Within these integrons, four
different trimethoprim resistance genes (dfrA1, dfrA12, dfrA14,
dfrA17), four streptomycin/spectinomycin resistance genes
(aadA1, aadA2, aadA5, aadA6/aadA10), two streptothricin
gentamicin/tobramycin/kanamycin resistance gene (aadB) were
detected. Six different cassette arrangements were identified
within class 1 integrons: aadA1 (21 isolates), dfrA1 + aadA1 (16
isolates), dfrA17 + aadA5 (9 isolates), dfrA12 + orfE + aadA2 (7
isolates), dfrA14 + recombined aadA6/aadA10 (1 isolate), and
aadB + aadA1 (1 isolate). Two different cassette arrangements
in class 2 integrons, dfrA1 + sat2 + aadA1 or estX + sat2 +
aadA1, were identified in 23 and 5 isolates, respectively.
Sequencing of the resistance genes revealed the presence of
novel aadA1, aadA5, dfrA1 and dfrA17 variants. Plasmid
location of the integrons was confirmed in 32 isolates. One
isolate harbouring a class 2 integron and six isolates with a class
1 integron were plasmid free.
Class 1 and/or class 2 integrons carrying resistance genes were
detected in 17.7 % of the isolates tested. In contrast to all other
types, the class 2 integron estX + sat2 + aadA1 was seen only in
porcine isolates. This molecular analysis complements the
phenotypic susceptibility testing conducted in the BfT-GermVet
monitoring study and also helps to explain the persistence of
resistance genes without direct selective pressure.
Molecular characterization of Yersinia strains harbouring a
type IV secretion system
B. Kraushaar1, D. Knabner1, A. Konietzny1, B. Appel1
B. Guerra Roman1, E. Strauch1
Bundesinstitut für Risikobewertung, Molekulare Diagnostik und
Genetik, Berlin, Germany
Antimicrobial susceptibility of Klebsiella spp. and Proteus
spp. from horses and small animals as determined in the
BfT-GermVet monitoring program 2004-2006
M. Grobbel1, A. Lübke-Becker1, E. Aleík2, S. Schwarz3
J. Wallmann4, C. Werckenthin2, L. Wieler1
Freien University Berlin, Institute for Microbiology and Animal
Epidemic, Department Veterinary Medicine, Berlin, Germany
Ludwig-Maximilians-University Munich, Institute for Medical
Microbiology, Infektions- and Epidemic Medicine, Munich
Institut für Tierzucht der Bundesforschungsanstalt für
Landwirtschaft (FAL), Neustadt-Mariensee, Germany
Bundesamt für Verbraucherschutz und Lebensmittelsicherheit
(BVL), Berlin, Germany
In Yersinia enterocolitica 29930 (O:7,8; biotype 1A) a plasmid
encoded type IV secretion system was discovered. To
investigate the distribution of this system in the genus of
Yersinia a multiplex PCR was developed to screen our strain
collection. We detected 24 out of 483 strains so far carrying the
transfer system. To elucidate the genetic relatedness among the
strains belonging to Yersinia enterocolitica, Y. frederiksenii and
Y. kristensenii we performed pulse field gel electrophoresis
(PFGE) analysis. Furthermore amplified fragment length
polymorphism (AFLP) was employed for genotyping and the
results were compared to PFGE analysis.In all Yersinia strains
the type IV system is located on plasmids with a size ranging
from 36 to 48 kb. A recombinant plasmid carrying the system is
transferable between E. coli and different Yersinia strains with
high efficiency. The type IV conjugation system consists of a
transfer region encoding a mating pair formation system (Mpf)
and a DNA transfer and replication system (Dtr). By subcloning
experiments the origin of transfer (oriT) was identified. An entry
exclusion function of the type IV system is encoded by the eex
gene located in the mpf region. Eex excludes the transfer of the
recombinant plasmid with high efficiency whereas the transfer
of unrelated plasmids is nearly unaffected.
DRG 2007/2008 - 5 Jahre DRG-AG - ein
ZwischenberichtDRG 2007/2008 - 5 years DRG-AG - a
L. Leitritz1, E. Kniehl2, T. Pietzcker3, B. Gaertner4, E. Straube5
H. Mauch6
Bioscientia, Microbiology, Ingelheim, Germany
Clinic Karlsruhe, Microbiology, Karlsruhe, Germany
University of Ulm, Ulm, Germany
University Homburg, Virologie, Homburg, Germany
University Jena, Microbiology, Jena, Germany
Helios Clinic Berlin, Microbiology, Berlin, Germany
1998 wurde ein Fallpauschalensystem zur Finanzierung
deutscher Krankenhaeuser beschlossen. 2002 hat sich die DRGAG der DGHM gegruendet und ihre Arbeit aufgenommen.
Durch eine Reihe von Maßnahmen der DRG-AG konnte das
DRG-System an spezielle deutsche Verhaeltnisse angepasst und
veraendert werden. Die Maßnahmen und Veraenderungen
werden dargestellt.
In 1998 a Fallpauschalensystem was decided upon for
reimbursement of Geramanys hospitals. In 2002 the DRG-WG
of the GSHM was founded and immediatly took up action. Due
to a couple of actions the DRG-WG was able to amend and fit
the DRG-system to specific german needs. These actions and
amendments are presented.
The BfT-GermVet monitoring is a complementary program to
the national veterinary resistance monitoring GERM-Vet of the
BVL. In the BfT-GermVet program, bacteria from selected
indications of cats, dogs, swine, horses and cattle were
investigated for their susceptibility to 24 antimicrobial agents by
broth microdilution according to CLSI standards.
Multiresistant strains of Klebsiellaspp. are known to cause
severe nosocomial infections, and production of extended
spectrum -lactamases (ESBL) is commonly reported in human
medicine. Also Proteus spp. are a frequent cause of hospital
acquired infections in humans and usually exhibit unfavourable
resistance patterns. To date, there are only few studies in
veterinary medicine dealing with resistance in this species.
The present study comprised Klebsiellaspp. from infections of
the genital tract (GT) of horses (n=36) and the urinary/genital
tract (UGT) of dogs and cats (n=17) were included. Proteus spp.
were isolated from infections of the UGT (n=37) and the skin
(incl. ear/mouth) (n=30) of small animal (dogs and cats).
Klebsiellaspp. are part of the normal vaginal flora in horses and
dogs, but can also cause infections in this organ system. In
horses, clinical inapparent infections of the genital tract are a
frequent cause of infertility or abortion. Among the Klebsiella
isolates, resistance appeared most frequently against ampicillin
(53-67%), sulfamethoxazole (19-29%), and potentiated
sulfonamides (19-24%). A considerable percentage (29%) of
enrofloxacin resistant isolates was detected among the UGT
isolates of small animals.
Proteusspp. are frequently isolated from infections of the
urogenital tract in animals. Even though these bacteria are part
of the normal skin flora, they can also cause serious infections,
particularly otitis externa in dogs. Amont the Proteus isolates,
highest percentages of resistance were seen against tetracycline
(90-92%). More than 20% of the isolates exhibited resistances to
potentiated sulfonamides (27-37%), sulfamethoxazole (2437%), and chloramphenicol (24-37%).
The results of this study provide for the first time minimum
inhibitory concentrations of representative Klebsiella spp. and
Proteus spp. strains of horses, dogs and cats from selected
indications of animals in Germany.
Antimicrobial susceptibility of Escherichia coli from swine,
horses, dogs and cats as determined in the BfT-GermVet
monitoring program 2004-2006
M. Grobbel1, A. Lübke-Becker1, E. Aleík2, S. Schwarz3
J. Wallmann4, C. Werckenthin2, L. Wieler1
Free University Berlin, Instituet for Microbiology and Animal
Epidemic, Department Veterinary Medicine, Berlin
Ludwig-Maximilians-University Munich, Institute for Medical
Microbiology, Infektions- and Epidemic Medicine, Munich,
Institute for Animal Breed of Bundesforschungsanstalt für
Landwirtschaft (FAL), Neustadt-Mariensee, Germany
Bundesamt für Verbraucherschutz und Lebensmittelsicherheit
(BVL), Berlin, Germany
The BfT-GermVet monitoring is a complementary program to
the national veterinary resistance monitoring GERM-Vet of the
BVL. In the BfT-GermVet program, bacteria from selected
indications of cats, dogs, swine, horses and cattle were
investigated for their susceptibility to 24 antimicrobial agents by
broth microdilution according to CLSI standards.
Escherichia coli is part of the commensal microflora of the
gastrointestinal tract, but pathogenic strains can also cause
infections in this organ system. Additionally, pathogenic E. coli
are the most frequently isolated bacteria from urinary tract
infections in humans and animals, and a possible cause of severe
infections of other organ systems including the central nervous
system, the respiratory tract, and the skin. Therefore, E. coli
isolates from infections of the urinary/genital tract from swine
(n=87), horses (n=102) and small animals (n=100) as well as of
the gastrointestinal tract (n=100) and the respiratory tract (n=17)
from small animals were included in this study. Regardless of
the animal species, resistance was detected most frequently
against sulfamethoxazole (18-59 %), tetracycline (14-54 %), and
ampicillin (14-39 %). Additionally, high percentages of
intermediate isolates were observed for cephalothin (39-46 %).
In general, low prevalences of resistance were detected for
amoxicillin/clavulanic acid (1-4 %), gentamicin (1-9 %), and
cefazolin (0-11 %).
In general, highest percentages of resistant isolates occurred in
pigs, followed by horses and small animals. E. coli from the
urinary/genital tract of small animals showed in comparison
with isolates from the gastrointestinal tract a higher prevalence
of resistance for ampicillin (24 % vs. 14 %) and more isolates
with MIC values 2 µg/ml for enrofloxacin (7 % vs. 2 %).
The results of this study provide for the first time minimum
inhibitory concentrations of representative E. coli strains of
horses, dogs and cats from selected indications of animals in
Germany, determined by an internationally accepted
methodology. Furthermore, susceptibility data of E. coli from
indications of swine not yet tested in the GERM-Vet program
were provided.
First tigecycline-resistant Enterococcus strain isolated from
a german ICU patient
G. Werner1, S. Gfrörer2, C. Konstabel1, W. Witte1, I. Klare1
Robert-Koch-Institute Wernigerode Branch, Wernigerode
Marienhospital, Institute for Laboratory Medicine, Stuttgart
Tigecycline is a promising new antibiotic of last resort active
against many bacteria including Enterococcus spp. Enterococcal
isolates displaying minimal inhibitory concentrations (MIC) of
0.25 mg/L are considered susceptible. The epidemiological
cut-off value (breakpoint) for tigecycline is > 0.5 mg/L for
We received the first tigecycline-resistant Enterococcus (strain
UW6940) from an urine sample of an ICU patient from a
German hospital. The patient had severe underlying diseases
and received intensive care treatment, several courses of
antibiotics that included tigecycline treatment for several weeks.
Species identification revealed Enterococcus faecalis. Initial Etest for tigecycline resulted in 6 mg/L. The strain was sent to us
for further characterizations.
Resistance to tigecycline was confirmed with E-test revealing 2
mg/L. MIC in broth media was 1 mg/L. Tigecycline MICs for
susceptible reference strains E. faecium ATCC19434 and E.
faecalis ATCC29212 were between 0.047 and 0.125 mg/L.
Non-susceptibility to tigecycline is associated with expression
of efflux porters in Acinetobacter baumanii or via mutations in
Tet(A) mediating tetracycline efflux, e.g. in E. coli. We tested
MICs for tigecycline in the presence and absence of several
efflux pump inhibitors, such as reserpin, verapamil, omeprazol,
and prochlorperazine. Concentrations were chosen as given in
the literature (20 - 60 mg/L) and 2 mg/L for prochlorperazine. In
all but one cases addition of inhibitor substances did not show
any effect, except with omeprazol where addition of it resulted
in a several-fold increased MIC. A single tetracycline resistance
gene, tetX, encodes an oxygen-dependent monooxygenase
conferring tigecycline resistance. MICs for tigecycline were not
influenced by aerobic or anaerobic growth conditions. In
addition, we failed to amplify a specific PCR product for tetX
with DNA from strain UW6940. We grew the strain for two
weeks (ca. 400-500 generations) on agar plates and in liquid
broth in absence of any selective pressure to test for stability of
resistance. MICs for tigecycline for all tested progenies
remained stable at 1 mg/L. The resistance trait was not
transferable in filter-mating experiments using tigecyclinesusceptible E. faecalis recipient strains JH2-2 and OG1X.
Preliminary results show stable tigecycline resistance in an E.
faecalis isolate from an ICU patient after prolonged tigecycline
therapy. The basis of tigecycline resistance is obviously not
efflux-driven and could not been identified so far.
Antimicrobial effects of different essential sandalwood oils
on Airborne microbes
M. Wosny1, S. Krist1, G. Buchbauer1
University of Vienna, Vienna, Austria
The antimicrobial potential of essential oils is a long known fact
[1,2]. As airborne microbes represent a constant challenge to the
human immunic system, we aimed to investigate the effect of
essential sandalwood oils on airborne microbes. Therefor we
determined the total air count in an exactly defined testing room
before and after application of different dilutions ( 1:100, 1:200,
1:350, 1:1000, 1:5000) of essential oils of Santalum album, L. (
Santalaceae), Santalum spicatum, R.Br. ( Santalaceae), and
Amyris balsamifera, L. (Rutaceae). The total microbial count in
the testing rooms was determined with an RCS Air Sampler (
Biotest). After vaporizing of each essential oil, the total air
count was determined again 15 min. later.
In our study all tested essential oils showed significant reduction
of airborne microbes. Especially effective was Australian
Sandalwood oil in the dilution of 1:200. It reduced the
totalcount of airborne microbes about 67.11%. The other tested
essential sandalwood oils showed a reduction ranging between
32.32% and 64.46%.
Concluding from these results it can be stated that the essential
oils of different sandalwood types can be useful for reduction of
airborne microbes.
Cost-effectiveness of tigecycline in the treatment of
complicated intra-abdominal infections in Germany
U. Kuchenbecker1, C. Runge1, W. A. Krüger2
Wyeth Pharma GmbH, Health Economics, Muenster, Germany
Eberhard-Karls-University , Anesthesiology and Intensive
Care, Tuebingen, Germany
Background: Increasing rates of resistance to antimicrobial
therapy increase the risk of therapeutic failure and impose a
challenge on therapy of infections. Thereby, resistant
microorganisms lead to a significant increase in patient
morbidity, mortality, and health care costs. Tigecycline, a
glycylcycline, offers a broad-spectrum of activity including
resistant pathogens such as methicillin-resistant Staphylococcus
aureus (MRSA). In order to assess the cost-effectiveness of
Tigecycline vs. selected standard antibiotics, we modeled its use
in the treatment of complicated intra-abdominal infections
Methods: A decision-analytic model was developed and adapted
to estimate expected outcomes and costs of initial antibiotic
imipenem/cilastatin and levofloxacin/metronidazole. We used
published data on pathogen prevalence, in-vitro eradication
rates, length of stay (LOS), failure rates and mortality in order to
populate the model. Information on inpatient costs and drug
costs were derived from official databases.
Results: Overall success rate of initial tigecycline therapy was
levofloxacin/metronidazol (76%) showed lower success rates.
LOS was shortest with tigecycline therapy (13.8 d vs. 15.1,
15.1, 14.3, 14.7 respectively). Cost-effectiveness of tigecycline
was better than for all comparators (6631.76 vs. 8542.42 ,
9200.67 , 7251.55 and 7925.48 per treatment success).
Tigecycline therapy was dominant, e.g. it was more effective
than all other regimens at lower total costs.
Conclusions: The model indicates that empirical therapy with
tigecycline is more cost-effective than standard antibiotic
Antimicrobial effects of jasmine absolute and the essential
Oils of clove leaf, palmarosa grass and siberian fir needles
on airborne microbes
P. Pasierb1, S. Krist1, G. Buchbauer1
University Vienna, Department for Clinical Pharmacy and
Diagnostic, Vienna, Austria
Airborne microbes surround us all day long and are partly able
to represent a danger for human beings. Therefore, in this study
we examined natural products with known anti-microbial
potential [1-4] for their possible use as air-disinfectants. First of
all the total germ count in the testing room was determined with
an RCS Air Sampler. Then the dilutions of Jasmine Absolute,
Clove Leaf-, Palmarosa Grass- and Siberian Fir Needles
Essential Oil, respectively were vaporized in five different
concentrations (1:100, 1:200, 1:350, 1:1000 and 1:5000). After
15 minutes the total microbial count was measured again, using
the air sampler.
All tested dilutions were able to reduce the airborne microbes in
the testing room. The essential oil of Siberian Fir Needles in a
concentration of 1:100 (51.60 % reduction) was the most
effective one. The average reduce of germ count of Jasmine
Absolute was 45.39 % (1:1000), of Clove Leaf essential oil
42.38 % (1:100) and of Palmarosa Grass essential oil 43.52 %
According to this results it can be stated that the tested absolute
and the three essential oils could possibly be used as an
alternative to established air disinfectants.
1 Jirovetz, L.; Buchbauer, G.; Schweiger, T.; Denkova, Z.;
Slavchev, A.; Stoyanova, A.; Schmidt, E.; Geissler, M.; Natural
Product Communications 2007; 2 (4); 407-412
2 Jirovetz, L.; Eller, G.; Buchbauer, G.; Schmidt, E.; Denkova,
Z.; Stoyanova, A.S.; Nikolova, R.; Geissler, M.; Recent
Research Developments in Agronomy and Horticulture 2006; 2;
3 Donaldson, J.R.; Warner, S.L.; Cates, R.G.; Young, D.G.;
Pharmaceutical Biology 2005; 3 (8); 687-695
4 Lopez, P.; Sanchez, C.; Batlle, R.; Nerin, C.; Journal of
Agricultural and Food Chemistry 2005; 53 (17); 6939-6946
Molecular and functional analysis of the trimeric
autotransporter Adhesin Yersinia Adhesin A (YadA)
M. Schütz1, U. Grosskinsky1, D. Linke2, I. B. Autenrieth1
University Hospital Tuebingen, Institute for Medical
Microbiology and Hygiene, Tuebingen, Germany
Max-Planck-Institute for Development Biology, Department
Proteinevolution, Tuebingen, Germany
Yersinia adhesin A (YadA) is a non-fimbrial adhesin which is
essential for the pathogenicity of Yersinia enterocolitica. YadA
mediates binding to eukaryotic extracellular matrix proteins and
host cells, triggers host cell responses, and protects Yersinia
from phagocytosis and killing by human serum. We have
focused on the membrane anchor of YadA particularly with
regard to the highly conserved glycine at position 389 (G389).
The corresponding amino acid residue is conserved in nearly all
related trimeric autotransporters (TAAs) including, e.g. H.
influenzae Hia (G1064). The YadA membrane anchor is a
trimeric b-barrel with each monomer contributing 4 b-strands to
the structure. G389 is supposed to be located in the inner
luminal part of the pore. The fact that G389 is strikingly
conserved suggests that this amino acid residue is important for
the biologic function of YadA. Consequently, our hypothesis
was that a mutation of G389 would disturb the transport of
YadA to the bacterial membrane.
We constructed an inducible expression vector for analysis of
YadA function in E. coli. By means of site directed mutagenesis
we then created G389A, G389S, G389T, G389N and G389H
Protein expression in E. coli was analysed by western blot.
Surface exposure and time course of YadA exposure were
investigated by immunofluorescence (IF) staining and flow
cytometry (FACS) analysis with specific antibodies. Adhesive
and invasive properties of E. coli expressing YadAwt or mutant
YadA were analysed by adhesion assays with collagen coated
coverslips, monolayer cells and gentamicin killing assays.
Western Blot analysis revealed that YadA mutant G389A
retained the ability to form stable trimers under denaturing
conditions, whereas the mutants G389S, G389T, G389N and
G389H were detected as monomers only. IF- and FACSanalysis indicated that even YadA mutants with clearly impaired
trimer stability present some YadA protein on the bacterial
surface. Nevertheless, serum resistance and autoagglutination
was significantly reduced in some of the YadA mutants
compared to YadAwt. Therefore we conclude that G389 is
essential for proper export and trimerisation of YadA.
Bifidobacterial adhesion to intestinal epithelial cells
correlates strongly with anti-inflammatory effects on
intestinal epithelial cells
H. Wei1, U. M. Samen1, B.J. Eikmanns1, C. U. Riedel1
University of Ulm, Institute of Microbiology and
Biotechnology, Ulm, Germany
Bifidobacteria are Gram-positive, anaerobic microorganisms
that used in probiotic intervention in inflammatory bowel
disease (IBD). IBDs are multifactorial disorders characterized
by chronic inflammation of the intestinal epithelium. Probiotics
containing bifidobacteria have been shown to be effective in
reducing the severity of inflammation in several rodent models
and patients of IBD. LPS induces inflammatory events through
toll-like receptor 4 (TLR4) and its co-receptor CD14. There is
increasing evidence that in chronic intestinal inflammation,
expression of TLR4 and CD14 on intestinal epithelial cells
(IECs) is abnormal.
We established in vitro models for LPS-induced inflammation in
cultured Caco-2 and T84 IECs. Using these models, we were
able to demonstrate that under normal conditions cultured IECs
are specifically unresponsive to challenge with LPS alone. By
contrast, when challenged with a combination of LPS and
CD14, IECs showed a dramatic increase in NF- B activation
and IL-8 secretion. Pre-treatment of IECs with different strains
of bifidobacteria revealed the same strain-dependent inhibition
of LPS-induced inflammatory events in both cell lines
confirming previous studies on bifidobacterial inhibition of
LPS-induced inflammation in HT-29 cells (Riedel et al., 2006).
Additionally, the same 10 strains of bifidobacteria were tested
for adhesion to cultured Caco-2 and T84 cells which revealed
the same strain-specific pattern of adhesion to both cell lines.
Interestingly, those strains that showed the highest levels of
adhesion were also those that performed best in inhibiting LPSinduced NF- B activation. Statistical analysis indicates a strong
correlation of adhesion with inhibitory activity for both cell
lines (Caco-2: r =0.97, p<0.0001; T84: r= 0.91, p = 0.0021).
Thus, direct or indirect blocking of LPS-induced inflammation
as a consequence of bifidobacterial adhesion could be a possible
mechanism by which bifidobacteria exert their antiinflammatory effects in probiotic intervention in IBD.
Quantitative resistance level of Pasteurella multocida
isolates from respiratory tract infections of cattle:
Current results of the national resistance monitoring by the
BVL 2005/2006
U. Schröer1, H. Kaspar1, J. Wallmann1
Bundesamt für Verbraucherschutz und Lebensmittelsicherheit
(BVL), Referat Biologische Untersuchungen
Antibiotikaresistenz, Berlin, Germany
In 2001 the representative national resistance monitoring was
put into service by the Federal Office of Consumer Protection
and Food Safety (BVL). The results made it possible to evaluate
the resistance situation, development and spread in pathogens
from food producing animals. As part of the monitoring
programme Pasteurella (P.) multocida is included in the
investigations, because P. multocida plays an important role as a
pathogen in a wide variety of animals, including pneumonia in
During the monitoring study 2005/2006 a total of 188 bovine P.
multocida-strains originating from cattle sufferingfrom
respiratory tract infections were tested with the brothmicrodilution method as given in the CLSI document M31-S1 to
determine the in vitro susceptibility (minimum inhibitory
concentration) to 24 antimicrobial agents.
95 strains were collected from calves, 64 strains from young
cattle and 29 strains from diary cows.
The P. multocida strains were susceptible to the most
antimicrobial agents tested. Low to moderate resistance rates
were determined against nine agents tested (cephalothin,
enrofloxacin, gentamicin, nalidixic acid, spectinomycin,
tetracycline, trimethoprim, chloramphenicole, the combination
trimethoprim/sulfamethoxazole). Differences could be noted
regarding the production levels: the strains from calves showed
resistance against eight, the strains from young cattle against
seven and the strains from diary cows only against three of these
antimicrobial agents. The highest resistance rates for all strains
were detected against spectinomycin (up to 20%).
The presented results show that it is crucial to analyse resistance
data according to the various cattle production levels. The data
of this study help to identify trends in resistance rates, to deduce
appropriate therapeutic management measures and provide
practising veterinary surgeons with valuable information for
establishing empirical therapy.
Pharmacoepidemiologic study in primary dysmenorrhea
M. Ostin1, T. Liliana1, M. Cosmina1, S. Mircea Gabriel1
"Gr.T.Popa" University of Medicine and Pharmacy
Pharmacology Department, Iasi, Rumania
Aim: Pharmacoepidemiologic investigation regarding the
intensity of pain, the associate symptoms, and the management
in primary dysmenorrhea. Dysmenorrhea is described as
menstrual pain that is severe enough to limit a woman’s normal
activities and is requiring medical attention. This study is a part
of a larger study that deals with prevalence and symptomatology
of dysmenorrhea in Iasi. This exploratory study was performed
on 266 volunteer females, with a ages between 19 and more than
40, employed in some factories, which completed a
questionnaire consisting of 21 questions about intensity of
menstrual-related distress, measured with the visual analogue
scale (range=0-10), systemic symptoms, medical adresability
and the drugs used to reduce pelvic pain. In summary, in this
study the prevalence of dysmenorrhea was reported by 61% of
the study subjects. Drug therapy plays an important role in pain
management. Analgesic therapy was realized especially by selfmedication (33%) or according to pharmacist’s recommendation
Regulation of the expression of the Lantbiotic mersacidin
structural gene in different genetic backgrounds
C. Szekat1, A. Hoffmann1, S. Schmitz1, G. Bierbaum1
University Bonn, Institute for Medical Microbiology
Immunology and Parasitology, Bonn, Germany
The lantibiotic (lanthionine-containing antibiotic) mersacidin is
an antimicrobial peptide that consists of 20 amino acids. The
producer strain of mersacidin is Bacillus sp. strain HIL Y85,54728. The structural gene (mrsA) and the genes for
modification enzymes, transporters, and producer self-protection
are encoded on a 12.3 kb biosynthetic gene cluster on the
chromosome of the producer strain. Moreover, three regulatory
genes are encoded on the gene cluster (1). The two component
system MrsR2/K2 is mainly involved in immunity and induction
of mersacidin biosynthesis in the presence of mersacidin by a
quorum sensing mechanism. A single regulatory protein MrsR1
is encoded downstream of mrsA. Mersacidin inhibits the growth
of gram-positive bacteria by binding to the cell wall precursor
lipid II and inhibiting cell wall biosynthesis. It shows good
Mersacidin production is strongly influenced by the growth
phase and composition of the culture medium, e. g. good
production yields are obtained in synthetic media, whereas in
LB or TSB no production of mersacidin is observed.
Correspondingly, in the presence of the putative operator
structure and mrsR1, transcription of mrsA was high in synthetic
media and low in complex media. Surprisingly, heterologous
expression of mrsA and mrsR1 in B. subtilis 168 yielded
different results: Here, transcription was high in complex media
and repressed in synthetic media. In the absence of the putative
operator and/or the regulator MrsR1, the repression was
abolished in B. subtilis 168. Furthermore, an in silico analysis of
the nucleotide sequence of the upstream sequences of mrsA
indicated a putative ScoC (hpr) binding motif that is present in
the promoter region. ScoC was detected as a regulator of
protease production in Bacillus, however the ScoC regulon
comprises at least 560 genes (2). Deletion of scoC in B. subtilis
168 resulted in complete loss of mersacidin transcription in
synthetic as well as in complex media, indicating that ScoC
(hpr) is involved in the regulation of mersacidin transcription in
this strain. The role of ScoC in the original host of mersacidin
production is currently under investigation.
(1) Altena K., Guder A., Cramer C., Bierbaum G., Appl Environ
Microbiol. 2000; 66 : 2565-71
(2) Caldwell R., Sapolsky R., Weyler W., Maile R.R., Causey S.
C., Ferrari E., J. Bacteriol. 2001 ; 183 : 7329-40
Cell-cell communication in spatially structured microbial
communities: The privacy of a microcolony
A. Dötsch1,2, L. Lardon3, B. A. Hense4, S. Häußler1
J. - U. Kreft2
Helmholtz-Center for Infection Research, CPI, Brunswich
University of Bonn, Theoretical Biology, IZMB, Bonn
E&R DTU, Microbial Ecology Research Group, Lyngby
GSF - National Research Center for Environment and Health
Institute of Biomathematics and Biometry, Neuherberg/Munich
Bacteria use autoinducers (diffusible signal molecules) to
regulate their gene expression, including genes involved in the
production of exoenzymes, antibiotics, and virulence factors.
Autoinducer (AI) signalling has been extensively studied in
well-mixed liquid cultures of single species. Under these
conditions, AI concentration depends primarily on cell density.
This is commonly known as Quorum Sensing. But since bacteria
only measure AI concentration rather than really counting their
fellows the question is: Which factors actually influence this
concentration in a structured, more natural environment? Nacyl homoserine lactones (AHL) are common AI signals in
gram-negative bacteria like Pseudomonas aeruginosa. Because
of the limited chemical diversity of AHLs, identical molecules
are often used by different species. Thus another question arises:
How could signalling be kept intraspecific in an environment
like soil where neighbouring colonies are likely to be
unrelated?In this work mathematical modelling is applied to
demonstrate how AI signalling is influenced by cell density,
mass transfer and spatial distribution and to evaluate the extent
of interspecific communication in a spatially structured
We developed a simple mathematical model of autoinducer
signalling. In this model cells are positioned in two-dimensional
space and produce a diffusible AI signal. The AI is produced at
a low basal rate and at a higher rate when cells are induced by
high AI concentration. Simulations were run on iDynoMiCs, a
newly developed software platform for individual-based
modelling of the dynamics of microbial communities.
Our results show that besides cell density and diffusibility of AI
molecules the spatial distribution of cells has a strong impact on
the AI concentration that a single cell experiences. Within and
around cell clusters - naturally arising as clonal microcolonies
from cell division - the concentration of AI is much higher than
in regions with more loosely distributed cells. Due to the
combined effect of clustering and positive feedback cells are
synchronously upregulated in microcolonies and their close
In conclusion, intercellular communication in structured
microbial communities is primarily within rather than between
microcolonies and the potential for cross-talk between species is
reduced if the bacteria are not located randomly but clustered.
Clustering and positive feedback enable a synchronized
response of microcolonies promoting intraspecific cooperative
On the way towards the entire proteome of Staphylococcus
K. Hempel1, S. Wolff1, C. Kohler1, K. Rogasch1, S. Engelmann1
D. Becher1, M. Hecker1
University of Greifswald, Institute for Microbiology
Greifswald, Germany
Staphylococcus aureus is a prevalent component of the human
microbial flora that can turn into a dangerous human pathogen
causing diseases ranging from wound infection to endocarditis,
osteomyelitis, and sepsis. For investigation of the entire
proteome of this organism we combined several analytical stateof-the-art tools, viz. gelbased and gelfree approaches in
combination with MALDI-ToF or ESI-FTICR mass
spectrometry resulting in a comprehensive description of the
proteome of exponentially growing S. aureus COL cells.
Analysis of the cytoplasmic proteome with a combination of
2D-PAGE and the gel-free approaches resulted in the
identification of 1123 cytoplasmic proteins representing twothirds of the predicted cytoplasmic proteome of S. aureus COL.
Six hundred fifty additional proteins were identified by the gelfree MDLC-MS/MS approach covering proteins undetectable in
a 2D gel such as alkaline and hydrophobic proteins (Kohler et
al., 2005). Because of their special relevance for host-pathogen
interaction, extra cytoplasmic proteins were prepared from the
supernatant and analysed by 2D-PAGE. This resulted in the
identification of 42 proteins (Rogasch et al., 2006). To gain
access to the hydrophobic membrane proteins, membranes of S.
aureus COL cells were purified and analysed by 1D-gel-LCMS/MS as well as 2D-LC-MS/MS. In summary 432 proteins
were identified by these two methods. Among them 204 proteins
possess transmembrane domains (TMDs), representing about 30
% of all theoretical proteins carrying TMDs. For analysis of the
pathogenicity relevant cell surface associated proteins intact S.
aureus cells were biotinylated with Sulfo-NHS-SS-Biotin
(Pierce) and the proteins enriched by affinity chromatography.
Subsequent 1D-gel-LC-MS/MS resulted in the identification of
70 proteins including 34 proteins known to be cell surface
Our investigation made clear that gel-based and gel-free
methods are highly complementary and that the combination
increases the amount of accessible proteins of different
subproteomes significantly (Wolff et al., 2006). In the study
presented here the identification of almost 50 % of the
approximately 2600 proteins encoded in the genome of S.
aureus COL is described in detail.
HCMV enters endothelial cells by macropinocytosis
N. Ettischer1, Y.-D. Stierhof2, D. Ripper2, K. Laib Sampaio1
C. Sinzger1
University Tuebingen, Institute for Medical Virology
Tuebingen, Germany
University Tuebingen, ZMBP, Tuebingen, Germany
Based on findings in fibroblast cultures, HCMV is supposed to
enter its target cells through fusion of the viral envelope with the
cell membrane. In contrast, electron microscopic analysis of
infected endothelial cells (EC) showed endotheliotropic HCMV
strains in vesicular structures. The aim of this work was to prove
that uptake of HCMV in endothelial cells occurs by endocytosis,
to test the relevance of this pathway for infection efficiency and
to identify the underlying mechanism. The role of clathrin-,
caveolin- and actin-dependent pathways was determined
quantitatively by inhibitor studies. A new improved in-situ
embedding method for the ultrastructural analyses allowed
identifying the virion-containing structures as vesicles by their
localisation in the cytoplasm and by serial sections. The
relevance of endocytosis for infection of EC was proven by
inhibitor studies with methyl-ß-cyclodextrin, latrunculin A,
chlorpromazine and bafilomycin A1. Colocalisation with marker
proteins of the most common endocytic pathways, caveolin-1
and clathrin, was also investigated.
In ultrastructural analyses at 10 minutes after penetration all
internalized particles were found within vesicular structures in
endothelial cells. Therefore, a direct fusion with the cellular
membrane as known from fibroblasts can be excluded. Methyl46
ß-cyclodextrin and latrunculin A reduced infection efficiency
from 80 % to 0-2 %. In contrast, chlorpromazine and
bafilomycin A1 had no influence on the infection efficiency of
HCMV in EC. Therefore, a role of clathrin- and pH-dependent
endocytosis of HCMV in EC was excluded. These findings
rather indicated the endocytic pathway used by HCMV is actinand cholesterol-dependent. The absence of colocalisation with
clathrin and caveolin during entry confirmed this result and also
excluded a contribution of caveolin-dependent endocytosis.
Remarkably, cellular protrusions were found close to virions in
further ultrastructural analyses. Taken together, these results
consistently indicated that HCMV infects endothelial cells by
using an actin- and cholesterol-dependent endocytic pathway
which resembles macropinocytosis.
Identification of functional sites in pUL128 of human
cytomegalovirus by Charge-cluster-to-Alanine-Scanning
A. Schüßler1, K. Laib Sampaio1, N. Ettischer1, B. Adler2
C. Sinzger1
University of Tuebingen, Institute for Medical Virology
Tuebingen, Germany
Max-von-Pettenkofer-Institute, Munich, Germany
The UL128 protein has been shown to be essential for
endothelial cell tropism of HCMV by generation of deletion
mutants. The functional sites within the protein however, have
not yet been identified because it was impossible to generate
single amino acid mutations in an intact viral genome with
conventional mutagenesis techniques. In this work, we aimed to
develop a method for the identification of functional sites within
HCMV proteins and apply it to pUL128. Charge-cluster-toalanine-scanning provides an approach to specifically identify
putative protein-protein interaction sites. The innovative “en
passant” mutagenesis technique allows the introduction of single
base pair exchanges without remaining marker sequences. This
method was applied for the mutation of the C-terminal part of
pUL128 in a highly endotheliotropic BACmid of HCMV.
Several charge clusters were mutated and the phenotype of the
resulting mutant viruses was characterized with regard to
endothelial cell tropism. Cell-free infectivity, cell-associated
spread in a monolayer and virus release from infected
endothelial cells were quantified. Two pentapeptide sequences
relevant for endothelial cell infection were identified. Mutation
of the amino acids HSLTR82-86 reduced endothelial cell tropism
to almost 0%, mutation of KKHKR155-159 reduced endothelial
cell tropism to under 10%. The mean focus expansion value was
reduced from 55 to only single infected cells or 18 infected cells
per focus, respectively. A third examined amino acid sequence
(EYDK137-140) had no influence on endothelial cell tropism,
proving the specificity of the method. A revertant virus for the
KKHKR-mutant showed the same phenotype as the wildtype,
thus excluding any phenotypic changes due to second site
mutations. This method can be used to examine other HCMV
proteins relevant for endothelial cell tropism. Based on the
identified amino acid sequences it is possible to design peptides
and test them for their inhibitory potential for HCMV infection.
Revision of the International Health Regulations (IHR 2005)
of the WHO-challenges for infectious diseases epidemiology
and medical care
D. Matysiak-Klose1, I. Mücke1, T. Eckmanns2
Robert-Koch-Institute, Department of Infectious Disease
Epidemiology, Berlin, Germany
Robert-Koch-Institute, Department of Infectious Disease
Epidemiology, Berlin, Germany
Background: In June 2005, the World Health Assembly of the
WHO adopted the revision of the International Health
Regulations (IHR) which will be legally effective for Germany
as of June 2007. After erroneous decisions and missing
communication had an impact on the rapid spread of the SARS
virus in 2003, and in expectation of a highly pathogenic human
influenza virus, the WHO decided to accelerate a revision of the
1969 version of the International Health Regulations. The 1969
version was no adequate instrument for public health
emergencies of international concern such as those caused by
new and re-emerging diseases with epidemic potential.
Objectives and contents of the IHR 2005 are introduced and
challenges discussed.
Objectives and Contents of the IHR 2005: Main objective of the
IHR (2005) is to prevent, protect against, control and respond to
the international spread of diseases, irrespective of their sources,
while avoiding unnecessary interference with international
traffic and trade. IHR 2005 are applicable on any natural as well
as intended incident. The IHR 2005 contain more notifiable
issues than the German Protection Against Infection Act in
2001. Concerning notifiable biological events, the IHR
differentiate between specific pathogens and infections caused
by unknown pathogens. Annex 2 provides a decision instrument
with uniform limited criteria for the evaluation of events. For
this evaluation it is substantial to assess the event’s impact on
the international level. Events that may pose public health
emergencies of international concern have to be notified to the
WHO via a nominated national focal point within 24 hours after
Consequences for the Public Health Service and Medical Care It
is necessary to revise the German Protection Against Infection
Act to accommodate to the IHR 2005. The usefulness of the
surveillance system is determined by its potential to locally
detect known pathogens as well as emerging ones in a timely
manner. This enables control in parallel to routine surveillance.
Prerequisites are a high sensitivity in the clinical area for
particular infection events and rapid and continuous reporting to
the Public Health Service. Specific communication structures
have to be available to allow for efficient crisis communication
and to provide all parties involved with non-formal data rapidly.
Regular further trainings are also prerequisites for the successful
processing. To implement the International Health Regulations
the necessary structures must be in place by the year 2012.
Functional domains of the Candida albicans Efg1 regulator
D. Kurtz1
Heinrich-Heine-University, Institute for Microbiology
Duesseldorf, Germany
Efg1 is a central transcriptional regulator in Candida albicans,
which controls multiple aspects of morphogenesis and
metabolism. It contains a central bHLH domain, flanked by
sequences conserved in fungal APSES proteins, as well as
polyglutamine (polyQ) stretches at the N- and C-terminal ends.
We performed a systematic deletion approach to specify
functional domains of Efg1p. The bHLH/APSES domain was
required for morphogenesis of the normal yeast and true hyphal
cell forms. Hypha formation on solid medium and
chlamydospore formation also required the C-terminal polyQstretch; chlamydospore formation, in addition, depended on the
presence of the entire N-terminal end. Pseudohypha formation
induced by overexpression of EFG1 only occurred if the
bHLH/APSES domain was intact and an EFG1 overexpressionforced switch from the opaque to the white cell type, in addition,
depended on the presence of specific N- and C-terminal
segments. Efg1p repressor activity in C. albicans required two
specific sequences outside of an bHLH/APSES domain, while
the APSES domain was responsible for Efg1p-phosphorylation
and for binding to a MCB cell cycle-box. These results suggest
that Efg1p has a dual specificity for binding to E- and MCB-box
sequences and reveal domain-specific functions of Efg1p,
providing a framework for detailed structure-function analyses.
Transcriptional and physiological adaptation to defective
protein-O-mannosylation in Candida albicans
P. Cantero1
Heinrich-Heine-University Duesseldorf, Institute for
Microbiology, Duesseldorf, Germany
Five Pmt isoforms O-mannosylate secretory proteins in Candida
albicans. Comparisons of genome-wide transcript patterns of
each pmt mutant revealed commonly downregulated genes
involved in glycolysis and glycerol production. Increased
phosphorylation of the Cek1p- but not the Mkc1p-MAP kinase,
as well as increased transcript levels for stress-related genes
were detected in the pmt1 strain but not in the other pmt
mutants. The transcriptomal pattern after short term-inhibition
of Pmt1p-activity confirmed such stress responses, but did not
indicate an alteration of glycolytic flow. Short- but not longterm adaptation to Pmt1p inhibition required signaling
components Cek1p, Mkc1p, Efg1p and Tpk1p, while lack of
Cna1p (calcineurin) was essential for survival during Pmt1pinhibition; accordingly, cyclosporin A strongly inhibited growth
of the pmt1 mutant. The lack of Pmt isoforms influenced
transcript levels for the remaining isoforms both positively and
negatively, suggesting complex cross-regulation among PMT
genes. These results confirm individual functions of Pmt
isoforms but indicate also a common biphasic adaptation
response to Pmt deficiency. While known signaling pathways
modulate or, in the case of calcineurin, are essential for shortterm adaptation, long-term adaptation likely occurs
independently of stress pathways but may require adjustments of
remaining Pmt activities and of glycolytic flow.
Antimicrobial peptides from insects and vertebrates Chemical modification and activity against multi-drug
resistant bacteria
D. Knappe1, A. Nimptsch1, C. Stegemann1, M. Cassone2
L. Otvos Jr.2, A. Kolobov Jr.3,4, E. Korableva3,4, O. Shamova3
V. N. Kokryakov3,4, R. Hoffmann1
University of Leipzig, Faculty of Chemistry and Mineralogy
Institute of Bioanalytical Chemistry, Center for Biotechnology
and Biomedicine, Leipzig, Germany
Temple University, Sbarro Institute for Cancer Research and
Molecular Medicine, Philadelphia, USA
Russian Academy of Medical Sciences, Institute of
Experimental Medicine, Saint-Petersburg, Russia
Saint-Petersburg State University, Saint-Petersburg, Russia
The presence of antimicrobial substances in secretions, blood
and leukocytes has been known since the end of the 19th
century. Later different classes of antimicrobial peptides (AMP)
with specific activities against bacteria, fungi and viruses were
isolated from vertebrates as well as from invertebrates and
proved to activate the innate immune system of the hosts [1,2].
These gene-encoded peptides differ in length from about 10 to
more than 100 amino acid residues showing a great variety in
structure and antimicrobial modes of action.
The emergence of bacterial and fungal pathogens resistant to
small molecule antimicrobial drugs demands the development of
new antibiotics with novel modes of action. Native
antimicrobial peptides kill bacteria by mechanisms different
than those employed by quinolones and tetracyclines. Short,
proline-rich AMPs, e.g., were isolated from various insects
during recent years possessing significant sequence homologies
and targeting similar intracellular proteins.
We synthesized several short proline-rich AMPs including
drosocin (Drosophila melanogaster), formaecin (Myrmercia
gulosa), metalnikowin (Palomena prasina), and heliocin
(Heliothis virescens) as well as mutated analogs. These
modifications were introduced to improve the pharmacological
profile of the native sequences, including strain specificity and
mammalian protease resistance. In broth microdilution efficacy
assays the minimal inhibitory concentrations (MIC) for
Escherichia coli, Klebsiella pneumoniae, Micrococcus luteus,
Pseudomonas aeruginosa, Staphylococcus aureus, and
Staphylococcus saprophyticus strains were determined.
Remarkably, drosocin derivatives with unnatural amino acid
substitutions showed improved serum stabilities and
significantly lower MIC values compared to the native peptide.
The in vitro activity of peptides isolated from the European
pond turtle (Emys orbicularis) and dog (Canis familiaris) was
also investigated. Some of these new peptides were also active
against all three clinical pathogen classes, e.g., the Grampositive Listeria monocytogenes, the Gram-negative Escherichia
coli and the fungus Candida albicans.
1) Bulet P., Stoecklin R., Menin L., Immunol Rev., 198 (2004)
169-184; 2)Ganz T., Lehrer R.I., Innate Immunity ed. Ezekowitz
R. A. B., Hoffmann J. A. (2002) 287-303, Humana Press,
Totowa, NJ
Inhibition of proliferation of superficial bladder cancer cell
after infection with mycobacterium BCG by cell cycle arrest
and apoptosis
K. Schwarzer1,2, A. Eitner3, M. Förster4, E. Straube1
FSU-University, Medical Microbiolgy, Jena, Germany
Friedrich-Schiller-University Jena, Institute of Medical
Microbiology, Jena, Germany
Friedrich-Schiller-University Jena, Institute of Anatomy II
Jena, Germany
Friedrich-Schiller-University Jena, Department of Internal
Medicine I, Division of Pneumology Medical Center, Jena
Intravesical instillation with Bacillus Calmette-Guerin (BCG),
an attenuated strain of Mycobacterium bovis (M. bovis) used for
anti tuberculosis immunization, is a clinically well-recognized
therapy for superficial bladder cancer and recurrence
prophylaxis. The underlying mechanisms have not yet been
fully elucidated. It is not clear why BCG induces inhibition of
proliferation in some bladder cell lines and not in others we
tested. In this study we evaluated if inhibition of proliferation is
mediated by cell cycle arrest and apoptosis.
Five permanent cell lines gained from different patients were
cocultured either with viable or with heat-inactivated BCG (S4Jena-strain). Proliferation was tested by cell proliferation
reagent WST-1. Cell cycle arrest (propidium iodide DNA
staining) and apoptosis (annexin V/propidium iodide staining)
were analyzed by flow cytometry. Additionally, annexin V
binding and activation of caspases 8/7/3 as well as
morphological hallmarks of apoptosis were shown by confocal
laser scanning microscopy.
Three of five bladder cancer cell lines tested showed cell cycle
arrest and features typical of apoptosis (such as annexin V
binding, caspase-activation) after incubation with BCG. In
contrast to viable BCG inactivated bacteria induced cell cycle
arrest but no apoptosis. Furthermore we showed that the two cell
lines which were not inhibited in their proliferation after
infection with BCG, exhibited no or only slight features of
apoptosis and cell cycle arrest.
These results show that BCG infection has a direct effect on the
cell cycle and viability of bladder cancer cells. We conclude that
BCG infection leads to cell cycle arrest followed by apoptosis
and finally results in cell death.Future studies will concentrate
on why some bladder cancer cells lines are resistent to BCGmediated cell cycle arrest and apoptosis.
Monitoring of nosocomial bacteremia/device-related
infection: How relevant are the data of the G-DRG-system?
E. Kniehl1, B. Gärtner2, L. Leitritz3, H. Mauch4, T. Pietzcker5
E. Straube6
Clinic Karlsruhe, ZLMT – Department of Microbiology and
Hygiene, Karlsruhe, Germany
University Hospital Saarland, Institute for Virology
Homburg, Germany
bioscientia Labor Ingelheim, Ingelheim, Germany
HELIOS Clinic Emil-von-Behring, Institute for Microbiology,
Immunology and Laboratory Medicine, Berlin
University Hospital, Ulm, Germany
Friedrich-Schiller-University Jena, Institute for Medical
Microbiology, Jena, Germany
ICD-codes were introduced for coding death causes (WHO) and
reimbursement-relevant diagnoses (G-DRG). However, there is
a trend to use the data of the G-DRG-system like "medical
documentation"; f.e., some benchmark-projects compare the use
of selected ICD-codes (as T82.7 device-associated infection)
between hospitals.
For nosocomial bacteremia and device-related infection, we
compared data of the G-DRG-system with data of infection
control surveillance and with data of the microbiology lab. The
encoded G-DRG-data have very low "sensitivity"; in this study,
less than 30% of cases documented by the surveillance system
were encoded with the corresponding ICD-code. The main
reasons for this difference may be: that the additional code was
not reimbursement relevant in this case or that "over-coding" is
The use of such data of the G-DRG-system in QM-benchmarkprojects may be misleading and should be regarded with
Homologous high-throughput expression and purification of
highly conserved E. coli proteins
A. Ergin1, K. Büssow2, J. Sieper1, A. Thiel3, R. Duchmann1
T. Adam1
University Medicine Berlin, Berlin, Germany
MPI Mol Genetik, Berlin, Germany
German Rheuma Research Center, Berlin, Germany
Background Genetic factors and a dysregulated immune
response towards commensal bacteria contribute to the
pathogenesis of Inflammatory Bowel Disease (IBD). Animal
models demonstrated that the normal intestinal flora is crucial
for the development of intestinal inflammation. However, due to
the complexity of the intestinal flora, it has been difficult to
design experiments for detection of proinflammatory bacterial
antigen(s) involved in the pathogenesis of the disease. Several
studies indicated a potential association of E. coli with IBD. In
addition, T cell clones of IBD patients were shown to cross react
towards antigens from different enteric bacterial species and
thus likely responded to conserved bacterial antigens. We
therefore chose highly conserved E. coli proteins as candidate
antigens for abnormal T cell responses in IBD and used highthroughput techniques for cloning, expression and purification
under native conditions of a set of 271 conserved E. coli
proteins for downstream immunologic studies.
Results As a standardized procedure, genes were PCR amplified
and cloned into the expression vector pQTEV2 in order to
express proteins N-terminally fused to a seven-histidine-tag.
Initial small-scale expression and purification under native
conditions by metal chelate affinity chromatography indicated
that the vast majority of target proteins were purified in high
yields. Targets that revealed low yields after purification
probably due to weak solubility were shuttled into Gateway
(Invitrogen) destination vectors in order to enhance solubility by
N-terminal fusion of maltose binding protein (MBP), N-utilizing
substance A (NusA), or glutathione S-transferase (GST) to the
target protein. In addition, recombinant proteins were treated
with polymyxin B coated magnetic beads in order to remove
lipopolysaccharide (LPS). Thus, 73% of the targeted proteins
could be expressed and purified in large-scale to give soluble
proteins in the range of 500 µg.
Conclusions Here, we report a cost-efficient procedure to
produce around 200 soluble recombinant E. coli proteins in
large-scale, including removal of LPS by polymyxin B coated
beads for subsequent use of the proteins in downstream
immunological studies.
Carbohydrate - Protein Interaction and antibody
engineering: Mutations in the primary structure of single
chain FV and their effect on binding properties
S. Gerstenbruch1, L. Brade1, P. Kosma2, R. MacKenzie3
S. Evans4, H. Brade1, S. Müller-Lönnies1
Research Center Borstel, Borstel, Germany
University of Natural Resources and Applied Life Sciences
Vienna, Austria
National Research Council, Ottawa, Canada
University of Victoria, Victoria, Italy
Lipopolysaccharide from Chlamydia contains a 3-deoxy- -Dmanno-oct-2-ulosonic acid (Kdo) trisaccharide of the sequence
Kdo(2 8)Kdo(2 4)Kdo. In C. psittaci additionally a linear
Kdo(2 4)Kdo(2 4)Kdo trisaccharide and a branched
Kdo(2 8)[Kdo-(2 4)]Kdo(2 4)Kdo
synthesized. The immunization of mice with the branched
structure led to the generation of antibodies which are crossreactive with the linear trisaccharide. After chemical synthesis
of the terminal branched trisaccharide Kdo(2 8)[Kdo(2 4)]Kdo and immunization we were able to isolate
antibodies by phage-display which bind with high affinity to
LPS oligosaccharides containing the branched structure and
with lower affinity to the linear trisaccharide. We have
biochemically characterized these antibodies in binding assays
with several natural and synthetic oligosaccharides.
Furthermore, we have generated mutant proteins in which amino
acid residues were altered which are important for high affinity
and specific binding as deduced from the previously reported
cocrystal structures of mAb S45-18 [1]. This mAb has been
shown to bind the Kdo(2 4)Kdo(2 4)Kdo epitope in the
linear and the branched structure. By a combination of amino
acid residues from the CDR3 VH of the high affinity mAb S4518 and the low affinity mAb S69-4, two highly homologues
antibodies, we were able to improve the affinity of the latter
considerably. The mutated residues were not suspected to be
directly involved in binding [1] and thus can only be explained
by an indirect conformational effect. Several of these antibodies
can be used for the serological diagnosis of C. psittaci
infections. Funding by Deutsche Forschungsgemeinschaft
(DFG) grant SFB470-C1 is acknowledged.
[1] Nguyen HP, Seto NO, MacKenzie CR, Brade L, Kosma P,
Brade H, Evans SV., 2003, Nat Struct Biol., 10, 1019-25.
Influence of the Lipid A Phosphorylation on the recognition
of enterobacterial LPS by the neutralizing monoclonal
antibody WN1 222-5
L. Heinbockel1, L. Brade1, B. Lindner1, H. Brade1
S. Müller-Lönnies1
Research Center Borstel, Medical And Biochemical
Microbiology, Borstel, Germany
Lipopolysaccharide (LPS, Endotoxin) elicit an immune reaction
which is responsible for many of the effects seen in Gramnegative septic shock. The LPS from Enterobacteriaceae can be
structurally subdivided into the O?polysaccharide (O-PS), the
outer and inner core regions and the lipid A. The latter is the
endotoxic principle of LPS. The mAb WN1 222-5 has been
shown to cross-react with LPS from E. coli and Salmonella
enterica, despite different O-PS and outer core structures.
Importantly, mAb WN1 222-5 has been shown to neutralize
LPS in different in-vivo models of Gram-negative sepsis. As a
prerequisite for the development of a carbohydrate based
vaccine, we have characterized the epitope of mAb WN1 222-5
and showed that the side-chain heptose and the phosphate on the
branched heptose of the LPS inner core are main antigenic
determinants (Fig. 1). Mild acid treatment, however, which
cleaves the ketosidic linkages of Kdo residues leads to a loss of
binding [1].
To investigate whether phosphates of the lipid A are important
for the recog-nition of the epitope by mAb WN1 222-5, which is
located distal to the lipid A, we have isolated LPS coreoligosaccharides from an RcP+ mutant of S. enterica sv
Typhimurium, which differed in their glycosylation and
phosphorylation. We have determined their structures by mass
spectrometry and nuclear magnetic resonance spectroscopy.
ELISA inhibition studies using these oligosaccharides revealed
unexpectedly that the lack of the 4’-phosphate in the lipid A
improved binding considerably. This can only be explained by a
long-range conformational effect on the LPS structure.
[1] Müller-Loennies,S., Brade,L., MacKenzie,C.R., Di
Padova,F., Brade,H., 2003. J. Biol. Chem., 278, 25618-25627
Funding by the Deutsche Forschungsgemeinschaft (DFG) grant
SFB470-C1 is acknowledged
Figure 1:
Mats of Antibacterial Nanofibers by Electrospinning
A. Greiner1,T. Röcker1, M. Distler1, J. Hehl1
Philipps-University Marburg, Department Chemistry, Marburg
Mats of antibacterial nanofibers were obtained by
electrospinning of polymers doped with silver nanoparticles,
quaternized polyethylene imine nanoparticles, or Maluka honey.
Antibacterial activity was probed against Escherichia coli and
Micrococcus luteus. Short term, instant antibacterial activity as
well as long term antibacterial activity over a prolonged period
of time was obtained.
Extraordinary endocytobionts in free-living amoebae
isolated from the contact lens storage cases of patients with
P. Scheid1, L. Zöller1, R. Michel1
Zentrales Institut des Sanitaetsdienstes der Bundeswehr
Koblenz, Labor für Med. Parasitologie, Koblenz, Germany
Introduction:Free-living amoebae (FLA) occur ubiquitously in
many aquatic habitats and humid soils. In addition to their role
as pathogens, e.g. as infectious agent of „Acanthamoeba
keratitis“, FLA are known to serve as natural hosts and vehicles
of various intracellular organisms (bacteria, viruses and
eucaryonts). Our FLA strains were recently isolated from the
contact lens storage cases of two different female patients with
keratitis. FLA as hosts of intracellularly replicating organisms
play also a role in both of the cases.
Materials and methods: Fluid from the storage case was
transferred to non-nutrient agar (NNA) plates and incubated at
29° and 35°C. The FLA strains harboring endocytobionts were
maintained on NNA. For electron microscopical investigations
amoebae were harvested from 3-5 days old NN agarplates and
from 5 to 7 days old Agar-culture and pelleted by centrifugation.
Results: Within a period of three days of incubation of the
sample, FLA could be detected by light microscopy at 100x
magnification. Some trophozoites appeared to harbor small
bacteria-like organisms of round-oval shape which replicated
intracellularly within the border of the shape of the disintegrated
amoeba. Both distinct endocytobionts appeared contained by a
massive electron-dense outer wall – distinct structures
characteristic for eucaryonts could be distinguished with the
help of electron – microscopy. One of theses endocytobionts
could be morphologically classified as a microsporidan-like
organisms due to the clear electron microscopic results.
Discussion: The detection of different FLA and other
accompanying organisms, especially endocytobionts, is limited
to culture methods. Whereas synergistic effects between
amoebae and bacterial contaminants as factors for corneal
infections have been described, the meaning of these
endoparasitic organisms with respect to the pathogenesis of
keratitis remains unresolved. As shown by morphology the
present isolats were different from many other endocytobionts
reported as yet, and identification on the genetic level is needed.
SERION MultianalytTM Diphtheria / Tetanus / Pertussis
Toxin IgG - A new multiplex immunoassay for vaccination
R. Skopek1, A. Schmiedl1, S. Reder1, I. Kühlmann1
G. Hermann1
Institute Virion/Serion GmbH, Wuerzburg, Germany
The SERION Multianalyt™ technology is based on the
principle of flow cytometry and allows the simultaneous
quantification of several analytes in a small liquid sample
The new SERION Multianalyt™ DIPHTHERIA / TETANUS /
simultaneous quantification of human IgG class antibodies
against the toxoids of diphtheria, tetanus and pertussis. The
particle-based immunoassay is recommended to be used as a
vaccination control and for the determination of the current
immune status in order to avoid hyperimmune reactions.
The application of the test is not limited to one cytometer
platform but can be achieved on all common flow cytometers.
The different antibody titers are quickly and easily calculated
from the raw data by using the SERION Multianalyt™
evaluation software. The test principle opens up new
possibilities for the generation of innovative bioanalytical
Identification of genetic difference between strains of
Campylobacter jejuni and Campylobacter coli by
polymerase chains reaction
F. M. Bin jasass1, S. F. Park2
King Abdulaziz City for Science and Technology, Riyadh
Saudi Arabiaf
University of Surrey, School of Biomedical and Molecular
Science, Guildford, Surrey, United Kingdom
The objectives of this study were to extract DNA from different
strains of Campylobacter jejuni and Campylobacter coli and the
use of PCR assay for concurrent detection of Campylobacter
jejuni NCTC 11168, Campylobacter jejuni NCTC 11322,
Campylobacter jejuni NCTC 11828, Campylobacter coli NCTC
12110, Campylobacter coli NCTC 11437, Campylobacter coli
NCTC 11350, Campylobacter coli NCTC 11366,
Campylobacter coli NCTC 11438, and Campylobacter coli
UA585 using probes derived from the genes SADC1, SADC2,
VAC1, VAC2, PPK1, and HEL2. Nine (9) bacterial strains
representing species of Campylobacter jejuni and
Campylobacter coli were lysed with guanidium thiocyanate.
Electrophoresis in agarose gels showed that the DNA obtained
was of a high molecular mass. PCR amplification of
Campylobacter coli UA585 DNA yielded one band of ~705 bp
while it did not succeed in the specific amplification of
Campylobacter coli NCTC 12110, Campylobacter coli NCTC
11437, Campylobacter coli NCTC 11350, Campylobacter coli
NCTC 11366, and Campylobacter coli NCTC 11438. Also,
PCR amplification of Campylobacter jejuni 11168 and
Campylobacter jejuni 11828 DNA yielded one band of ~1750
bp while it did not succeed in the specific amplification for
Campylobacter jejuni 11322. The primers of PPK1, and HEL2
were not successful in the specific amplification for
Campylobacter jejuni.
Early steps in intestinal infection by enteropathogenic E. coli
(EPEC) leading to barrier disruption: Esp-independent
functional integration of translocated intimin receptor (Tir)
of enteropathogenic Escherichia coli (EPEC) into host cell
C. Rüter1
University Hospital Muenster, Institute of Infectiology, ZMBE
Muenster, Germany
The pathogenesis of enteropathogenic Escherichia coli (EPEC)
is characterized by the type III secretion system-dependent
exploitation of target cells that results in attaching and affacing
(A/E) lesions, actin rearrangements, pedestal formation and a
breach in gastrointestinal barrier integrity. This pathology is
mediated by effector proteins, such as the translocated intimin
receptor (Tir) and several E. coli secreted proteins (Esp), that are
translocated by the type III secretion system into the host cell.
Secretion of virulence proteins of EPEC is tightly regulated. In
response to low Ca2+, Esp-secretion is drastically reduced,
whereas secretion of Tir is increased. Under these conditions the
membrane insertion of Tir is independent of Esps. Furthermore,
espB and espD mutant strains of EPEC, unable to form the
translocation pore, still translocate Tir into host cells
membranes. This autointegrated Tir is functional, as it is able to
complement a tir mutant strain in recruiting actin to bacterial
contact sites. The uptake of Tir into the host cell appears to be
dependent on the C-terminal part of the protein, as deletions in
this part of Tir prevent autointegration. Taken together our
results suggest, that under conditions of limited Ca2+ an
alternative mechanism of functional Tir integration might
trigger the first steps in infection resulting in the induction of
A/E-lesions and barrier disruption.
Molecular subdifferentiation of enterohemorrhagic
Escherichia coli isolates from Bavaria, 2006
U. Busch1, C. Schreiber1, M. Garcia Diez1, I. Huber1
S. Hörmansdorfer1, A. Sing1
Bavarian Health and Food Safety Authority, Oberschleissheim
Enterohemorrhagic Escherichia coli (EHEC) cause diarrhea,
hemorrhagic colitis and hemolytic-uremic syndrome (HUS) in
humans. HUS is defined by a triad of features: acute renal
failure, thrombocytopenia and microangiopathic haemolytic
anemia. It is a leading reason for acute renal failure in
childhood. Shigatoxins (Stx) are regarded to be the cardinal
virulence factors of EHEC. They can be divided into two
groups, Stx1 and Stx2. Subtypes are known from each group, so
Stx1, Stx1c, Stx1d, Stx2, Stx2c, Stx2d, Stx2e and Stx2f.
Stx2-variants, expecially Stx2 and Stx2c, are considered to be
more often associated with clinical symptoms in human as Stx1subtypes. Besides the Stx, EHEC often carry the eae-gene,
which encodes for intimin, an important virulence factor for
severe illness in humans. Other virulence factors are EHECenterohemolysin encoded by the hly-gene and the novel
subtilase cytotoxin (SubAB). SubAB was first describes from an
Australien non-O157- STEC-strain linked to an outbreak of
HUS. In mice it causes microangiopathy, suggesting a direct
role in endothelial damage.
In the year 2006 stool samples of 154 persons were tested
positive for stx1 and/or stx2 by real-time-PCR (LightCycler®,
Roche Diagnostics) after growing up on ENDO-agar-plates
(37°C, 18h) and suspending in sterile NaCl. These probes were
investigated for further virulence factors: for the eae- and hlygene also by real- time-PCR (LightCycler®, Roche Diagnostics)
and for the subAB-gene by PCR. This method was described by
Paton and Paton (2005) (1).
Furthermore a subtyping was done by melting-curve analyses,
as first described by Reischl et al. (2002) (2).
We found 77 samples (50%) positive for stx1 only, 56 (36%)
positive for stx2 only and 21 samples positive for stx1 and stx2
(14%). 4 samples were subAB positive.
(1) Paton AW, Paton JC (2005). Multiplex PCR for Direct
Detection of Shiga Toxigenic Escherichia coli Strains Producing
the Novel Subtilase Cytotoxin. J Clin Microbiol 43 (6), 29442947.
(2) Reischl U, Youssef MT, Kiliwinski J, Lehn N, Zhang WL,
Karch H, Stockbine NA (2002).
Real- Time Fluorescence PCR Assays for Detection and
Charakterization of Shiga Toxin, Intimin, and Enterohemolysin
Genes from Shiga Toxin- Producing Escherichia coli. J Ciln
Microbiol 40 (7), 2555- 2565.
Immunization with H. pylori lysate in combination with
CpG-ODN and cholera toxin in the Mongolian Gerbil model
of H. pylori-infection
S. Denk1, N. Lehn1, A. Hartmann2, Schneider-Brachert1
University Ratisbon, Institute for Medical Microbiology and
Hygiene, Ratisbon, Germany
University Ratisbon, Institute for Pathology, Germany
CpG oligodeoxynucleotides (ODNs) stimulate Th1-type
immune responses and activate the innate immune system to
produce proinflammatory cytokines. In contrast, cholera toxin
(CT) is a potent mucosal adjuvant to co-administered antigens
resulting in a Th-2 immune response. A combination of H.
pylori lysate, CpG-ODN and CT has been shown to result in
sterile immunity towards H. pylori infection in mice. We
examined the immunostimulatory effects of both adjuvants in
combination with H. pylori lysate in the Mongolian Gerbil
model of H. pylori-infection.
Starting ten weeks before infection with H. pylori all gerbils
received five i.n. immunizations at intervals of two weeks with
CpG and/ or CT all in combination with H. pylori lysate. After
four weeks and after eight weeks animals were killed, blood was
collected for antibody titer determination and the stomachs were
removed for analysis of cytokine and chemokine response,
histology and mucosal colonisation.
All infected gerbils showed increased antibody titers against H.
pylori compared to non-infected animals, but those treated with
lysate, CpG-ODN and/or CT had even higher titers. Histological
signs of inflammation could be seen in all infected animals
however gerbils treated with cholera toxin showed more severe
histopathological changes in the gastric antrum. The mucosal
colonisation in the stomach decreased significantly after four
weeks in those animals who received the combination of lysate,
CpG-ODN and CT which was not observable after eight weeks
of infection. Expression of cytokines and chemokines was
analysed by quantitative PCR (TaqMan). After four weeks of
infection the proinflammatory cytokines IL-1 beta, TNF, IL-6,
the regulatory cytokine IL-10 and the chemokines MIF, KC,
Mip-2 showed elevated secretion especially in those animals
treated with lysate, CpG-ODN or CT. The combination of
lysate, CpG-ODN and CT resulted in an additional increase in
expression levels. This effect was only transient and could not
be detected after eight weeks of infection.
The combination of lysate, CpG-ODN and CT in the Mongolian
Gerbil model of H. pylori-infection resulted in a transient
immunostimulatory effect which did not result in sterile
immunity as has been shown in mice. However we observed a
longer period of infection. Although CT is known as a potent
mucosal Th-2 adjuvant, it was able to increase all cytokine and
chemokine expression levels and leading to more severe
histological changes in the gastric mucosa. But despite the
enhanced inflammation it does not seem to clear H. pylori
Genetic background and mobility of the non-LEE encoded
effector a of pathogenic E. coli
S. Stingel1, K. Creuzburg1, H. Schmidt1
University of Hohenheim, Institute of Food Sciences and
Biotechnology, Stuttgart, Germany
Enterohemorrhagic Escherichia coli (EHEC) can damage
epithelial cells by injecting type III effector proteins through a
type III secretion system (T3SS) into the cytoplasm of their host
cell. The T3SS and some effector proteins are encoded within a
chromosomal pathogenicity island termed the “locus of
enterocyte effacement” (LEE). It was shown, that genes of other
type III effector proteins are localized outside the LEE in the
genome of cryptic or intact prophages. One of these effector
proteins is NleA (“Non-LEE encoded Effector A”), the function
of which is still unknown. We detected nleA in 150 out of 170
EHEC and enteropathogenic E. coli (EPEC) strains. Up to now
15 different nleA-variants are known, which show between 71%
to 96% identity at the deduced amino acid level.
Plaque-hybridization was used to prove the hypothesis, that
nleA-variants are generally encoded within inducible prophages.
Moreover, we investigated the linkage of nleA and Shiga Toxin
(stx). We could detect three of the nleA-variants in the genome
of inducible bacteriophages. In addition, all stx2-positive isolates
carried stx2 on an inducible phage, whereas Stx1-converting
phages were just partly inducible. The already characterized
prophage BP-4795 of E. coli O84:H4 strain 4795/97 harbors
beside nleA also stx1. In contrast, the genes nleA and stx are
encoded in the genome of different phages in the so far tested
The nleA-variants are located downstream of the tail fiber genes
in the already sequenced prophages. In the majority of cases, ISelements and genes of further type III effector proteins are
located in the neighborhood of nleA. Among these, nleH
occurred most frequently.
This study supports the assumption, that bacteriophages
contribute essentially to the evolution of virulence factors of
pathogenic E. coli.
Leucine-responsive regulatory protein (Lrp) is involved in
transcriptional regulation of the adherence/lymphocyte
inhibitory factor gene efa/lifA in EHEC strain RW1374
L. H. Wieler1, U. Böhnke1, J. Jores1, K. Tedin1
Free University Berlin, Institute of Microbiology and
Epizootics, Berlin, Germany
EHEC are the most common cause of hemorrhagic colitis,
haemolytic-uremic syndrome (HUS), a leading cause of acute
renal failure in children [1]. Ruminants, especially cattle, are the
main transient reservoir of EHEC, which are transmitted to
humans primarily via contaminated food and water [2, 3, 4]. In
addition to the locus of enterocyte effacement (LEE), Shigatoxin and haemolysin, these pathotypes may express several
other potential virulence genes. One of these virulenceassociated genes, the EHEC factor for adherence/lymphocyte
inhibitory factor (efa1/lifA), was described as part of the LEE
pathogenicity islands of the bovine O103:H2 EHEC strain
RW1374 (PAI IRW1374); [5]. Although efa1/lifA is the largest
virulence-associated E. coli gene known so far, its function and
regulation are currently not studied well.
Here we identified the transcriptional start site of the efa1/lifA
gene and determined the promoter activities of an efa1/lifA
promoter-lacZ fusion in E. coli K-12. While the strongest
promoter activity was observed in complex medium, the
promoter was most active during the early logarithmic phase in
all media tested. This regulation parallels the expression patterns
of two global regulatory proteins, Leucine-responsive regulatory
protein (Lrp) and Factor for inversion stimulation (Fis),
suggesting either regulation by these factors or a similar growth
phase control. We therefore examined the effects on efa1/lifA
expression in lrp and fis mutant strain backgrounds as well as
the influence of different growth media. The results suggest that
Lrp plays a role in the regulation of efa1/lifA expression,
possibly in a complex with Fis.
1. Karch, H. et al. 2005. Int J Med Microbiol 295:405-18.
2. Karch, H., et al. (1999) Infect. Immun. 67:5994-6001.
3. Wieler, L. H. et al. (1998) J. Clin. Microbiol. 36, 1604-1607
4. Wieler, L. H. et al. (2007) Berl. Muench. Tieraerztl.
Wochenschr. (im Druck).
5. Jores, J. et al. (2005) Int. J. Med. Microbiol. 294, 417-425
E. coli Nissle 1917 inhibits EPEC infection by its adherence
via F1C fimbria
S. Kleta1, M. Nordhoff1, K. Tedin2, L. H. Wieler2, S. Oswald3
T. Ölschläger3, W. Bleiß4, G. Holland5, P. Schierack1
Free University Berlin, Institute of Microbiology and
Epizootics, Berlin, Germany
Free University Berlin, Institute of Microbiology and
Berlin, Germany
University Wuerzburg, IMIB, Wuerzburg, Germany
Humboldt-University, LMP, Berlin, Germany
Robert-Koch-Institute, Berlin, Germany
The probiotic E. coli strain Nissle 1917 (EcN) is thought to be
effective in the treatment of chronic inflammatory intestinal
diseases and shows strong inhibitory effects on adherent and
invasive intestinal bacterial pathogens in in vitro studies.
In this study we determined the ability of EcN to influence host
cell infection by enteropathogenic E. coli (EPEC), an important
diarrheogenic pathogen in humans and animals, in an in vitro
porcine intestinal epithelial cell model (IPEC-J2). Preincubation of IPEC-J2 with EcN was found to drastically reduce
the infection efficiency of EPEC in a concentration dependent
manner, suggesting EcN interferes with early interactions of
EPEC with host cells. While attachment and formation of microcolonies was reduced, adherent bacteria still formed attaching
and effacing lesions.
Further studies revealed that EcN adherence to porcine epithelial
cells is largely mediated via F1C fimbria. This adhesion
mediates the probiotic effect since a non-adherent EcN foc
mutant did not reduce EPEC infection. A commensal E. coli
wild type strain lacking F1C fimbriae with low adherence and
no probiotic effect showed strong adhesion and an inhibitory
effect on EPEC infection after introduction of a complementing
plasmid harbouring the foc gene cluster. In addition to F1C
fimbriae, we also show that EcN flagellae are also involved in
the adhesion process and probiotic action. The H1 flagellae of
EcN forms a bacterial network on the host cell surface. A nonflagellated EcN fliA mutant showed reduced adhesion and a
reduced capacity to inhibit EPEC infection.
In conclusion, the inhibitory effect of EcN on the infection of
porcine intestinal epithelial cells with EPEC is mediated by its
adherence via F1C fimbriae and H1 flagellae.
Escherichia coli O26 strains: influence of geography, host
animal and stx gene on virulence characteristics
I. Aktan1, B. Carter1, H. Wilking2, R. M. La Ragione1
M. J. Woodward1, L. H. Wieler3, M. F. Anjum1
Veterinary Laboratories Agency, Department of Food and
Environmental Safety, Weybridge, United Kingdom
Friedrich-Loeffler-Institute, Institute for Epidemiology
Wusterhausen, Germany
Free University Berlin, Institute of Microbiology and
Epizootics, Berlin, Germany
In this study, the influence of geography, host animal and
presence of the stx gene on the virulence characteristics of
Escherichia coli O26 strains was determined. A clear
association was found between the virulence profile and
geographic origin of shiga-toxigenic E. coli (STEC) O26 strains,
with UK STEC O26 strains harbouring virtually identical
profiles whilst central European strains showing considerable
heterogeneity in plasmid encoded genes. The former group were
also more likely to be non-motile and katP gene positive.
Comparison of UK STEC and aEPEC O26 strains showed
presence of the stx1 gene was positively correlated with
presence of espP and katP genes, and negatively associated with
presence of the yagP-yagT region and rhamnose fermentation.
In contrast to the uniform profiles of STEC O26 strains from the
UK, aEPEC O26 strains of bovine and ovine origin showed very
diverse profiles both within and between groups, and could not
be separated into discrete groups. Therefore this study has
shown the characteristics of UK O26 isolates are distinct from
O26 strains from other regions in Central Europe, arguing for a
recent clonal spread of O26 (or: emerging O26 clone) in the UK.
Such differences are expected to influence the zoonotic potential
of this pathogen and the incidence of O26 associated human
Role of protein kinase C in Helicobacter pylori-induced
actin cytoskeletal rearrangements.
S. Brandt1, S. Wessler2, R. Hartig3, W. König1, S. Backert1
Otto-von-Guericke-University, Department of Medical
Microbiology, Magdeburg, Germany
Paul-Ehrlich-Institute, Junior Research Group, Langen
Otto-von-Guericke-University, Department of Immunology
Magdeburg, Germany
Helicobacter pylori translocates the CagA effector protein
through type IV secretion system (T4SS) into AGS gastric
epithelial cells. Tyrosine phosphorylation of CagA by Src and
Abl family kinases triggers global changes in the actin
cytoskeleton leading to the ‘scattering’ phenotype and AGS cell
elongation but the signalling events involved are not fully
understood. Time-lapse video microscopy revealed that AGS
cells infected by CagA-positive H. pylori become elongated
because they fail to release their back ends during cell
locomotion due to a retraction defect. Consistent with a model
in which CagA causes host cell elongation by inhibiting the
disassembly of adhesive cell contacts at migrating cells lagging
ends, immunohistochemical analysis revealed that focal
adhesions complexes persist at the distal tips of elongated cell
projections. Thus, our data implicate a different set of signaling
molecules in the elongation phenotype than those previously
suspected. We found that protein kinase C (PKC) is another
crucial mediator essential of the H. pylori-induced elongation
phenotype. PKC comprises a large family of serine/threonine
kinases which are activated by many extracellular signals. To
gain insights into the role of PKC during H. pylori infection we
evaluated in a time course of infection the activation-dependent
phosphorylation state of individual PKC members and
compared our data with PMA (phorbol myristate acetate), a
common cell-permeable activator of PKCs. Finally, we used
specific siRNA and a set of at least 10 pharmacological
inhibitors to analyze the effect of the individual PKC family
isoforms on the H. pylori- induced actin-cytoskeletal
rearrangements involved in the elongation phenotype.
Characterization of the genes associated with motility of
Campylobacter jejuni.
J. I. Dasti1, V. Simon1, R. Lugert1, R. Schmidt-Ott1, U. Gross1
University of Goettingen, Medical Microbiology, Goettingen
Campylobacter jejuni is known as the most frequent bacterial
cause of foodborne-illness in the developed world including
Germany, where the reported number is more than 60,000 cases
in a year. Despite the recent completion of its genome sequence,
little is known about the pathogenesis of the disease caused by
this bacterium. Different factors are reported to be contributing
in this lack of understanding of the pathogenesis of
campylobacteriosis, likewise, the unavailability of an efficient
system of experimental genetics, the lack of appropriate animal
models for the disease, and the genetic diversity of C. jejuni
strains. By considering all these factors we collected lab strains,
NCTC11168, NCTC11828, 81-176 and eighty-three clinical
isolates and more precisely determined them by combining
biochemical and molecular markers. Transposon mutagenesis of
C. jejuni chromosomal DNA was performed by using the in-vivo
transposition method, which produced a random transposon
mutant library consisting of 660 individual mutants. The
BALB/c mouse model was optimized for an in-vivo genetic
screen of the random mutants. The genetic screen of C. jejuni
mutant library identified 3 mutants defective for their flagellar
motility, an important virulence determinant of C. jejuni. In
addition, electron microscope analysis revealed flagellated and
non-flagellated non-motile mutants of C. jejuni.
Irritable bowel syndrome - A pharmacoepidemiologic
M. Ostin1, T. Liliana1, S. Mircea Gabriel1, M. Cosmina1
"Gr.T.Popa" University of Medicine and Pharmacy
Pharmacology Department, Iasi, Romania
This article presents a pharmacoepidemiologic investigation in
patients suffering from irritable bowel syndrome. The study was
carried on 25 patients with irritable bowel syndrome using an
questionaire about abdominal pain, other symptoms and the
pharmacological and non-pharmacological treatment. From
analysis and statistical processing of data results that a half of
patients presents abdominal pain with medium intensity,
according to a visual analog scale. The results of the study
shows that pharmacological treatment consists especially of
using metoclopramide and anticolinergic drugs.
Probiotic Therapy of Prolonged Non-specific Diarrhoea in
Infants and Toddlers by Treatment with E. coli Nissle 1917
J. Henker1, B. M. Blokhin2, Y. K. Bolbot3, V. G. Maydannik4
C. Wolff5, J. Schulze5, U. Sonnenborn5
University Hospital Carl-Gustav-Carus, Department of
Paediatrics, Dresden, Germany
Russian State Medical University, Moscow, Russia
Dnepropetrovsk State Medical Academy, Dnepropetrovsk
National Medical University, Ukraina
Ardeypharm GmbH, Herdecke, Germany
Background: Infants and toddlers with prolonged diarrhoea are
in danger of developing dehydration and a rapid deterioration of
their general state of health. Since no effective causal therapy
exists so far, the efficacy of the probiotic E. coli strain Nissle
1917 (EcN) for the treatment of prolonged diarrhoea was
compared to placebo in a confirmatory, randomized, doubleblind clinical trial.
Patients and Methods: 151 children aged 1 to 47 months (mean:
25 months) with prolonged non-specific diarrhoea (>3 watery or
loose stools without blood in 24 hours of a diarrhoeal episode,
persisting for more than 4 consecutive days but not longer than
14 days) were randomized in a double-blind design to receive
either the probiotic EcN suspension (n = 75) or placebo (n = 76).
All children were slightly dehydrated (5 - 10% loss of body
weight) and received oral rehydration (according to WHO) once
at the beginning of the study. Then, depending on the age of the
infants, 1 to 3 ml verum (containing 108 viable EcN bacteria/ml)
or placebo suspension were given orally per day for 21 days.
Results: The response rate (number of patients showing a
decrease of stool frequency to less than 3 watery or loose stools
in 24 hours over a period of at least 4 consecutive days) was
higher in the EcN group than in the placebo group and increased
continuously from day 7 (EcN 78.7%, placebo 59.2%) and day
14 (EcN 93.3%, placebo 65.8%) to day 21 (EcN 98.7%, placebo
71.1%). Therapeutical superiority for EcN compared to placebo
was statistically significant on days 14 (p=0.0017) and 21
(p<0.0001). The duration of diarrhoea was shortened by 3.3
days. Treatment with EcN was very well tolerated by the
Conclusion: Due to its clinical efficacy and its excellent
tolerability the probiotic EcN is a suitable remedy for the
treatment of prolonged non-specific diarrhoea.
Effects of different commensal bacteria on the repopulation
of donor T cells in an adoptive T cell transfer model
K. Fink1, F. Leithäuser2, M. Müller1, F. Kahl1, J. Reimann3
I. B. Autenrieth1, J. - S. Frick1
University Tuebingen, Medical Microbiology, Tuebingen
University Ulm, Pathology, Ulm, Germany
University Ulm, Internal Medicine I, Ulm, Germany
The adoptive transfer of CD4+ T cells into immuno-deficient
hosts is a well known model of inflammatory bowel disease.
The development of colitis in this model is associated with the
accumulation of activated CD4+ T cells of the Th1 phenotype in
the colonic lamina propria and results in severe colitis within 46 weeks. Factors driving the Th1-biased T cell responses like for
example the CD4+ T cell subset and the bacterial antigen
inducing inflammatory responses, as well as the APC
stimulating the response are still not defined. We
monocolonized germfree RAG1-/- with either B. vulgatus or E.
coli mpk. In previous studies B. vulgatus revealed in the IBD
model of IL-2 deficient mice a protective, E. coli mpk a
colitogenic potential. The RAG1-/- mice were transplanted with
CD4+ T cells from healthy BL/6 donor mice and monitored for
signs of intestinal inflammation. Furthermore histopathological
changes and the phenotype of T cells and DCs from spleen,
MLN and cLP of monocolonized transplanted RAG1-/- mice
were analysed.
Surprisingly, only mice colonized with E. coli mpk showed a
repopulation and accumulation of donor T cells in spleen, MLN
and cLP. However no symptoms of colitis were observable in E.
coli colonized mice. This might be due to the fact that the
repopulated T cells showed enhanced expression of CD103,
suggesting that colonization with E. coli mpk promotes
proliferation of Tregs which might contribute to the maintenance
of immune homeostasis in the intestine thereby preventing
inflammatory bowel disease.
This data indicate that certain bacterial strains might account for
repopulation of donor T cells but further additional colitogenic
stimuli are necessary for the induction of inflammatory bowel
Functional characterization and mutagenesis of the
Helicobacter hepaticus motility regulator FliA
T. Sterzenbach1, J. Schulze1, W. Behrens1, B. Brenneke1
E. Katzowitsch1, S. Sürbaum1, C. Josenhans1
Hanover Medical University, Institute for Medical
Microbiology, Hanover, Germany
The enterohepatic Helicobacter species Helicobacter hepaticus
is a model organism belonging to the large group of
enterohepatic Helicobacters which persistently colonize the
intestinal tract of humans and various animals including mice.
H. hepaticus has originally been identified as a cause of liver
cancer in wild type mice, and naturally colonizes the upper
bowel of mice. It is a quite prevalent infectious agent in research
laboratory mouse colonies. In several strains of
immunocompromised mice, it may also cause chronic
inflammation of the intestinal tract, similar to inflammatory
bowel disease (IBD) in humans, colitis and colon carcinoma,
and therefore is used as a natural model for possible infectious
causes of these diseases. Properties of immune evasion by H.
hepaticus have been implicated in persistent colonization and in
the diseases that those bacteria may trigger, but little is known
about the natural requirements for the bacteria to persistently
colonize their intestinal niche. Specific factors of host
susceptibility for infection, or bacterial properties which may
aid to establish the infection or be essential for persistence,
except for the toxin CDT, have not been investigated. According
to the published genome sequence of H. hepaticus, the motility
system of H. hepaticus is quite similar to the one of
Helicobacter pylori, which has been studied in some detail. A
central regulator of late motility genes, FliA, is also present in
H. hepaticus. The aim of the present study was to characterize
the function of FliA in H. hepaticus in vitro and in vivo for its
role in mouse colonization.
Results: A mutant in H. hepaticus fliA was successfully
constructed by allelic exchange mutagenesis. The mutant was
non-motile, but displayed truncated flagella and a slightly
altered cellular morphology. H. hepaticus fliA mutants did not
express the flagellin FlaA, which is the main flagellin protein of
the bacterium and expressed by two identical gene loci. H.
hepaticus mutants deficient in fliA were assayed in a noncompetitive mouse model for their colonization ability in
comparison to wild type-infected mice. FliA mutants showed a
strongly diminished ability to initially colonize mice compared
to wild type. Regulatory features of the fliA mutant in vitro were
studied by H. hepaticus whole genome microarray and will be
discussed. In conclusion, this study established FliA of H.
hepaticus as a central motility regulator and as an essential
factor to establish initial colonization.
Characterization of Shiga toxin 1 receptors by mass
C. H. Schweppe1, M. Bielaszewska1, H. Karch1
J. Peter-Katalinic2, J. Müthing2
University of Muenster, Institute for Hygiene, Muenster
University of Muenster, Institute for Medical Physics and
Biophysics, Muenster, Germany
The combination of mass spectrometry with immunochemical
overlay detection is a convenient tool for the full structural
characterization of glycosphingolipids (GSLs). Sugar-binding
proteins such as GSL-specific antibodies or toxins give
structural information about the oligosaccharide type. Mass
spectrometry provides detailed information about the type and
sequence of the monosaccharides and the ceramide moiety
consisting of sphingosine and a fatty acid of variable chain
length. GSLs of the globo-series separated by high-performance
thin-layer chromatography (HPTLC) are detected with an
immunoassay comparable to an ELISA using Shiga toxin (Stx)
1, anti-Stx1 antibodies and alkaline phosphatase conjugated
secondary antibodies. GSLs are extracted from Stx1-positive
HPTLC-bands and applied to mass spectrometry.
GSLs were isolated from mammalian cells which express the
immunodetection, Gb3Cer species were extracted from the silica
gel and applied to mass spectrometry. Substitution of the
constant sphingosine moiety of Gb3Cer with dominating short
and long chain fatty acids results in a double band on the
HPTLC-plate. This ceramide heterogeneity is shown for various
Stx1-detected Gb3Cer species permitting their full structural
characterization. The mass spectra obtained revealed full series
of Y- and Z-type and B- and C-type fragment ions, indicating
the sequential loss of the three hexose moieties from the Gb3Cer
molecules. Thus, the MS data together with the overlay assay of
Gb3Cer-specific Stx1 provide the complete receptor structures.
Interaction of EHEC-Hemolysin with endothelial and
intestinal epithelial cells
T. Aldick1, M. Bielaszewska1, W. Zhang1, J. Brockmeyer1
A. W. Friedrich1, K. S. Kim2, M. A. Schmidt3, H. Karch1
University Muenster, Institute of Hygiene, Muenster, Germany
John-Hopkins-University School of Medicine, Division of
Pediatric Infectious Diseases, Baltimore, MD, USA
University Muenster, Institute of Infectiology, Center for
Molecular Biology of Inflammation (ZMBE), Muenster
Shiga toxin (Stx)-producing Escherichia coli (STEC) cause
hemorrhagic colitis and hemolytic uremic syndrome (HUS)
which is the leading cause of acute renal failure in children. In
the pathogenesis of HUS a microvascular endothelial damage, in
particular in renal glomeruli, plays the central role. Stxs are
believed to be the major precipitants of this vascular injury.
Recently, we isolated from patients with HUS E. coli strains of
serotype O26:H11 which contained no stx-genes; the patients
had no other evidence of STEC infection. Because this finding
was unexpected, further investigation of these strains for other
potential virulence markers and their interaction with human
intestinal epithelial cells and endothelial cells was performed.
We could demonstrate that all isolates possessed several genes
(eae, efa1, iha) coding for adhesins. These strains adhered
efficiently to HCT-8, Caco-2 and T84 intestinal epithelial cell
lines. Furthermore, all isolates harboured the operon (EHEChlyCABD) encoding for EHEC hemolysin (EHEC-Hly) and
displayed the corresponding enterohemolytic phenotype. They
caused a time- and dose-dependent cytotoxicity to human brain
microvascular endothelial cells (HBMEC) and the human
umbilical vein endothelial cells (HUVEC)-derived cell line
EA.hy926. A spontaneous EHEC-hly negative derivate of one of
these strains displayed a non-toxic pattern. Therefore we
assessed the ability of recombinant EHEC-Hly cloned from an
E. coli O26 strain to cause LDH-release as an overt marker for
cytotoxicity. The EHEC-Hly caused similar cytotoxic effects
towards the endothelial cells which was observed for the EHECHly-producing bacteria, indicating that the cytoxicity of the stxnegative EHEC-Hly-positive E. coli O26 strains was induced by
EHEC-Hly. We conclude that their adherence capacity to the
intestinal epithelium as well as the expression of a non-Stx
endothelium-damaging factor might add to the ability of these
stx-negative E. coli O26 to cause severe disease.
Characterization of bacteria-binding glycosphingolipids by
mass spectrometry
A. Müsken1, W. Zhang1, J. Souady2, K. Dreisewerd2
M. Bielaszweska1, A. W. Friedrich1, J. Peter-Katalinic2
H. Karch1, J. Müthing2
University of Muenster, Institute of Hygiene, Muenster
University Muenster, Institute for Medical Physics and
Biophysics, Muenster, Germany
The presented method allows the full structural characterization
of bacteria-binding glycosphingolipids (GSLs) directly on the
high-performance thin-layer chromatography (HPTLC) plate
with low amounts of receptors. The bacterial adhesion assay was
established with bacteria with well known GSL binding
specificity. After the separation of a mixture of GSLs on silica
gel-precoated HPTLC plates the binding-specificity of bacteria
can be determined in an overlay assay. For this purpose the
HPTLC plates are incubated with the bacteria of interest, and
the GSL-receptors can be identified in a solid phase binding
assay similar to common ELISA methodologies. Binding of
bacteria to GSL receptors was proved with a polyclonal
antibody and the appropriate alkaline phosphatase labeled
secondary antibody, which mediates the color reaction (BCIP)
to visualize the bound GSLs. The positive GSLs were further
structurally characterized directly on the HPTLC plate by use of
soft-ionization mass spectrometry. Their complete structures,
including composition details of the ceramide part and the
oligosaccharide moiety of various GSL species, were obtained.
This combination of overlay assay and mass spectrometry
should be applicable to identify the receptors of any type of
GSL-binding bacteria.
Investigation of Shiga-toxin 1 induced cellular damage
A. Bauwens1,M. Bielaszewska1, H. Karch1, H. Schweppe1
R. Reichelt2, B. Kemper3, P. Langehanenberg3, G. von Bally3
J. Peter-Katalini2, J. Müthing2
University Clinic, Institute for Hygiene, Muenster, Germany
University of Muenster, Institute for medical Physics and
Biophysics, Muenster, Germany
University of Muenster, Laboratory of Biophysics, Muenster
Shiga toxin (Stx) producing E. coli (STEC) are responsible for
life-threatening zoonotic food- or water-borne illness. STEC
infections result in a spectrum of outcomes ranging from
asymptomatic carriage to uncomplicated diarrhea, bloody
diarrhea, and the hemolytic uremic syndrome (HUS). Stx 1 is an
AB5-toxin produced by several STEC-strains. The B-subunit
pentamer of Stx1 interacts via 15 binding sites for its
(Gb3Cer/CD77). Binding of Stx1 to Gb3Cer on endothelial cells
is postulated to be a critical event underlying the vascular injury
during STEC-mediated diseases. In this study we explored Stx1mediated cytotoxic action and cellular damage of endothelial
To determine the cytotoxic potential of Stx1, serial dilutions of
the toxin were incubated with confluent monolayers grown in 96
well microtitre plates. Scanning electron microscopy revealed
cellular damage leading to cell irregularity, cellular injury,
detachment, and partial disruption of cell monolayers. The most
obvious effect of Stx1 was a drop of surface microvilli. Stx1caused cell death occurred after 20 to 50 h as shown by long
term time lapse measurements of dynamic changes of the cells’
shape using digital holographic microscopy of single cells.
Immunofluorescence microscopy demonstrated the expression
of the Stx1-receptor Gb3Cer and globotetraosylceramide
(Gb4Cer). The presence of Gb3Cer apparently underlies the
Stx1 sensitivity and might be related to cell death.
Vascular injury during STEC infections most likely results from
the action of Stxs on vascular endothelial cells. An increasing
knowledge about the molecular mechanisms by which Stxs
interact with their target cells is important not only for the
elucidation of pathways of intracellular transport of Stx, but also
for the development of preventive and therapeutic measures for
STEC-mediated diseases.
Forschungsgemeinschaft (grant SPP 11330) and the IZKF
Muenster (Ka2/061/04).
Chromatin modification and regulation of p21 WAF1
promoter in Helicobacter pylori infected gastric epithelial
G. Xia1,2, A. Diestel3, C. Habold3, S. Krüger3, A. Rössner3
M. Naumann2, U. Lendeckel2, R. Schneider-Stock3
University Hospital Tuebingen, Medical Microbiology and
Hygiene, Tuebingen, Germany
Otto-von-Guericke-University Magdeburg, Institute for
Experimental Medicine, Magdeburg, Germany
Otto-von-Guericke-University Magdeburg, Institute for
Pathology, Magdeburg, Germany
Aims: Pathogenic bacteria have acquired the capacity to
modulate the transcriptional machinery of host cells. Only little
is known about the ability of the human pathogen H pylori to
modify the chromatin architecture. We aimed to determine the
influence of H pylori infection on chromatin modifications in
gastric epithelial cells. As an example the epigenetic regulation
at the promoter of the cell cycle regulator p21WAF1 was
Methods: H pylori strain P1 was used to infect gastric epithelial
tumor cell line NCI-N87 and mRNA and proteins were prepared
after 3, 6, and 24h. The modulation of chromatin modifying
enzyme such as methyltransferases (DNMT1, 3A, 3B), histone
acetyltransferases (p300, PCAF), and histone deacetylase
(HDAC1), acetylated histones H3 and H4 (acH4, acH3), p53,
and p21WAF1 were analyzed by western-blot or real-time RTPCR. Chromatin immunoprecipitation was performed to study
the epigenetic regulation at the p21WAF1 promoter. The
formation of -H2A.X foci as a sign of DNA-damage was
studied by immunofluorescence microscopy.
Results: Chromatin modifying enzymes were modulated in H
pylori infected cells. An up-regulation of p21WAF1 was detected
both in NCI-N87 cells and primary cells 6h post infection. This
was associated with a higher p53 and acH4 binding at the
p21WAF1 Sp1-promoter region. Simultaneously, HDAC1 was
released from this promoter site. P21WAF1 up-regulation was not
associated with H pylori induced ROS-dependent DNA-damage
which was confirmed by treatment with the ROS scavenger Nacetylcystein. Conclusions: We show for the first time, that H
pylori infection results in global modification of chromatin in
gastric epithelial cells and thus influences gene transcription by
direct epigenetic effects on gene promoters.
Helicobacter pylori controls serine and tyrosine
phosphorylation of cortactin to induce actin-cytoskeletal
rearrangements and host cell scattering
N. Tegtmeyer1, R. Wittelsberger1, S. Brandt1, W. König1
F. Meyer2, N. Martinez-Quiles3, S. Backert1
Otto-von-Guericke-University, Department of Medical
Microbiology, Magdeburg, Germany
Otto-von-Guericke-University, Department of Surgery,
Magdeburg, Germany
University of Madrid, Institute for Microbiology, Madrid, Spain
Cortactin is an actin-binding protein and central regulator of the
actin cytoskeleton. In vitro, cortactin binds and activates NWASP via its SH3 domain to stimulate actin polymerization by
the Arp2/3 complex. Cortactin is a target for phosphorylation by
Src kinases and by serine/threonine kinases that include Erk1/2,
however, the mechanism by which cortactin phosphorylation
events modulate the architecture of the actin cytoskeleton in
vivo is unknown. Interestingly, cortactin is also a common target
exploited by microbes during infection. We have used
Helicobacter pylori as a model system to study the role and
function of cortactin during infection of gastric epithelial cells.
H. pylori translocates the CagA effector protein into the host
cell cytoplasm of target cells where it induces both the
activation of Erk and the inactivation of Src signaling. This
induces the redistribution of cortactin to actin-rich cellular
protrusions and global rearrangements of the host cell actin
cytoskeleton leading to cell scattering. We monitored the
phosphorylation status of cortactin over the time course of
infection using phosphospecific antibodies. In addition, we
produced a series of phosphorylation-deficient and
phosphorylation-mimicking cortactin constructs fused to GFP to
study their role in H. pylori-induced phenotypical outcome. We
show that the serine-phosphorylated form of cortactin strongly
increased its ability to trigger host cell elongation and scattering.
By contrast, overexpression of cortactin mimicking its tyrosinephosphorylated form strongly reduced the latter activity and
even suppressed serine-phosphorylated cortactin. Thus, Src
phosphorylation terminates cortactin activation and blocks cell
scattering during H. pylori infection. These findings support an
in vivo model where Erk or Src phosphorylation of cortactin acts
as a molecular switch modulating its ability to trigger host cell
elongation and scattering. Taken together, injected CagA
specifically triggers the activation of cortactin by two
independent pathways, the stimulation of Erk and inactivation of
Src. This has an important impact on how H. pylori abuses
cortactin to control the architecture of the host actin
cytoskeleton and cell scattering. These processes may contribute
to the development of gastric cancer by H. pylori infection.
Time course analysis of Helicobacter pylori colonization in
Mongolian gerbils: Role of cag-pathogenicity island on
physiological and immunological markers
T. Wiedemann1, M. Stoeckelhuber2, M. Stolte3, R. Haas1
G. Rieder1
Max-von-Pettenkofer-Institute, Hygiene and Medical
Microbiology, Munich, Germany
Ludwig-Maximilians-University Munich, Institute of Anatomy
Munich, Germany
Institute of Pathology, Clinic Bayreuth, Bayreuth, Germany
Colonization of H. pylori in the stomach is associated with the
development of several gastroduodenal diseases. Recently, we
and others have shown that the development of precancerous
conditions in Mongolian gerbils is associated with the
colonization of the corpus mucosa by H. pylori type I strains
expressing the cag-pathogenicity island (cagPAI). Thus strains
inducing a severe corpus dominant gastritis after seven months
of infection.
Aim: Therefore, we continued to investigate the effect of the
cagPAI on physiological and immunological markers of the
gastric mucosa of H. pylori-infected gerbils during a long-term
colonization experiment.
Methods: Mongolian gerbils (n = 150) were infected with H.
pylori B128 (WT) or an isogenic cagY-mutant for 2, 4, 8, 16,
32, and 64 weeks. Paraffin sections of antrum and corpus
mucosa were graded for H. pylori infection, histological changes
stained specifically, and verified by immunohistochemistry.
Physiological markers like colonization density, pH, gastrin, and
somatostatin were measured. Frozen biopsies were used for
qRT-PCR of several inflammatory markers.
Results: In comparison to the mutant, WT-infected corpus
mucosa revealed a severe inflammation already after several
weeks accompanied with a significant up-rise of
proinflammatory cytokines like IL1-ß, TNF-a, IFN-g, and KC.
Physiological markers (pH, gastrin) as well as histological
changes of the mucosa towards atrophy, metaplasia, and
dysplasia were occurring in a later phase of the experiment.
Interestingly, the colonization density of WT-infected gerbils is
uprising in the corpus continuously over the time period. After
14 months of WT infection we found severe histological
changes of the corpus showing typical precancerous markers
and prominent gastritis cystica profunda.
Conclusion: Our data reveal that an intact cagPAI is responsible
for an early and severe corpus inflammation associated with an
increased expression of cytokines. These cytokines regulate the
physiology of the stomach followed by severe histological redifferentiation towards the intestinal type. In general, our study
indicates early markers for late gastric diseases that represent
important risk factors.
Genetic analysis of the functional interplay between SurA,
Skp and PpiD in the maturation of integral outer membrane
proteins in Escherichia coli
Y. Matern1, S. Behrens1
Georg-August-University Goettingen, Institute for
Microbiology and Genetics, Dep. Molekular Genetics and
preparative Molecular Biology, Goettingen, Germany
Integral beta-barrel outer membrane proteins (OMPs) contribute
to the pathogenicity of many virulent Gram-negative bacteria.
Several folding factors have been proposed to facilitate the
folding and assembly of these proteins in Escherichia coli. A
main player in OMP maturation, which has recently been shown
to also affect virulence of uropathogenic E. coli [1], is the
periplasmic chaperone and peptidyl-prolyl isomerase (PPIase)
SurA. Lack of SurA chaperone activity results in reduced levels
of the major integral OMPs, the porins, concomitant with a
defective outer membrane and a constitutively induced sigmaEdependent extracytoplasmic stress response. In addition, the
small periplasmic chaperone Skp and the inner membraneanchored PPIase PpiD have been suggested to affect OMP
folding. Most importantly, the combined deletion of SurA with
either Skp or PpiD was reported to cause synthetic lethality, and
overproduction of PpiD to substitute for SurA function [2,3,4].
This prompted us to more closely examine the functional
interplay between SurA, Skp and PpiD. Our data call the
previously suggested role of PpiD in OMP maturation into
question. Yet, while PpiD in our system neither substitutes for
SurA nor for Skp function, we found moderate overproduction
of PpiD to complement the lethal phenotype of a surA skp
double mutant in a PPIase-independent manner. We currently
examine whether this effect is caused by an indirect mechanism
or by the weak chaperone-like activity we observed for PpiD in
[1] Justice et al. (2006) Infect. Immun. 74, 4793-4800.
[2] Behrens et al. (2001) EMBO J. 20, 285-294.
[3] Rizzitello et al. (2001) J. Bacteriol. 183, 6794-6800.
[4] Dartigalongue and Raina (1998) EMBO J. 14, 3968-3980.
Host cell invasion of Campylobacter jejuni: importance of
the small Rho GTPases Rac1 and Cdc42 and the bacterial
fibronectin-binding protein CadF
M. Krause-Gruszczynska1, M. Rohde2, W. König1
M. E. Konkel3, S. Backert1
Otto-von-Guericke University, Medical Microbiology
Magdeburg, Germany
Helmholtz-Center for Infection Research, Microbial
Pathogenesis, Brunswich, Germany
Washington State University, School of Molecular Biosciences
Pullman, USA
Host cell invasion of the food-borne pathogen Campylobacter
jejuni is one of the primary reasons of tissue damage in humans
but molecular mechanisms are widely unclear. Here, we show
that C. jejuni strains 81-176 and F38011 trigger membrane
ruffling in the eukaryotic cell followed by invasion in a very
specific manner first with its tip followed by the flagellar end.
To pinpoint important signaling events involved in the C. jejuni
invasion process, we examined the role of small Rho family
GTPases. Using specific silencing RNA (siRNA), GTPasemodifying toxins, inhibitors and GTPase expression constructs
we show that Rac1 and Cdc42, but not RhoA, are involved in C.
jejuni invasion. In agreement with these observations, we found
that internalization of C. jejuni is accompanied by a timedependent activation of both Rac1 and Cdc42. We also show
that C. jejuni-induced GTPase activation involves the bacterial
fibronectin-binding protein CadF, but GTPase activation is
independent of the cytolethal distending toxin (CDT), adhesin
PEB1, virulence plasmid pVir and certain genes such as kpsS
and waaF which play a role in the biosynthesis of capsular
polysaccharide and lipooligosaccharide, respectively. Thus,
CadF is a bifunctional protein which triggers bacterial binding
to host cells as well as signalling leading to GTPase activation.
Finally, we show that the activation of these GTPases by CadF
involves different host cell kinases and guanine-exchange
factors (GEFs). Collectively, our results suggest that C. jejuni
invade host target cells by a unique mechanism and the
activation of the Rho GTPase members Rac1 and Cdc42 plays a
crucial role in this entry process.
Shiga toxin-specific immunity and STEC-shedding in
naturally infected calves
J. Fröhlich1, G. Baljer1, C. Menge1
Justus-Liebig-University, Institute for Hygiene and Infektion
Deseases of Animals, Giessen, Germany
Calves become infected with Shiga toxin-producing E.coli
(STEC) early in life. Previous data suggest that, in the course of
this first infection, Shiga toxins (Stx) prevent the development
of a STEC-specific cellular immune response resulting in longterm STEC shedding. As a first step towards a vaccination
strategy counteracting the immunosuppressive effect of Stx, we
aimed at assessing the quantity of maternal (colostral) and
endogeneous Stx-antibody titers in naturally STEC-infected
calves. Fecal and serum samples were taken weekly from 27
calves at a local dairy herd from birth until the 24th week of life.
Colostrum and serum samples of dams were also assessed.
STEC shedding was investigated by detection of stx-genes in the
feces using PCR. Stx1- and Stx2-specific antibodies in sera and
colostrums were quantified by Vero-cell neutralization assay.
Shedding of stx1-positive and stx2-positive STEC was detected
in 24 and 26 calves, respectively, and at up to 13 sampling times
per animal. Stx1-specific antibody titers were observed in serum
and colostrum samples of all dams and all postcolostral serum
samples of newborn calves but with individual differences.
Calves´serum titers rapidly decreased within the first 6 weeks
of life down to the detection limit. Even though 22 calves shed
stx1-STEC within the first 14 weeks, only 5 calves developed a
Stx1-specific seroconversion. Only low Stx2-specific titers were
detectable in serum and colostrum of 3 dams and the
postcolostral serum samples of the respective calves. Despite
frequent stx2-STEC shedding, Stx2-specific seroconversion
could not be detected in any of the calves. In conclusion, first
STEC infections of calves coincide with a rapid decline of
maternal and lack of endogeneous Stx-specific seroconversion.
These findings encourage the use of Stx as an antigen for
vaccination strategies in calves aimed at preventing long-term
STEC shedding in cattle.
Shifts towards pro-inflammatory bacteria in the intestinal
flora of Helicobacter pylori-infected Mongolian gerbils
M. M. Heimesaat1, A. Fischer1, U. B. Göbel1, S. Bereswill1
G. Rieder2
Charité-University Medicine Berlin, Microbiology und
Hygiene, Berlin, Germany
Ludwig-Maximilians-University Munich, Max-von-PettenkoferInstitute, Munich, Germany
Inflammatory bowel diseases in humans, such as Crohn`s
disease or ulcerative colitis, are frequently accompanied by
shifts of the gut flora towards pro-inflammatory bacteria. We
and others have recently shown that Escherichia coli and Bacteroides/Prevotella
inflammation and aggravate experimental ileitis as well as
colitis via Toll-like-receptor signaling. Furthermore, H. pyloriinfection causes alterations of the gastric microflora in mice.
Stomachs of H. pylori-infected animals were colonized by
bacteria, which are naturally restricted to the lower intestinal
tract. This indicates that H. pylori-infection increases the
microbial diversity in the rodent stomach. In order to investigate
if H. pylori-infection is associated with microflora changes of
the intestine, we analyzed the gastric and intestinal flora in
Mongolian gerbils, which were colonized with H. pylori for 14
months and developed severe gastric ulcer, metaplasia, and
gastritis cystica profunda. The microflora in the stomach,
duodenum, jejunum, ileum, caecum, and colon was analyzed
using cultural as well as molecular methods (denaturing gradient
gel electrophoresis (DGGE)). A global cultural survey of the gut
flora revealed that luminal loads of E. coli, Proteus spp.,
Enterococcus spp., and Bacteroides/Prevotella spp. were
increased in the ileum, caecum, and colon of animals with
gastric ulcerations, as compared to control animals without
clinical signs of H. pylori-infection. Levels of lactobacilli
remained constant. H. pylori and disease-associated shifts in the
flora of the lower intestine were further confirmed by molecular
analysis, which did provide evidence that other bacteria
disappear in H. pylori-infected animals. We conclude that H.
plyori-infection can influence the gut flora composition. The
fact that the accumulating bacteria show a strong proinflammatory potential in inflammatory bowel diseases indicates
that the gut flora shifts may contribute to the development of
intestinal diseases and raise the risk for inflammatory processes
in the lower gastrointestinal tract.
Characterization of the translocation factors CagF and Cag
of the Helicobacter pylori Cag type IV secretion system
I. Pattis1, E. Weiss1, R. Laugks1, R. Haas1, W. Fischer1
Ludwig-Maximilians-University Munich, Max-von-PettenkoferInstitute, Munich, Germany
The virulence factor CagA is so far the only effector protein
known to be transported by the Helicobacter pylori Cag type IV
secretion system into eucaryotic cells. We have previously
defined a set of 14 essential components of the secretion
apparatus and 4 additional proteins that are necessary for CagA
translocation, but little is known about the molecular details of
CagA recognition as a type IV secretion substrate. Recently, the
examination of translocated effector proteins in different type IV
secretion systems has shown that their respective C-terminal
regions are necessary and sufficient for translocation. Substrate
recognition via these C-terminal regions is thought to be
mediated by the so-called coupling proteins. Two components
that are specifically necessary for CagA translocation are the
coupling protein homologue Cag and the CagF protein that has
been shown to interact with CagA. The aim of this study was to
characterize the roles of these proteins for the type IV secretion
process of CagA.
We show that CagF is a secretion chaperone-like protein that
interacts with CagA at the bacterial cytoplasmic membrane.
GST pulldown experiments indicate that CagF binds to an
approximately 100 amino acid domain located in the C-terminal
region of CagA, which is adjacent to, but distinct from the Cterminal translocation signal. Although Cag binding to CagA
could also be demonstrated, our data indicate that CagF binding
precedes recognition of the C-terminal CagA translocation
signal, and that both steps are required to recruit CagA to the
type IV translocation channel. Moreover, they suggest that at
least two sequential steps are involved in CagA recognition as a
type IV secretion substrate. Thus, CagA recognition as a type IV
secretion substrate seems to be more complex than that of other
type IV effector proteins.
Incidence of EPEC and EAEC in patients with diarrhoea in
a university hospital detected by use of a novel multiplex
Real-Time PCR
C. Hardegen1, S. Messler1, B. Henrich1, K. Pfeffer1
J. Würthner1, C. MacKenzie1
Heinrich-Heine-University, Medical Microbiology and Clinic
Hygiene, Duesseldorf, Germany
Accurate measurement of the incidence of enteropathogenic
E.coli in patients with diarrhoea is hindered by the current
methods of detection and varies from country to country
(particularly the incidence of EAEC). In order to improve the
diagnosis we developed a multiplex TaqMan Real-Time PCR
designed to detect the respective pathogens from an overnight
stool culture. In addition to this, we noted that clinicians seldom
requested detection of enteropathogenic E. coli in patients with
diarrhoea and therefore set out to determine how many such
infections were being missed at a university hospital. Over a
period of 12 months (Jan 2006-Jan 2007) all stool specimens
received were investigated for EPEC and EAEC using the newly
developed multiplex Real-Time PCR.
The targets for the PCR were the eae gene and the EAF plasmid
(EPEC) and pCVD432 (EAEC). Additionally, two separate
multiplex PCRs were developed for the other known
enteropathogenic E. coli with the targets slt1 and slt2 (EHEC),
ipaH (EIEC) and lt and st (ETEC).
A total of 1981 specimens were investigated over the period. Of
these 371 specimens had no growth of Enterobacteriaceae
(mostly due to selective gut decontamination). Of the remaining
1610 specimens 144 (8,9 %) were positive for EPEC and 78 (4,8
%) positive for EAEC as the identified pathogens in the
respective samples.
We divided the requesting departments into three groups:
Paediatrics, Tropical diseases and the remaining wards of the
hospital. Among the EPEC positive stool specimens 28 (20 %)
were from the tropical diseases unit, 49 (33,8 %) from the
paediatric dept. and 67 (46,2 %) from the remainder of the
wards. The EAEC were distributed as follows: 39 (50 %) –
tropical diseases, 19 (24,4 %) –paediatrics and 20 (25,6 %) other
Analysing the age groups, we found a cluster of EAEC in
children under 3 years of age. For the EPECs a cluster was seen
in children under 3 and then a relatively constant incidence
through the eighth decade of life. In only 22 % of the detected
EPECs and 23 % of EAEC was the investigation requested.
This is the first study, to our knowledge, using a multiplexed
Real-Time PCR for the detection of enteropathogenic E. coli.
We conclude from this that the PCR is a useful investigation for
the detection of enteropathogenic E.coli, the DGHM MIQ
recommendation that all stool specimens for children under 3
years be investigated for EPEC is valid, that it may be useful to
additionally screen for EAEC in this age group and finally that
clinicians do not look for enteropathogenic E.coli routinely and
perhaps they should.
Allelic variation of the H. pylori adhesin genes babA and
sabA during chronic infection is associated with quantitative
differences of adhesion to Lewisb and sialyl Lewisx
S. Schwarz1, E. Katzowitsch1, S. Cleland1, P. Olbermann1
S. Sürbaum1
Hanover Medical School, Institute of Medical Microbiology
and Hospital Epidemiology, Hanover, Germany
The remarkable genomic plasticity, and the dynamic and
multifactorial binding properties of H. pylori are both believed
to be important for persistent infection. BabA and SabA are
outer membrane proteins of H. pylori which mediate specific
adherence to the histo blood group antigens Lewisb (Leb) and
sialyl Lewisx (sLex), respectively. In order to test the hypothesis
that H. pylori might modify its adhesin genes during chronic
infection, possibly adapting to the host individual, we sought to
sequence babA and sabA from eight pairs of H. pylori that were
sequentially isolated at intervals from three months to three
years. Three pairs lacked a babA gene, for all other strains,
complete babA and sabA sequences were obtained. In two pairs,
babA alleles differed by a single nucleotide polymorphism
(SNP) and three pairs showed multiple nucleotide
polymorphisms (MNPs), possibly representing recombination
events. All pairs were sabA positive with three pairs harbouring
identical sequences, two pairs differing by SNPs, and two pairs
showing MNPs. In one pair, the second isolate had a frameshift
mutation in sabA leading to a premature stop codon. Binding of
the paired strains to Leb and sLex was quantified by a glycoblot
procedure. In 2 out of 4 Leb adherent pairs, the second strain
exhibited significantly weaker binding than the first strain. Of
the five pairs that showed binding to sLex, one pair showed
significantly reduced binding of the second strain. One of the
pairs comprised a first isolate adherent to Leb and sLex and a
second isolate that did not bind either glycan. Transformation of
the non-adherent strain with a complete babA gene amplified
from the adherent isolate could restore Leb adhesion. Sequence
analysis of the recombinant babA genes from clones with
restored adherence is in progress and should permit to identify
domains within babA critical for Leb binding.
Chronic enteric Salmonella infection in mice leads to severe
and persistent intestinal fibrosis
G. Grassl1, Y. Valdez1, K. Bergstrom2, B. Vallance2, B. Finlay1
University of British Columbia, Michael Smith Laboratories
Vancouver, Canada
University of British Columbia, BC'
s Children'
s Hospital
Vancouver, Canada
Background: Intestinal fibrosis and stricture formation are
serious complications of Crohn’s disease, often requiring
surgical intervention. Unfortunately the mechanisms underlying
intestinal fibrosis development are poorly understood, in part
because of the lack of relevant animal models. In this study, we
present a novel murine model of severe and persistent intestinal
fibrosis caused by chronic bacterial induced colitis. Methods:
Mice were treated with streptomycin 24 h prior to oral infection
with Salmonella enterica serovar Typhimurium, and euthanized
at various times post infection. Tissues were analyzed for
bacterial colonization and inflammation, while fibrosis was
assessed by Masson’s trichrome staining and collagen
quantification. Expression of the pro-fibrotic cytokines
transforming growth factor- 1 (TGF- 1) and connective tissue
growth factor (CTGF) was determined by ELISA and RT-PCR
and the cell types present in fibrotic tissues were assessed by
immunohistochemistry. Results: Infection led to chronic
Salmonella colonization of the caecum and colon followed by
edema, mucosal ulcerations and severe transmural
inflammation. This pathology was accompanied by significantly
elevated expression of TGF- 1 and CTGF along with extensive
collagen deposition in the caecal mucosa, submucosa, and
muscularis mucosa of infected mice. This fibrosis was evident
by 7 days post infection, peaking at day 21 and still present at
day 70. The fibrotic regions were found to be rich in fibroblasts
and myofibroblasts. Conclusions: These data demonstrate that
chronic Salmonella infection of the murine GI tract leads to
severe tissue fibrosis. Since this model is highly reproducible
and easy to perform, it provides great potential for investigating
both host and bacterial contributions to intestinal fibrosis.
Surveillance of nosocomial infections in patients undergoing
hematologic stem-cell transplantation - five years ‘ONKOKISS’
M. Dettenkofer1, E. Meyer1, R. Babikir1, H. Bertz2, A. Widmer3
H. Rüden4, T.5
Universtiy Medical Center Freiburg, Institute of Environmental
Medicine and Hospital Epidemiology, Freiburg, Germany
University Medical Center Freiburg, Department of Medicine
I, Hematology and Oncology, Freiburg, Germany
University Hospital Basel, Division of Infectious Diseases and
Hospital Epidemi, Base, Switzerland
Charité Berlin, Institute of Hygiene and Environmental
Medicine, Berlin, Germany
ONKO-KISS, Study Group
For surveillance of nosocomial bloodstream infections (BSI)
and pneumonia during neutropenia in adult patients undergoing
bone marrow transplantation (BMT) or peripheral blood-stem
cell transplantation (PBSCT), an ongoing multicenter
surveillance project was initiated by the German National
Reference Center for Surveillance of Nosocomial Infections in
2000 (ONKO-KISS).
Methods: Nosocomial Infections are identified using CDC
definitions for laboratory-confirmed BSI and modified criteria
for pneumonia in neutropenic patients [for detailed information
Results: Over the 60-month period up to June 2006, 4.203
patients with 62.338 neutropenic days were investigated. Of
these, 2.492 (59%) had undergone allogeneic and 1.711 (41%)
autologous BMT or PBSCT. The mean length of neutropenia
was 14.8 days (9.4 d after autologous and 18.6 d after allogeneic
transplantation). In total, 776 bloodstream infections and 353
cases of pneumonia were identified. Site-specific incidence
densities (pooled mean) were: 12.4 BSIs (10.6 for allogeneic vs.
17.8 for autologous transplantations) and 5.7 cases of
pneumonia (5.8 for allogeneic vs. 5.3 for autologous
transplantations) per 1,000 neutropenic days, respectively. There
was a trend to lower incidence densities over the five years
reported. Following allogeneic transplantation, 19.7 BSI/100
patients and 10.8 cases of pneumonia / 100 patients occurred
whereas following autologous transplantation 16.7 cases of BSI/
100 patients and 5.0 cases of pneumonia /100 patients were
observed. The main pathogens associated with BSI were
coagulase-negative staphylococci (52%).
Conclusions: The ongoing ONKO-KISS project adds to the
improvement of quality of care in patients undergoing
Hematologic Stem-Cell Transplantation. Since 2006,
surveillance is extended to neutropenic patients with acute
leukemia to allow participation for Centers not performing
Nasal carriage of MRSA in third-year medical students
U. Vogel1, W. Witte2, K. Engelhard1, M. Frosch1
University Wuerzburg, Institute for Hygiene and Microbiology
Wuerzburg, Germany
Robert-Koch-Institute, Wernigerode, Germany
The incidence of carriage and infection with MRSA is
increasing in German hospitals. Undetected colonization of
health care personnel might cause MRSA clusters. In University
hospitals, medical students throughout their education will have
increasing contact to patients of a variety of different clinics
without necessarily being trained in MRSA prevention and
without microbiological control for carriage. As part of the third
year medical education, students were trained in medical
microbiology and obtained swabs from their own nose to screen
for S. aureus carriage. Since 2003 1174 students attended the
course. MRSA was found in four female subjects (point
prevalence of 3.4/1000 course students). The following spatypes were identified: t008 (n=1); t003 (n=1); t667 (n=1; t044cc;
PVL-positive); t032 (n=1). In three cases, MRSA was
eradicated by topical antiseptic treatment of the nose, skin, and
oral mucosa. In one case, the students´mother was also MRSA
positive with the identical clone, whereas flatmates of all
students were not colonized. Two strains were associated to skin
infections with delayed healing and contact to ambulant surgery.
We suggest that MRSA rates found here underestimate reality
due to the low sensitivity of the procedure applied; furthermore,
MRSA rates might be higher in students of the fourth to sixth
year with increasing clinical involvement. We suggest enhanced
surveillance of medical students and intensified training in
MRSA prevention.
Monoclonal outbreak of catheter-related septicaemia by
Ralstonia mannitolilytica in two oncology departments
S. Gröbner1, P. Heeg1, I. B. Autenrieth1, B. Schulte1
University Tuebingen, Institute of Medical Microbiology and
Hygiene, Tuebingen, Germany
Background: Ralstonia mannitolilytica is a non-fermentative,
Gram-negative bacterium isolated infrequently from clinical
samples. However, within a period of 11 weeks five inpatients
developed septicaemia and R. mannitolilytica was cultivated
from blood samples of these patients suggesting an outbreak.
Methods: Blood cultures and one catheter tip were analysed by
standard microbiological procedures. Genetic relatedness of the
isolates was investigated by pulsed field gel electrophoresis. To
ascertain the possible source of the outbreak, environmental
sampling and challenge-recovery experiments to test filters used
for multi-dose solution bottles were performed.
Results: In the present study a monoclonal outbreak with R.
mannitolilytica causing septicaemia of five haematological
patients is reported. Underlying severe diseases with
intravenous devices, multiple intravenous applications, and
chemotherapy were possible risk factors promoting septicaemia.
Challenge-recovery experiments revealed that R. mannitolilytica
to a high extent even passed through Mini-spike Plus® filters of
pore size 0.2 µm.
Conclusion: Although the source of the outbreak could not be
identified, it is possible that solutions given intravenously were
contaminated. Since R. mannitolilytica had never been isolated
in our laboratory before and environmental testings performed
were negative, it cannot be excluded that commercial products
like drugs, saline solutions or infusion systems (filters) were
Nationwide Study on Moxifloxacin susceptibility among
pneumococcal isolates from invasive disease in Germany
M. van der Linden1, R. R. Reinert1, C. Heeg1, I. Seegmüller1
University Hospital RWTH Aachen, National Reference Center
for Streptococci, Aachen, Germany
Background: In the last two decades, the worldwide increase in
antibiotioc resistance in Streptococcus pneumoniae has
developed into a serious problem.
Method: In this study 679 Streptococcus pneumoniae isolates,
collected in Germany in the year 2006 were examined. Minimal
inhibitory concentration (MIC) testing was performed using the
broth microdilution method as recommended by the Clinical
Laboratory Standards Institute. The pneumococcal strains were
isolated from blood (n = 568, 83,7%), Cerebrospinal fluid (CSF;
n = 86; 12,7%), broncho-alveolar lavage (n = 12; 1,8%) und
pleural fluid (n = 13; 1,9%).
Results: MIC-values of moxifloxacin towards pneumococci
were between 0,03 und 0,5 µg/ml, independent of -lactam or
macrolide susceptibility. All isolates were susceptible towards
moxifloxacin. A rate of macrolide resistant isolates was
obeserved. The results of the antibiotic susceptibility testing are
summarized in the table.
Conclusion: Our data show that moxifloxacin is a promising
therapeutic option for the treatment of pneumococcal infections,
including those which are caused by penicillin- or macrolideresistant strains.
Table 1:
Susceptibility of S.pneumoniae towards Moxifloxacin
Moxifloxa Penicillin Clarithromyc Clindamyc
<= 0,015
<= 0,12
<= 0,12
MIC 50 0,12
<= 0,016
<= 0,13
MIC 90 0,25
Association of resistance, hospital type and hospital
department in E. coli
K. Heckenbach1, T. Eckmanns1
Robert-Koch-Institute, Infectious Disease Epidemiology
Berlin, Germany
Resistance to Escherichia (E.) coli is a rising problem in
Europe. We identified different risk factors in reference to the
hospital type and the departments of the hospitals.
Antimicrobial susceptibility data on E.coli collected by the
European Antimicrobial Resistance surveillance system
(EARSS) is based on a voluntary notification from the
laboratories. The resistance of E. coli is monitored for following
antimicrobial groups: aminoglycosides, aminopenicillines and
fluoroquinolones. The analysis by logistic regression modelled
single resistance to the three antibiotic groups as a function of
hospital type, departments (medical, immune suppression,
surgical and intensive care), age over 65 and sex.
During the period from 2001 to 2005, overall 41 hospitals
participated in different phases. E. coli isolates (N=2209) from
blood cultures had an overall resistance of 68%, 46% had a
single, 18 % a double and 4% a triple resistance. 65% of the
isolates were resistant to aminopenicillins, 21% to
fluoroquinolones, and 7% to aminoglycosides. Double
resistance occurred in 14% of the 18% as aminopenicillin
/fluoroquinolone 3% as aminopenicillins/aminoglycosides and
1% in other combinations.
The risk for a bacteraemia caused by an E. coli resistant to
aminopenicillins was lower in hospitals other than tertiary care
hospitals. In comparison to the medical wards, wards with
immune suppressed patients had a twofold higher risk (OR 2.1,
95%CI[1.4 – 3.1]) and intensive care units had an OR of 1.3
(95%CI[1.4 – 3.1]). Risk factors for aminoglycoside resistance
were tertiary care hospitals and immune suppressed patients
(OR 2.3, 95%CI[1.5 – 3.7]). Risk factors for fluoroquinolone
resistance were tertiary care hospitals, immunological (OR3.2
95% CI [2.3– 4.5]) and surgical departments (OR 1.6, 95% CI
[1.2 – 2.1]). Male patients had a higher risk of an infection with
a resistant E. coli (OR 1.3 to 1.6).
The analysis of routine data from the laboratories identified
hospital type and departments as risk factors for resistant E. coli
involved in bacteremia. As expected the risk of a resistance is
lower in hospitals other than tertiary care hospitals. In contrast
to other investigations intensive care units are not a general risk
factor for resistant E. coli.
The impact of imported MRSA for a hospital
A. Petzold1, R. Fleck1, C. Kupfahl1, G. Geginat1, H. Hof1
University Hospital Mannheim, Institute for Medical
Microbiology and Hygiene, Mannheim, Germany
Staphylococcus aureus (MRSA) in Germany has increased
dramatically during the last few years. The major reservoirs for
MRSA in the hospital setting are colonized patients. The Danish
example shows, that the implementation of screening, isolation,
and antibiotic use policies decrease the MRSA prevalence.
In our study, we explored the MRSA situation in the University
hospital Mannheim, a 1400-bed tertiary care hospital. Compared
to the MRSA study 2004 of the Paul-Ehrlich-Gesellschaft, the
hospital had a high ratio of MRSA to methicillin-sensitive
Staphylococcus aureus. The prevalence of MRSA among
admitted patients was unknown.
Methods: Two regular wards from the departments of surgery
and internal medicine screened every patient on admission and
discharge. The regular swab was taken from the nostril of each
patient. Additional swabs from wounds or other regions were
encouraged. All swabs were tested, using PCR and/or selective
agar. Positive results were communicated immediately and
contact isolation precautions were taken.
Results: From May to December 2006, 781 patients were
screened, and 48 MRSA-positive patients were detected. From
all 48 MRSA-positive patients, 45 patients were detected on
admission. Of the remaining 3 MRSA-positive patients, 2
patients missed the screening on admission and only one
colonization was hospital-acquired. The outcome of this datais a
ratio of 5.8% MRSA-positive patients compared to all admitted
patients. Of the 781 patients screened, 442 and 339 were
hospitalized on the surgery and on the internal medicine ward,
respectively. On the surgical ward 23 patients (5,2%) and on the
internal medicine ward 22 patients (6.5%) were found to be
MRSA-positive on admission.
Conclusion: Our results show that the vast majority of MRSA
cases are imported. Screening and isolation precautions are
effective to prevent the spread of MRSA in the hospital.
Silver coated textiles reduce the contamination in water
C. Unger1, P. C. Lück1
Technical University Dresden, Institute of Medical
Microbiology and Hygiene, Dresden, Germany
Silver cations (Ag+) show an antimicrobial effect on all
microorganisms, but they are not cytotoxic to human cells.
Therefore silver compounds are used as antimicrobial agents in
medicine, e. g. for treatments of burns or for coating of
catheters. This effect is also used in the production of sport
clothing or socks with silver coated textiles cockle.
The antimicrobial effect of silver coated textiles in water
systems was tested with different strains of bacteria (Legionella
pneumophila, Pseudomonas sp. and Staphylococcus aureus) in
laboratory experiments. For the detection of a minimal
inhibitory concentration (MIC) strains were incubated in
aqueous solutions with different concentrations of silver. The
killing of the bacterial count was tested after 1, 3 and 24 hours
of incubation. After 24 hours all strains were killed at a
concentration about 16 µg/l silver. Already, after 3 hours
incubation a reduction of 2 to 4 logarithmic magnitudes with a
concentration of silver of 4 to16 µg/l was attained.
Due to the antimicrobial effect, which was laboratoryconfirmed, the silver coated textiles were tested in different
practical applications. In these experiments three warm water
reservoirs with a volume of 1m3 lined with the silver coated
textiles were investigated. In addition the silver coated textiles
were installed in a industrial applied water system in a paper
Silver coated textiles reduce the number of viable bacteria about
at least 10 to 100 fold in the warm water reservoirs. This effect
could be observed for at least 6 month. This makes the
application cost-effective and low-maintenance. In the water
supply system in the paper mill there was a reduction of bacteria
about 2 to 3 logarithmic magnitudes. In both systems the silver
coated textiles are a effective method to reduce the
contamination by Legionella and other water borne pathogens in
applied water systems.
Molecular evidence of pneumocystis jirovecii transmission
among 16 patients after kidney transplantation
S. Schmoldt1, L. Bader1, I. Huber2, R. Schuhegger2
H. Söllner2, J. Heesemann1, A. Sing2
Ludwig-Maximilians-University, Max von Pettenkofer-Institute,
Munich, Germany
Bayerisches Landesamt für Gesundheit und
Lebensmittelsicherheit, Oberschleissheim, Germany
In recent years, nosocomial clusters of Pneumocystis jirovecii
immunocompromised individuals were reported. Mostly, the
source of infections was suspected within the clinical settings
when transplant recipients and PCP patients shared hospital
facilities. We report a cluster of 16 renal transplant recipients
positive for P. jirovecii. None of them received antiPneumocystis prophylaxis prior to P. jirovecii detection. 15 of
them were kidney transplanted at a German university hospital
and attended the same inpatient and outpatient ward from
January through September, 2006. Genotyping of P. jirovecii
isolates was performed using DNA sequencing of multiple loci
(MLST): ITS1, -tub, 26S and mt26S. P. jirovecii DNA was
available from 14 patients and revealed identical MLST types
among these renal transplant recipients. Surprisingly, one
patient who was treated at a different nephrological Center and
negated personal contacts to patients from the renal
transplantation cluster harboured the same P. jirovecii MLST
type. Three HIV patients and one bone-marrow transplanted
hematologic malignancy patient – all treated at two different
medical Centers - were used as controls and revealed different
MLST types. Interestingly, in three of the four previously
described regions new alleles were detected and one new
polymorphism was observed in the mt26S region. The
genotyping results and previous reports of outbreaks strongly
suggest a patient-to-patient transmission of P. jirovecii as one
predominant transmission route.
policies of each individual hospital. So the stratification
according to the frequency of nares culture screening will
become a routine method in MRSA-KISS.
MRSA rates of 53 german hospitals in relation to their
screening frequency
I. F. Chaberny1,2, M. Behnke3,2, H. Rüden3,2, P. Gastmeier1,2
Hanover Medical School, Inst. of Med. Microbiology and
Hospital Epidemiology, Hanover, Germany
German National Reference Center for Surveillance of
Nosocomial Infections, Berlin, Germany
Charité-University Medicine Berlin, Inst. of Hygiene and
Environmental Medicine, Berlin, Germany
Characterization of the role of IS256 in the development of
intermediate vancomycin resistance in a clinical
Staphylococcus aureus isolate
M. Nagel1, A. Jansen1, M. Türck1, G. Bierbaum1
University Bonn, Institute for Medical Microbiology
Immunology and Parasitology, Bonn, Germany
Background:MRSA is one of the most relevant resistant
pathogens. The German national nosocomial surveillance
infection system (KISS) established a hospital based prospective
surveillance system for MRSA to calculate the reference data
for Germany. However, the MRSA rates are dependent on the
hospitals screeening activities.Objective:To determine the
MRSA rates according to the screening activities of German
hospitals.Methods:KISS prepared a surveillance protocol and a
standardised case report form for MRSA-KISS. The
participating hospitals are spread all over Germany. The data
were recorded during the routine surveillance by the infection
control team of each hospital. Screening for MRSA carriage was
mainly performed using nares cultures. The number of nares
cultures was received from the microbiology and calculated per
1000 patient days. Results:Data from 101 hospitals, 10,647
MRSA case-patients with 180,158 MRSA patient days and with
14,409,799 patient days were analysed for the year 2005. 6,744
(63.3%) patients of the total number of MRSA case-patients
were colonized with MRSA and 4,016 (37.7%) had a MRSA
infection. 7,053 (66.2%) were imported and 3,707 (34.8%) were
nosocomial. The number of nares cultures was recorded in 53
hospitals. These data were stratified into 4 categories (s. table)
according to the different MRSA-rates.
The data from 2006 were currently calculated and will be
demonstrated.Conclusion:The amount of screening samples of a
hospital should be considered to evaluate MRSA rates.
Recording the number of nares cultures per 1,000 patient days
seems to be a good method for reflecting the actual screening
Table 1:
Number (MRSA
casecultures per
hospitals patients
per 1,000
<= 2
> 2 and <= 4 11
> 4 and <=
> 10
MRSA casepatient per
1,000 patient
MRSA days-associated
nosocomial MRSA rate
(nosocomial MRSA casepatients per 1,000 MRSA
patient days)
Staphylococcus aureus tends to acquire multiple antibiotic
resistance determinants and therefore vancomycin has been the
treatment of choice for serious infections caused by methicillinresistant strains. However, therapeutic failure has been
described. There is growing evidence that environmental stress,
which may be exerted by the presence of antibiotics themselves,
leads to an induction of mutational mechanisms in bacteria. The
bacterial SOS response, for example, is a physiological reaction
that is induced by the failure to replicate DNA and double strand
breaks and is caused by fluorochinolones and UV light, as well
as by cell wall biosynthesis inhibitors like vancomycin. Since
the SOS response is known to induce delocalization of ISelements, this study was conducted (i) to establish a system that
allows mapping of the copies of IS256, (ii) to locate the copies
of IS256 in the genome of the VISA isolates and (iii) to check
the influence of antibiotic stress and the SOS response on the
Mapping and localization of the IS256 elements were performed
by digestion of chromosomal DNA with XmnI, dilution and
religation. Circular fragments comprising a copy of IS256 were
amplified by reverse PCR, using outgoing primers that anneal in
IS256, and sequenced. The intermediately vancomycin resistant
S. aureus strain pair employed in this study carries at least 14
copies of IS256. In the selection mutant displaying higher
resistance, part of the resistance is caused by an insertion of
IS256 into the gene tcaA [2], whereas IS256 was found instead
in the promoter of yycFG in the clinical parent isolate leading to
an overexpression of this two-component regulatory system [1].
In addition, apart from several insertions in intergenic regions, a
copy of IS256 was found to interrupt orf SA1848 which encodes
a putative ammonium transporter in both strains. In conclusion,
mobile genetic elements may play a role in the acquisition of
resistance against antimicrobial agents.
[1] Jansen, A. et al., Int. J. Med. Microbiol. (2007)
[2] Maki, H. et al., Antimicrob. Agents Chermother. 48, 19531059 (2004)
Prevalence of multidrug-resistant bacteria (MRSA, VRE
and ESBL producing enterobacteria) in geriatric clinics,
nursing homes and ambulant care
T. A. Wichelhaus1, I. Gruber1, U. Heudorf2, C. Brandt1
R. Püllen3, V. Brade1
University, Institute for Medical Microbiology und Clinical
Hygiene, Frankfurt am Main, Germany
Local Health Authority, Frankfurt am Main, Germany
Deaconry-Clinic, Frankfurt am Main, Germany
Colonization/infection with multidrug-resistant bacteria (MRB)
such as methicillin-resistant Staphylococcus aureus (MRSA),
vancomycin-resistant enterococci (VRE), and extended
spectrum betalactamase (ESBL) producing enterobacteria, is an
increasing problem in hospitals but also in homes for the elderly
and in ambulant care.
Objective: The aim of this study was to determine the
prevalence of colonization/infection with MRSA, VRE and
ESBL producing enterobacteria in geriatric clinics, nursing
homes and ambulant care facilities in Frankfurt am Main.
Materials and methods: 255 patients and 62 staff members from
2 geriatric clinics (n = 61), 8 nursing homes (n = 206), and 2
ambulant care facilities (n = 50) were screened for MRB. If a
consent by a participant was given, swabs were taken from nose,
throat, rectum and if present wounds.
Results: 23 patients (9%) and 2 staff members (3.2%) were
colonized with MRSA, 8 patients (3.1%) were screened positive
for VRE, and 24 patients (9.4%) and 2 staff members (3.2%)
were found to be colonized with ESBL producing
Conclusion: This is the first report to our knowledge presenting
data on the prevalence of VRE and ESBL producing
enterobacteria in homes for the elederly and in ambulant care in
Germany. With respect to previous studies about MRSA, the
prevalence in Frankfurt am Main has risen from 2.3 to 9%. We
assume that existing hygiene recommendation concerning MRB
are sufficient, however, staff members and patients should
receive better education on how to implement these guidelines
in routine practice in order to avoid spread of MRB.
Can it be true? What does a positive MRSA-PCR result
stand for?
S. Schütt1, A. Carstens-Slim1, C. Wendt1
University Hospital, Institute of Hygiene, Heidelberg, Germany
Modern PCR methods allow the rapid identification of MRSA
patients. However, PCR-positive results can not always be
confirmed by conventional culture. This may be explained by
false positive results due to characteristics of the test kit, false
positive results due to contamination or true positive results
from non-viable microorganisms.
We conducted a retrospective analysis of our positive test results
to identify factors that may be associated with positive results
that could not be confirmed by culture.
University Hospital of Heidelberg is a 1500-bed tertiary care
Center. Patients who are admitted to an intensive care unit are
screened for MRSA with the BD GeneOhm™ MRSA Assay
using nasal specimens. The qualitative multiplex real-time PCR
assay is based on the detection of the mec right extremity
junction (MREJ) of the staphylococcal cassette chromosome
PCR-positive swabs were cultured in enrichment broth.
Additionally swabs from nose, throat, inguinal and anal were
requested and processed by conventional culture.
Between January and December 2006 swabs from 97 different
patients were PCR-positive. These patients were divided in 3
groups depending on the results of culture.
Group A “true positives”: 69 patients with positive MRSA
culture from nares (55) or other sites (14); Group B “false
positives”: 9 patients with culture positive for Methicillinsensible Staphylococcus aureus (MSSA) which contains MREJ;
Group C “unconfirmed”: 19 patients without any culture of
MRSA or MREJ-containing MSSA.
Patients of group C were not different from patients of group A
in terms of underlying diseases, presence of typical risk factors
for MRSA colonization or application of antibiotics. 17 of the
19 unconfirmed results were the only positive result in the batch
In our study nearly 10% of positive results were due to MREJ
positive MSSA as described previously. Almost 20 % of PCRAssay positive nasal swabs could not be confirmed by cultural
detection of MRSA from any patient site. The patients showed
typical risk factors for MRSA colonization and thus the results
can be considered genuine. Previous antibiotic therapy or local
antiseptic treatment can be a reason for lack of positive cultures.
Preventive hospital hygiene – measurments of airborne mold
spores during and after demolition work at an University
Childrens Hospital
S. Harpel1, A. Knaust1, T. Eikmann1, C. Herr1
Institute of Hygiene and Environmental Medicine, University
Medical Center Giessen and Marburg , Giessen, Germany
From February toApril 2007 part of the University Childrens
Hospital in Giessen was torn down. Extensive measures (taped
windows and irrigation of site) were applied to prevent spread of
dust from the demolition site into the adjacent remaining
hospital buildings. Nevertheless there was considerable concern
of possible spread of airborne spores into the wards, especially
the ward for children’s oncology (bone marrow transplantation).
Therefore, it was decided to accompany the demolition work
with measurements of airborne mold.
Four of the five samplings took place during the demolition
work and one after. Sampling locations: one in a patient’s room,
one on the ward, one outside of the ward in the hall, one on the
outside balcony of the ward and one outside control sample.
Viable mold spores were incubated at 20°C and 36°C on DG 18
agar. Additionally, humidity and temperature were assessed.
Inubation at 36°C yielded on ward mold spore concentration
between 0 and 384 cfu/m³, while respective concentrations on
the ward balcony (closest to the construction work) were
between 8 and 104 cfu/m³. The lowest outdoor value on this
balcony was measured, when on-ward concentration was highest
and the highest value on this balcony was found after
completion of the demolition work, when identical values were
determined at the outdoor control point. Mold spore
concentrations after incubation at 20°C showed some correlation
with simultaneously measured humidity. They were always
considerably lower on the ward (0-28 cfu/m³) compared to
oudoors (8-260 cfu/m³) and in the same range on the ward
balcony and the control point.
Mold concentrations in the outdoor ambient air near the
oncology ward were not related to the ongoing demolition work,
most likely due to the extensive preventive measures. On-ward
air spore concentrations were usually considerably lower and
not related to outdoor concentrations.
Different MRSA screening strategies – experiences, outcome
and costs
A. Knaust1, K. Madlener2, H. Wagner3, N. Tschernych3
J. Barekzai3, T. Eikmann3, C. Herr3
Institute of Hygiene and Environmental Medicine, Giessen
Kerckhoff Clinic, Bad Nauheim, Germany
Justus-Liebig-University Giessen, Institute for Hygiene and
Environment Medicine, Giessen, Germany
Screening of incoming patients is discussed to be an effective
method to decrease rates of MRSA colonization and infection in
hospitals. In previous studies various designs were used, either
screening patients with established risk factors in their history
and/or patients who are admitted to certain areas of health care
facilities, i.e. intensive care units.
The current study aimed to compare two different MRSA
screening strategies according to: i) effort of invention, ii)
sensitivity of identifying MRSA positive patients, iii) costs.
The study was based in a university hospital (UH) and a cardiosurgical hospital (CH) which used two different MRSA
screening strategies: A) in UH: screening of patients which
belong to risk groups (history or MRSA, wounds,
haemodialysis, submission from health care facilities)
andpatients who were admitted to selected departments or wards
(intensive care units), B) in CH all patients were screened upon
admission. Microbiological diagnostics were performed either
by bacterial cultures or by a real time PCR based method.
More than 4000 patients were screened during a 12-month (UH)
and 6-month period (CH) respectively. Invention of a selective
screening (strategy A) in UH turned out to require substantial
administrative efforts. Despite of communication via hygiene
standard and intranet and extensive coaching of staff the quality
of implementation was still suboptimal. Strategy A was
performed with relatively low costs but missed a significant
number of MRSA positive patients. Screening of all patients
upon admission was associated with high costs.
To establish MRSA screening strategies in health care facilities
careful cost-effectiveness calculation is necessary, taking into
account local MRSA epidemiology, risk of patients to suffer
MRSA infection and costs of screening procedures and
following barrier precautions. Therefore, standard strategies for
MRSA screening as recommended by various guidelines require
critical assessment with respect to their effectiveness and costs.
Test system for examination of the barrier effectiveness of
sterilised medial products in health care systems.
U. Schmelz1
University Goettingen, Human Medicine - Department
Gerneral Hygiene and Environmental Medicine, Goettingen
Up to now, there is no suitable system by which the barrier
effectiveness and the quality of the sterilised medical devices in
the final packaging can be examined after transport and storage.
Aim of study:
The purpose of our investigation consisted in developing a test
kit as a suitable test system for this task and in starting in a
s scheme.
The test kit exists of a dry Caso-Agar disk (soil for cultivateable
micro-organisms) and of an ampoule with 8 milliliters of water.
The ampoule is surrounded by a break-proof plastic cover and is
provided with a pipette.
The test kits were placed in see-through pouches, sterilised
under application terms analogously to normal medical devices
and stored.
For the controlling of the barrier effectiveness, the kit was
activated by breaking open the ampoule, without damaging the
Afterwards, the kits were incubated with 36°C. The packages
were still closed while incubating. Non-sterility is proved by
colony growth after incubating.
In each case, 100 test kits in packages for medical devices from
paper and plastic were exposed (according to EN 868-5) at 4
different sites for 8 weeks.
The packages with the kits were stored in closed shelves from
wicker network. For the additional simulation of frequent
pressure changes, exposition took place in a lift in 3 of the 4
As control, we used 100 systems, which were examined directly
after sterilization.
In dependence of the storage conditions, a recontamination rate
of 50 to 80% was proved with the exposed test kits.
The test kit provides a suitable procedure for the supervision and
quality assurance of sterilised medical devices taking into
account existing storage and environmental conditions.
Acquisition of MRSA in a geriatric rehabilitation Center
M. Michelmann1, T. Biundo2, K. Oberdorfer1, C. Wendt1
University Heidelberg, Department Hygiene, Heidelberg
Clinic of Rehabilitation Hockenheim, Hockenheim, Germany
Colonization with MRSA is often considered as a
contraindication for treatment in rehabilitation facilities. But
most facilities do not screen all admitted patients and there may
be an unknown number of MRSA carriers in such programs.
We conducted a study to assess the number of unknown MRSAcarriers treated in a rehabilitation Center for geriatric patients
and to identify risk factors for MRSA transmission in the
Setting: A geriatric rehabilitation Center in Southern Germany.
Patients were treated in 3 different locations with 38, 35 and 28
beds respectively.
Patients: All patients entering the program were screened for
MRSA at admission and discharge. The origin of swabs was
blinded until the end of the study.
Microbiology: Swabs taken from nose, throat and wounds were
cultured on chromogenic media (Biorad) and in caso broth.
MRSA was identified by coagulase and confirmed by PCR. The
isolate were typed by spa-typing.
Data analysis: A case-control study was performed to identify
risk factors for MRSA acquisition during the stay in the clinic.
Patients who acquired MRSA were matched with patients
admitted directly before or after the case patients to the same
house. Data were abstracted from the patient’s chart and
analysed by SPSS.
Of 990 patients screened 70 were colonized with MRSA. 18
patients acquired MRSA during their stay and 50 patients were
already positive at admission. 2 MRSA positive patients were
screened at discharge only and therefore the mode of acquisition
could not be evaluated. Spa typing revealed that 16 patients of
the newly colonized patients had MRSA probably acquired from
other patients. Two patients were colonized with unique spa
types and a source of transmission could not be detected.
Patients who had acquired MRSA during their stay were not
different from other patients in terms of gender, age, underlying
disease, functional status, or contact to therapists. There was a
weak association between housing in a room adjacent to a
MRSA carrier and acquisition of MRSA.
We detected an unexpected high number of unknown MRSA
carriers who were admitted to a rehabilitation program. 1.9% of
the patients of the rehabilitation Center acquired MRSA during
their stay. Because preventable risk factors for MRSA
transmission could not be identified specific prevention
measures can not be recommended. However, rehabilitation
therapy is an important therapeutic procedure that should not be
denied. Thus compliance with standard hygiene precautions is
of major importance.
Extracellular protease of the nosocomial pathogen
Stenotrophomonas maltophilia: Crystal structure as
prerequisite for drug design
W. Weber1, S. Windhorst1, U. Larsen1, M. Perbandt2
A. Gabdoulkhakov2, C. Betzel1
University Hospital Hamburg-Eppendorf, Institute for BioChemistry und Molekular Biology I , Hamburg/Eppendorf
University of Hamburg, Department of Bio-Chemistry
Hamburg, Germany
Stenotrophomonas maltophilia is a multiresistant pathogen
increasingly emerging in the hospital environment. In
immunosuppressed patients these bacteria may cause severe
infections associated with tissue lesions like pulmonary
haemorrhage. This indicated proteolysis as a possible
pathogenic mechanism in these infections. Indeed, a new
protease with broad specificity has been found to be secreted by
S. maltophilia [1]. The gene, termed StmPr1, was analyzed; it
codes for a 63 kDa precursor, which is processed to the mature
protein of 47 kDa. The enzyme is an alkaline serine protease
which, by sequence homology and enzymic properties, can be
further classified as a new member of the family of subtilases.
However biochemical studies with the purified protein indicated
that the StmPr1 protease differs substantially from so far known
subtilisins in molecular size and in substrate specificity.
In order to gain insights into the structure-function-relationship
the StmPr1 gene was overexpressed in E. coli and processed to
the active enzyme. Crystals of the native and inhibited enzyme
could be grown after the protease was allowed to truncate from
the C-terminus to a 36 kDa active core protein. Diffraction data
sets of the native and inhibited enzyme were collected to 1.5 Å
resolution applying synchrotron-radiation, and the 3D-structure
was solved. Compared to known baterial proteases it revealed a
new overall fold and architecture of the active site. The high
resolution structure can serve to develop specific agents for a
targeted therapy of St. maltophilia infections.
S. Windhorst, E. Frank, D.Georgieva, F. Buc, P. Borowski, N.
Genov, and W. Weber, W. J Biol Chem. 277, 11042 (2002)
Pseudomonas aeruginosa-contaminated manufactured
synthetic saliva solution caused nosocomial outbreaks in
several German hospitals
L. Bader1, A. Walter1, J. Heesemann1
Ludwig-Maximilians-University Munich, Max von PettenkoferInstitute, Bacteriology and Hygiene, Munich, Germany
During March and April 2007, at least 13 post-cardiac surgery
ventilated ICU-patientsin a Bavarian university hospital
wereinfected with early-onsetnosocomial pneumonia or
colonized inthe respiratory tract by a single Pseudomonas
aeruginosa (PSAE)-strain.Cause of this outbreak was a
synthetic saliva solution (SSS) used as lubricant for preoperative endotracheal intubation following the indicationsfor
this medical product given byits German manufacturer. PSAE
with identical genotype by RAPD-PCR was detected from these
13 patients andfrom originalSSS samples containing >
105CFU/ml in three lots produced in Octoberand December
2006, respectively. The outbreak was officially notified to the
appropriate authorities like the Federal Institutefor Drugs and
Medical Products (BfArM) and the regional governmental
pharmaceutical supervisor responsible for the manufacturer of
this SSS. As a result of the notifications performded inthis case,
we got informations concerning comparable nosocomial
outbreaks in severalGerman hospitals - both, in other Bavarian
regions and in other federal states - due to PSAE-contamination
of further lots from 2006 since Augustof this SSS, too. Although
the above mentioned pharmaceutical authorities were informed
recently by severalusersabout suspected or detected microbial
contaminationin manufacturing of this medical product - for
example in November 2006 and January 2007 - a Germanywide recall compaign for alldelivered SSS lots from 2006 and
2007 wascarried out by the manufacturer not earlier than in May
2007. The local reccalls until January 2007 performed in
hospitals which notified PSAE-contamination in SSS lots
produced for themselves onlywere not sufficientto
preventfurther outbreakscaused bythis medical productin other
hospitals. In our presentation we will discuss our concern for
hygienic safety in manufacturing ofmedical products like SSS.
An outbreak of Clostridium difficile-related diarrhoea
(CDAD) in an adult surgery unit
A. Cohrs1, P. Gastmeier1, A. Kola1, F. Mattner2,3, M. Kist4
I. F. Chaberny1
Hanover Medical School, Inst. of Med. Microbiology and
Hospital Epidemiolog, Hanover, Germany
University of Luebeck, Luebeck, Germany
University of Luebeck, Institute of Medical Microbiology and
Hygiene, Luebeck, Germany
Freiburg University Hospital, Institute of Medical
Microbiology and Hygiene, Freiburg, Germany
Background: From January 25th through April 27th 2007
several patients in a surgical unit acquired Clostridium difficilerelated diarrhoea in an university hospital. This outbreak was
Methods: A case was defined as the occurrence of diarrhoea and
either the detection of
C. difficile toxins/ toxin-producing C. difficile in a diarrhoeal
stool specimen or the detection in bacterial culture. All other
patients admitted in the same unit from January until April, who
developed diarrhoea, were included in the control group.
Preventive measures such as isolation, cohorting, education of
staff members as well as a change of disinfection procedures
were performed.
Epidemiological and clinical information as age, gender,
underlying disease, nosocomial or non-nosocomial acquisition
of diarrhoea, prior antimicrobial treatment were collected.
A multivariant analysis was performed to determine risk factors.
All 12 culture positive samples were evaluated by pulsed-field
gel electrophoresis, a PCR ribotyping investigation was carried
out in one of them exemplarily.
Results: In total 31 patients appeared with CDAD. 87% of these
cases were nosocomial acquired. 28 patients formed the control
group; 19 of them showed a negative result in microbiological
testing, the remaining 9 were lacking a sample.
Due to the implementation of preventive measures no further
nosocomial cases occurred.
No severe cases were found.
The pulsed-field gel electrophoresis of the 12 samples were
compared by GelCompar II and showed the identical strain in 11
cases; only one strain mismatched.
The PCR investigation of one sample of the predominant strain
showed the ribotype 001, which is the most common type.
According to multivariate analysis age > 78 years (95% CI,
1.57-50.37; OR 7.05) and the treatment with fluoroquinolones
(95% CI, 1.42-182.4; OR 9.23) were risk factors to acquire
Conclusion: A continuous prospective surveillance is needed to
discover cases of Clostridium difficile-related diarrhoea early.
This enables the implementation of preventive measures at an
early stage to avert outbreaks proactively.
Do seasonal variations of nosocomial infections exist?
B. Piening1, H. Rüden1, P. Gastmeier2
Charité-University Medicine Berlin, Institute for Hygiene and
Environmental Medicine, Berlin, Germany
Hanover Medical School, Institute of Medical Microbiology
and Hospital Epidemiology, Hanover, Germany
Background: Several infectious diseases are known to have a
seasonal variation in their incidence but it is not clear, if these
differences have to be considered for evaluating surveillance
data of nosocomial infections.
Objective: To investigate seasonal influences for the most
important nosocomial infections and their causing pathogens.
Method: The data from the surveillance components for ICU
patients (ICU-KISS) and operated patients (OP-KISS) of the
German Nosocomial Infection Surveillance System (KISS)
between January 1997 and December 2006 were considered for
the analysis.
Infection rates (infections/1000 patient days for ICU-KISS or
infections/100 patients for OP-KISS) for the winter period
(December – February) and the summer period (June – August)
as well as the resulting incidence density ratio (IDR) for ICUKISS or risk ratio (RR) for OP-KISS were calculated. As test of
independence two-sided p values were calculated using mid-p
exact method for IDR’s and Fisher’s exact test for RR’s.
Results: A total of data from 694 185 ICU patients from 401
ICUs and 267 537 operated patients from 382 departments were
analyzed. The results for the 4 most important infection types
and some pathogens with a significant difference are shown in
table 1.
Conclusion: Minor seasonal variations do exist but it seems not
to be necessary to consider these differences for evaluation of
surveillance data.
Table 1:
half year
10637 4.34
ICU-KISS Pneumonia
5248 2.09
3107 1.36
P. aeruginosa
2717 1.16
1861 0.82
Enterobacter spp. 1372 0.62
OP-KISS SSI / 100 patients 5215 2.17
Infection type /
Infection with
half year
No significant influence of the air quality in the operating
room on the Surgical Site Infections rate in abdominal
C. Brandt1, U. Hott2, D. Sohr2, H. Rüden2
Clinic of J.-W.-Goethe-University, Med. Microbiology and
Clinic Hygiene, Frankfurt am Main, Germany
Charité - University Medicine Berlin, Institute for Hygiene and
Environmental Medicine, Berlin, Germany
Introduction: There is no evidence, that ventilation systems in
operating rooms (OR)contribute to the prevention of SSI in
abdominal surgery. On the other hand, ventilation systems are
frequently used in Germany and account for high investment
costs and operating expenses. The aim of this study is to
evaluate, whether there is an influence of the OR air quality on
the SSI rates after abdominal surgery in hospitals participating
to the German nosocomial infections surveillance system KISS
or not.
Methods: Data here was included from surgical departments
participating to KISS, with each having at least 100 operations
(from the years 2000 to 2004) registered for the operative
procedure categories appendectomy, cholecystectomy and colon
surgery and having provided detailed information about the
ventilation system in the OR.
Results: Appendectomy: Among the 23 departments none has
natural OR ventilation, 9 had turbulent high-efficiency air
filtration (n = 3765 operations; SSI rate 1.9% <1.4; 2.3>) and 14
had laminar-air-flow (n = 7897 operations; SSI rate 2.5% <1.8;
Cholecystectomy: Included were the data from 36 surgical
departments, 1 had natural OR ventilation (n = 416 operations,
SSI rate 0.2% <0.0; 0.7>). Turbulent high-efficiency air
filtration was used in 14 departments (n = 6317 operations; SSI
rate 1.3% <1.0; 1.5>) and 22 departments had laminar-air-flow
(n = 13805 operations; SSI rate 1.5% <1.2; 1.8>).
Colon surgery: Out of the 23 included departments, all had
artificial ventilation. Turbulent high-efficiency air filtration was
used in 7 departments (n = 3248 operations; SSI rate 6.2% <5.4;
7.1>) and 16 departments had laminar-air-flow (n = 13805
operations; SSI rate 5.4% <4.8; 6.1>).
Discussion: This data does not support the current requirements
by German industrial standards. Neither the benefit of any
artificial ventilation, nor the propagated laminar-air-flow
supply-air diffuser have been shown any protection against SSI
inthis data.
How safe are sterilisation processes for complex surgical
J. Goldberg1, S. Harpel1, C. Herr1, T. Eikmann 1, A. Knaust1
Institute of Hygiene and Environmental Medicine, Giessen
Testing of sterilisation processes is commonly performed by
assessment of thermo-physical parameters. For complex surgical
instruments thermo-physical testing might not ensure
sterilisation, either because of special characteristics of material
or geometry of the instrument. Therefore, microbiological
testing might be asked to complete validation of sterilisation
processes of certain instruments.
In the presented study different instruments, which failed
validation after assessment of the sterilisation process by
thermo-physical parameters, were tested by microbiological
methods. The selection of instruments comprised instruments
with plastic surfaces and instruments with extreme tight lumens.
Instruments were charged with a suspension of Geobacillus
stearothermophilus spores. Positive controls were charged with
dilutions of the spore suspension to demonstrate the rate of
recovery. Sterilisation was performed by vapour sterilisation at
134oC under vacuum fractionation. Spores were recovered with
sterile swabs which were incubated in Caso bouillon at 56oC for
seven days.
Recovery of spores or growth of G. stearothermopphilus
respectively was shown even after charge with a 6-log dilution
of spore suspension. So far, no bacterial growth was shown after
performance of vapour sterilisation. Allthough, for tight lumens
sterilisation effectiveness seems to range in a borderline area,
which was shown by assessments performed under “half cycle”
Uncertainties of thermo-physical assessment of sterilisation of
complex surgical instruments require systematic assessment. To
avoid these uncertainties, which might also implicate forensic
consequences, validation of sterilisation processes by
manufacturers of instruments including microbiological testing
should be discussed.
Patient care quality parameters and central venous catheter
associated bloodstream infection rates: An international
S. Hansen1, F. Schwab1, M. Behnke1, H. Carsauw2, P. Heczko3
O. Lyytikaeinen4, A. Savey5, P. Gastmeier6
Charite - University Medicine Berlin, Institute of Hygiene
Berlin, Germany
Scientific Institute of Public Health , Unit of Epidemiology
Brussels, Belgium
Jagiellonian University, Department of Microbiology, Cracow
National Public Health Institute, Department of Infectious
Disease Epidemiology, Helsinki, Finnland
C. Clin Sud-Est, Lyon, France
HanoverMedical School, Institute of Medical Microbiology and
Hospital Epidemiology, Hanover, Germany
Background: Prevention of central venous catheter (CVC)
associated bloodstream infections (BSI) is an important part of
infection control activities. Corresponding measures and CVCBSI rates show a great variety in Europe.
To determine data on quality of infection control practices
concerning BSI prevention in Intensive Care Units (ICU) and to
evaluate the influence of the country on the CVC-BSI rate.
Methods: A survey embedded in the European surveillance
network HELICS (Hospitals in Europe Link for Infection
Control through Surveillance) was conducted. ICUs
participating in the ICU surveillance networks of 5 countries
were asked to fill in questionnaires which contained questions
about the structure of the ICU, surveillance methods, CVC
insertion techniques as far as further management of CVCs. The
results were associated with the corresponding CVC-BSI rates.
Univariate and multivariate analyses were carried out.
Results: Data from 288 ICUs of 5 countries (Belgium, Finland,
France, Germany, Poland) with 1.383.444 patient days, 969.897
CVC days and 2095 CVC-BSIs were analysed. The monthly
number of taken blood samples per 1000 bed days varied
between the participating ICUs (median=82, Q1=36; Q3=164).
The median CVC utilization rate was 68.8%, the median CVCBSI rate was 1.73. CVC-BSI rates showed a variety between the
countries as shown in table 1. Multivariate analysis showed the
categorical variable country as a significant risk factor for the
development of a CVC-BSI. Furthermore university hospitals
were a risk factor for CVC-BSI (OR:2.55, CI95:1.17-5.58;
Conclusion: Whether surveillance methods or infection control
policies are responsible for the countries influence on CVC-BSI
rates remains unclear. Probably differences in cultural, social
and legal perspectives as well as differences in health care
systems are crucial for explaining these differences.
Table 1:
CVC associated BSI rates of the participating countries
Country Country Country Country Country all
1,73 (0,78;
median 0,94
BSI rate 6,57)
Nosocomial outbreak of carbapenem-resistant pseudomonas
aeruginosa in a surgical intensive care unit
A. Kohlenberg1, D. Weitzel-Kage1, D. Sohr1
P. van der Linden1, H. Rüden1, K. Weist1
Charité Berlin, Institute of Hygiene and Environmental
Medicine, Berlin, Germany
surveillance activities for the German Nosocomial Infection
Surveillance System for Intensive Care Units noticed a cluster
of patients with isolates of carbapenem-resistant Pseudomonas
aeruginosa in the surgical intensive care unit.
Methods: An outbreak investigation including a descriptive
analysis, a case-control study and PFGE typing of P. aeruginosa
isolates was carried out. Cases were defined as patients with a
carbapenem-resistant isolate of P. aeruginosa in the surgical
intensive care unit during the outbreak period from 01.07.2006
to 31.10.2006. Controls were defined as patients with a
carbapenem-sensitive isolate of P. aeruginosa in the same
intensive care unit during the same time period. Odds ratios
were calculated in a univariate analysis. To identify independent
risk factors a multiple logistic regression analysis was
Results: 15 cases and 18 controls were identified. Seven cases
had a nosocomial infection according to CDC criteria. In the
univariate analysis isolation of P. aeruginosa from a wound
culture, presence of an abdominal or pleura drain, abdominal
surgery, abdominal lavage, an operation with a wound
contamination class
3 and therapy with aminoglycosides,
quinolones or carbapenems were significantly associated with
carbapenem-resistant P. aeruginosa. Presence of an abdominal
drain (odds ratio 26.37, 95% CI 1.90 – 999) and therapy with
quinolones (odds ratio 39.12, 95% CI 3.17 – 999) were
independent risk factors for the isolation of carbapenemresistant P. aeruginosa. Seven of ten available isolates from case
patients with carbapenem-resistant P. aeruginosa showed an
identical PFGE-pattern.
Conclusion: This polyclonal outbreak of carbapenem-resistant
P. aeruginosa in surgical patients was associated with the
presence of abdominal drains as an indicator of improper wound
management and antibiotic therapy with quinolones.
Increase of VRE in a german university hospital.
A. Kola1, F. Schwab2, S. Sürbaum1, P. Gastmeier1
Hanover Medical School, Medical Microbiology and Hospital
Epidemiology, Hanover, Germany
Charité-University Medicine, Hygiene and Environmental
Medicine, Berlin, Germany
Background: Starting in 2004, an increase of vancomycinresistant E. faecium (VREfm) was observed at HanoverMedical
School (MHH), a 1 400 bed university hospital with 40 000
admitted patients per year: The proportion of VREfm raised
from 1.2 % (first half-year of 2004) to 20,4 % (second half-year
of 2006) in spite of isolation precautions.
Methods: VREfm were typed by PFGE and the simple diversity
index SD (different genotypes / N isolates * 100) was
calculated. Results were compared to those obtained for 239
isolates of Vancomycin-susceptible E. faecium (VSEfm). In
addition, multiple-locus variable-number tandem repeat analysis
(MLVA) and PCR amplification of the esp-gene were
Results: 171 primary isolates of 166 patients hospitalised on 30
wards were analysed: PFGE revealed 57 different genotypes, of
which 36 were unique and 21 appeared in clusters of 2–31
isolates. With the exception of three clusters of two, three and
five isolates, there was no genotype that was related to a specific
ward. Sixty-one patients (37 %) with VREfm were in the
general surgery unit and 38 patients (23 %) in the
haematological oncology unit. In these units, 30 % of VREfm
were due to patient-to-patient transmissions. SD was
significantly different between VREfm and VSEfm (33.3 vs.
44.8, RR VREfm/VSEfm = 0,745, CI95: 0,58-0,96, p = 0.024),
as were the cluster sizes (number of isolates belonging to one
genotype) of VREfm and of VSEfm (mean: 6.47 vs. 4.77,
median: 3 vs. 2, p = 0.028). MLVA-analysis of the two most
prominent PFGE-clusters revealed the involvement of the
epidemic clonal complex-17 (MT 1 and 7), but only the isolates
of one of these two cluster were esp-positive.
Conclusion: The lower SD and the greater median cluster size of
VREfm express a higher genetic relationship of VREfm in
comparison to VSEfm. Considering the 36 unique genotypes
and the occurrence of identical genotypes on different wards
without epidemiological linkage, this is not explained by simple
patient-to-patient transmission of VREfm, which accounted for
approximately 30 % of VREfm. Further analysis has to be done
to clarify the causes of the increase in VREfm at MHH.
Long-term MRSA-persistence determined by MRSA
surveillance of subsequent hospital stays
F. Mattner1, F. Biertz2, H. Hecker2, M. to Baben-Yang2,3
S. Ziesing4, S. Sürbaum4, P. Gastmeier4, I. F. Chaberny4
University Luebeck, Institute for Medical Microbiology and
Hygiene, Luebeck, Germany
Medical University Hanover, Biometry, Hanover
Medical University Hanover, Electronic Data Processing
Centre, Hanover, Germany
Medical University Hanover, Medical Microbiology and
Hygiene, Hanover, Germany
Objective: To estimate the duration of the persistence of MRSAcolonized patients.
Methods: From 1st Jan 2001 to 31st October 2005 all patients
admitted to a Northern German university hospital who had at
least one MRSA-positive culture were included in our study.
Readmissions of MRSA-positive patients were identified by an
alert system. All microbiological materials submitted to the
laboratory during all subsequent hospital stays were tested for
MRSA. A patient was regarded MRSA-negative, if for three
subsequent days specimens taken at initially MRSA - positive
body sites were tested negative in absence of MRSA targeting
Results: A total of 1032 MRSA-positive patients yielded to
2038 admissions. 403 (39%) patients were readmitted at least
once. The mean follow-up time was 136±219 days for all 1032
patients. 339 Patients had a follow-up time of at least 90 days
(range 90; 1325 days)
For the frequence of MRSA-positivity at discharge of
subsequent hospital stays see table 1. The kaplan-meier survival
analysis of all MRSA-positive admissions revealed that at 362
days post admission 50% of the patients lost the MRSA.
Patients colonized only in the nares were significantly more
likely to loose the MRSA compared to those colonized at other
or a combination of body sites (OR 2,6; 95% CI 1,2; 5,9).
Decolonization during the first admisssion showed a trend
towards a long-term effectiveness (log-rank-test p = 0,07).
Conclusion: The high readmission rate (40 %) of MRSApositive patients underlines the importance of an alert
system.MRSA-carriage is partially lost dependend on time
course, initial colonization sites and decolonization procedures.
Table 1: Long-term MRSA-carriage of patients hospitalized at
least twice
MRSA-carriage at
Patients readmitted at Patients with a followleast once (%) (N=403) up of > 90 days (%)
At all subsequent
238 (59,1%)
admissions positive
At at least three
alternating MRSAstatus
25 (6,2%)
187 (55,2%)
24 (7,1%)
Only at the last
58 (14,4%)
admission negative
55 (16,2%)
82 (20,3%)
73 (21,5%)
During at least two
EUREGIO-project MRSA-net twente/Muensterland:
Prevalence screening and other network activities to control
MRSA dissemination in the dutch-german border region
A. W. Friedrich1, I. Daniels-Haardt2, R. Köck1, F. Verhöven3
A. Mellmann1, J. E. W. van Gemert-Pijnen3, F. Kipp4
K. Becker4, M. G. R. Hendrix5
University Hospital Muenster, Institute for Hygiene
Muenster, Germany
State Institute of Puplic Health Service North RhineWestphalia, Muenster, Germany
University Twente, Fakulty of Behavioral Science
Twente, Germany
University Hospital Muenster, Institute for Medical
Microbiology, Muenster, Germany
Laboratory Microbiology Twente-Achterhoek, Enschede
Against the background of remarkable differences in MRSA
prevalence between Netherlands and Germany, recent efforts to
reinforce cross-border cooperations in healthcare spotlight the
need to monitor and reduce MRSA spread in the Dutch-German
border region. Since July 1st 2005, the EUREGIO-project
MRSA-net Twente/Muensterland has created a vivid network of
healthcare providers aiming at setting up a regional quality
group which controls MRSA dissemination and enables reliable
cross-border cooperation in the long run. Here we report on
current network activities.
1. Until today, 44 hospitals, representing 95% of all patient beds
procured in the region, are participating actively in the project.
On the German side of the border a MRSA admission screening
and risk factor assessment was performed in November 2006.
Therefore, a collaboration of the regional microbiological
laboratories was established. All isolates were spa typed.
Screening of patients yielded a proportion of 7.4 MRSA/100 S.
aureus. Risk factor analysis led to the implementation of a
graduated regional screening recommendation.
2. In ambulatory healthcare, general practitioners were invited to
informative meetings conveying an educated handling of MRSA
patients. In cooperation with health insurers and the regional
Association of Statutory Health Insurance Physicians (KVWL)
the refunding of MRSA-associated treatment costs was
3. Standardized MRSA surveillance data are collected by all
participating hospitals using a standardized protocol and a
computerized database for transmitting the data annually to the
public health departments enabling them to control regional
MRSA dissemination according to §23 of the German Infection
Prevention Act.
4. MRSA guidelines for patient transport services and nursing
homes have been developed in cooperation with the public
health departments.
5. For improving the presentation of information on MRSA,
behavioural scientists analyzed MRSA guidelines with special
regard to their user-friendliness and target-group-orientation. A
bilingual web-based question-answering tool was developed on
the basis of an interview study.
Active crossborder collaboration strengthens the regional
network between the healthcare providers. Prevalence screening
enlightens the local prevalence of MRSA and the local
importance of MRSA-associated risk factors.
Molecular protein A (spa)-typing for excluding possible
MRSA transmission episodes among detected MRSA
I. F. Chaberny1, S. Marsch1, P. Gastmeier1
Hanover Medical School, Inst. of Med. Microbiology and
Hospital Epidemiology, Hanover, Germany
Background: A routine surveillance for MRSA was established
since 2001 at HanoverMedical School. In order to identify
nosocomial transmission episodes spa-typing was introduced.
Method: MRSA-isolates from all possible epidemiological
associated patients in 2004 (stay on the same unit and during the
same time interval (± 9 days)) were included in spa-typing
analysis. When two different spa-types were identified, a
possible MRSA transmission episode was excluded.
Results: 445 patients and their MRSA isolates were included in
the analysis. With 62.2% was the spa type t032 or so called
“Barnimer” strain predominant followed by t003 (7%), t456
(2.9%), t004 (2.6%) and other. Because of no epidemiological
associations 75 patients were excluded for further
epidemiological analysis. Hence, 370 patients were examined.
Due to the definition 317 possible transmission episodes were
revealed. Of these 105 (33%) were identified as definite nontransmissions by comparing spa typing results. 21 (6.6%) were
determined as definite transmission episodes. Due to the
predominant strain in our setting 193 (60.9%) episodes could
not categorized.
Conclusion: The molecular spa typing provided additional
information to the epidemiological associations during routine
MRSA surveillance.
Results of a prospective one year follow-up study with the
Methicillin-resistant Staphylococcus aureus intelligent
operating system (METHIOS) – The awarded computer
based real-time MRSA surveillance
D. Krickhahn1, C. Krickhahn1, M. Herrmann1, U. Geipel1
University Hospital des Saarlandes, Institute for Medical
Microbiology and Hygiene, Homburg, Germany
Objective: Evaluation of the computer-based surveillance
METHIOS and presentation of the actual prospective follow-up
study results.
Design: We compared the efficiency of MRSA eradication with
or without the assistance of thecomputer based surveillance
system. This study was performed to determine differences in
MRSA detection and decolonization procedures as well as
additional infection control interventions in the two groups.
Patients and Setting: A total of 290 MRSA colonized patients
with 367 hospital stays in a 1.355-bed university hospital
between January 2006 and May 2007.
Results: From the 367 in-patient treatments the duration of the
hospital stay after the initial MRSA detection was evaluated. In
194 cases the hospital length of stay was less than 11 days, so
the minimal time spent for a proved MRSA eradication trial in
our study protocol was not given. 173 cases could be observed
for MRSA eradication. Of these 36 were included in the
software based MRSA surveillance METHIOS, 137 patients
were on hospital units which were in this period in the normal
personnel based MRSA surveillance. We found a high
significant difference in verified MRSA eradication between the
METHIOS group (83.3%) and the control group (20.4%);
Conclusions: The here described Computer-based surveillance
system is an effective and also attractive option to optimize
MRSA infection control. This METHIOS surveillance software
package might also be a useful interactive platform for an
MRSA network of different hospitals optimizing surveillance
and intervention of infections due to MRSA and other
multiresistant nosocomial pathogens.
C.K. won the „IKOP-Innovationspreis für angewandte
Infektionspraevention 2007“ for the developmentand the
implementation of the computer-based surveillance of MRSA.
Oral continuous exposure to environmental mycobacteria
renders germfree but not conventional mice susceptible to
M. tuberculosis infection
S. Behnck-Knoblau1, O. Goldenberg2, U. Steinhoff1
MPI for Infektion Biology, Immunolgy, Berlin, Germany
Transgenomic Ltd., Berlin, Germany
We investigated the exposure to environmental mycobacteria
(MOTT, mycobacteria others than tuberculosis) and the impact
of the intestinal flora on the ability of the vaccine BCG to
protect against M. tuberculosis (M. tub.) infection.
Dissemination and persistence of MOTT and BCG were
analyzed by culture and identity of bacterial isolates was
determined by denaturing high performance liquid
chromatography (DHPLC) on the WAVE® Microbial Analysis
System (Transgenomic Ltd). To evaluate BCG efficiency,
animals were challenged 11-12 weeks after vaccination with
virulent M. tub.
When given via the drinking water, MOTT disseminated to
multiple organs but did not persist. Continuous administration of
MOTT had no influence on dissemination and persistence of
oral BCG in conventional (SPF) and germfree (GF) mice.
Furthermore, in SPF animals, MOTT exposure had no influence
on BCG mediated protection against subsequent aerosol M. tub.infection. In contrast, GF-mice pre-sensitized with MOTT were
highly susceptible to aerosol M. tub.-infection despite
vaccination with BCG. Although dissemination and persistence
of BCG was not affected, aerosol challenged mice showed high
titers of M. tub. in spleen and lung.
These findings indicate that i) in the absence of an endogenous
microflora oral exposure with MOTT interferes with BCG
induced immunity and ii) that these suppressive effects are
independent of the spread and multiplication of the BCG
vaccine strain.
C5a/C5a-receptor modifies the course of experimental colitis
in mice
M. Martin1, K. Johswich2, A. Bleich3, E. Bleich3, E. Gessner4
M. Kracht5, O. Dittrich-Breiholz5, S. Sürbaum2, C.
Rheinheimer2, A. Klos2
Lund University, University Hospital Malmoe, Department of
Laboratory Medicine, Lund, Denmark
Medical School Hanover (MHH), Medical Microbiology
Hanover, Germany
Medical School Hanover (MHH), Institute for Laboratory
Animal Science , Hanover, Germany
Medical School Hanover (MHH), Clinical Immunology
Hanover, Germany
Medical School Hanover (MHH), Institute of Pharmacology
Hanover, Germany
Crohn´s disease and ulcerative colitis show increasing incidence
and impact in industrial countries. Animal models are useful
tools to study the pathogenesis of these multifactorial diseases.
The complement system is activated in colitis - most likely by
bacteria originating from the gut due to an impaired mucosal
barrier. Because the application of a C5a-receptor (C5aR,
CD88) antagonist in rats indicated a crucial role of this receptor,
we questioned if a knockout of C5aR modifies the course of
dextran-sulfate-sodium (DSS)-induced colitis in mice.
Experimental colitis was induced in C57BL/6J-C5ar1tm1Cge
(C5ar1-/-) and wild-type (wt) mice by oral administration of 2.5
% (w/v) DSS ad libitum for eight and four days, respectively.
Control mice received normal drinking water. The mice were
scored daily for the following parameters: body weight,
spontaneous as well as induced behaviour, habitus, stool
consistency/faecal blood, food and water intake, as well as
overall health appearance. All these parameters were
incorporated into a disease activity index. On day four or eight,
respectively, mice were sacrificed and blood was withdrawn by
heart puncture. Colons were collected to assess the development
of colitis by histological score and measured to determine the
colitis-induced shortening. Myeloperoxidase activity was
quantified in colon homogenates, and plasma C3a-levels were
measured to determine the degree of complement activation.
DSS-treated wt mice showed significantly higher weight loss,
diminished water and food intake, shorter colons, as well as
higher disease activity indices from day five on compared to the
corresponding water controls, demonstrating that the DSStreated mice developed the typical colitis symptoms. Symptoms
were milder and appeared later in C5ar1-/- than in wt mice upon
DSS-application. Furthermore, C5ar1-/- mice developed less
severe colitis as determined by histology on day four of DSStreatment. Thus, our results strongly suggest that C5a/C5aR
modify the development of experimental colitis in mice and
could serve as a new therapeutic target in colitis. Additional data
will be presented at the meeting.
Analysis of the role of iNOS in chronic Burkholderia
pseudomallei infection
P. Wongprompitak1,2, K. Breitbach1, I. Steinmetz1
University of Greifswald, Friedrich-Loeffler-Institute of Med.
Microbiology, Greifswald, Germany
Mahidol University, Department of Immunology, Siriraj
Hospital, Bangkok, Thailand
The gram-negative soil bacterium Burkholderia pseudomallei is
the causative agent of melioidosis, an infectious disease which
has emerged as an important cause of severe communityacquired infections in certain areas of the tropics.
Burkholderiapseudomallei is able to multiply intracellularily
within the cytosol and can directly spread from cell to cell. The
clinical manifestations of melioidosis are extremely variable
being either localized or disseminated, either chronic or acute
infections with fulminant septicemias. Long periods of latency
are characteristic of the disease, but the mechanisms leading to
clinical infections are unknown. We could recently demonstrate
that iNOS was not essential for the early control of acute
B.pseudomallei infection in relatively resistant C57Bl/6 mice. In
contrast, C57Bl/6 iNOS-/- mice seemed to be even slightly
protected from infection within 48 h after infection (Infect
Immun 2006. 74:6300). In this study, we address a potential
function for iNOS at later stages during chronic murine
melioidosis. Our experiments showed that there was no
significant difference in mortality between C57Bl/6 wildtype
and C57Bl/6 iNOS-/- mice within three months after infection.
Moreover, C57Bl/6iNOS-/- bone-marrow derived macrophages
revealed no essential role for iNOS-mediated mechanisms to
eliminate B.pseudomallei in these cells over a period of 120 h.
However, NO inhibition in macrophages derived from
susceptible BALB/c mice led to a reduced bactericidal activity
compared to control cells. In ongoing experiments, we are
comparing and analyzing the role of iNOS in C57Bl/6 and
BALB/c mice in controlling B.pseudomallei infection in various
organs several months after infection.
Similar T cell-activating properties of egc-encoded and
classical Staphylococcus aureus superantigens
D. Grumann1, S. Holtfreter1, C. Kohler2, S. Scharf3, M. Hecker2
U. Völker3, S. Engelmann2, B. M. Broeker1
University of Greifswald, Institute for Immunology and
Transfusion Medicine, Greifswald, Germany
University of Greifswald, Institute for Microbiology
Greifswald, Germany
University of Greifswald, Institute for Functional Genomics
Greifswald, Germany
Neutralizing serum antibodies against S. aureus superantigens
are common in the healthy population with one exception:
superantigens of the enterotoxin gene cluster (egc). These are by
far the most prevalent superantigens in clinical S. aureus isolates
but only rarely elicit a neutralizing antibody response, even in
carriers of egc-positive S. aureus strains. We hypothesized that
this egc-gap in the antibody response is due to i) different T cellactivating properties of classical and egc superantigens and/or ii)
their differential regulation. Three classical (SEB, SEQ, and
TSST-1) and three egc superantigens (SEI, SEM, and SEO)
were overexpressed in E. coli, purified and LPS-depleted, and
their T cell activating properties were studied. We found no
major differences between the T cell-mitogenic potency of
classical and egc superantigens. In contrast, egc-encoded
proteins were secreted by S. aureus during logarithmic growth,
while classical superantigens were released in the stationary
phase. In conclusion, we propose that, due to their different
regulation, classical and egc superantigens contact the host’s
immune system under conditions, which either favour or, in the
case of egc superantigens, prevent efficient antibody production.
“Live carrier” therapeutic tumor immunization independent
of tumor antigens. Do we need tumor antigens?
K. Panthel1, S. Jellbauer1, B. Köhn1, G. Pfaffinger1, E. Roider1
H. Rüssmann1
Ludwig-Maximilians-University Munich, Max von PettenkoferInstitute, Munich, Germany
Today tremendous efforts are undertaken to identify and
characterize tumor antigens. Different immunization schedules
applying diverse immunization procedures such as antigen
loaded dendritic cells are examined. Because of the complexity
of the Tumor microenvironment, and the complex and highly
polymorphic nature of the patients immune system, for example
the MHC-polymorphism, it is difficult to identify optimal
antigens in different patients.
Here we show that a combined prophylactic / therapeutic
immunization with Listeria monocytogenes and Salmonella
typhimurium against a highly aggressive murine fibrosarcoma is
effective as a therapeutic treatment independent of a predefined
tumor antigen. Data suggest that potent and “MHC-haplotype
adapted” but tumor unrelated antigen may be transported to the
tumor cells by intracellular bacteria provoking a local anti tumor
immune response. This is achieved through colonization of
tumor tissue by bacteria after preimmunization against the
antigen. Utilizing the Type Three Secretion System of
Salmonella and the intrinsic properties of Listeria it is possible
to induce antigen specific cytotoxic CD8+ T cells that may be
responsible for the therapeutic effect observed in immunized
animals. Optimization of therapeutic immunization may be
achieved in the near future by simultaneously addressing antigen
specific immunity and characteristic properties of tumor
microenvironment through “live carrier” mediated therapy.
Today it is not clear whether direct or indirect effects account
for the observed therapeutic effects.
Clonal distribution of superantigen genes in clinical
Staphylcoccus aureus isolates
H. Silva1, D. Grumann1, M. Schmudde1, H. Thi Thu Ngyuen1
P. Eichler1, B. Strommenger2, K. Kopron3, J. Kolata1, S.
Giedrys-Kalemba3, I. Steinmetz4, W. Witte2, B. M. Bröker1
University of Greifswald, Immunology, Greifswald, Germany
National Reference Center for Staphylococci, Wernigerode
Pomeranian Medical University , Dept. Microbiology and
Immunology, Sczcecin, Poland
University of Greifswald, Friedrich-Loeffler-Institute for
Medical Microbiology, Greifswald, Germany
Staphylococcus aureus is both a successful human commensal
and a major pathogen. The elucidation of the molecular
determinants of virulence, in particular the assessment of the
contributions of the genetic background versus those of mobile
genetic elements (MGEs), has proved difficult in this variable
species. To address this, we have simultaneously determined the
genetic background (spa-typing) and, as markers for MGEs, the
distribution of all 19 known superantigens as well as the
exfoliative toxins A and D (multiplex PCR). Methicillinsensitive S. aureus strains from Pomerania, 107 nasal and 88
blood culture isolates, were investigated. All superantigenencoding MGEs were linked more or less tightly to the genetic
background. Thus, each S. aureus clonal complex was
characterised by a typical repertoire of superantigen and
exfoliative toxin genes. However, within each S.aureus clonal
complex and even within the same spa-type, virulence gene
profiles varied remarkably. Therefore, virulence genes of nasal
and blood culture isolates were separately compared in each
clonal complex. The results indicated a role in infection for the
MGE harbouring the exfoliative toxin D gene. In contrast, there
was no association of superantigen genes with blood stream
invasion. In summary, we show here that the simultaneous
assessment of virulence gene profiles and the genetic
background increases the discriminatory power of genetic
investigations into the mechanisms of S. aureus pathogenesis.
IFIT-2 – A putative novel negative regulator of
proinflammatory responses
S. Berchtold1, E. Fahl1, M. Hornef2, J. Geisel1, J. - S. Frick1
E. Bohn1
University Hospital Tuebingen, Institute for Medical
Microbiology, Tuebingen, Germany
MedicalUniversity Hanover, Institute for Medical
Microbiology, Hanover, Germany
Interferon-induced tetratricopeptide repeat protein (IFIT-) 2 is
induced upon acute infection with Yersinia enterocolitica in
CD11b-positive cells of the spleen of mice as well as in the
colon of IL-2 deficient mice which develop inflammatory bowel
disease. IFIT-2 is induced by type II and type I interferons or
indirectly by LPS in an type I interferon dependent manner.
Recently it was reported that mouse IFIT2 (P54) affects protein
synthesis by interaction with the translation initiation factor
eIF3c. Therefore we speculated that this gene could represent a
negative regulator of host responses by down regulating protein
synthesis. To address the role of IFIT2, stably transfected RAW
264.7 macrophages were established overexpressing IFIT-2.
These cells were viable and showed similar proliferation as
control cells. IFIT-2 overexpression did not alter LPS triggered
p38, ERK, JNK and I-kappaB phosphorylation indicating that
IFIT-2 does not affect LPS mediated signal transduction.
Overexpression of IFIT-2 in RAW 264.7 macrophages reduced
LPS induced protein expression in a selective manner at
posttranscriptional levels. Thus, TNF- , IL-6 and MIP-2
secretion but not protein expression of IFIT-1 or early growth
reponse 1 were affected by IFIT-2. IFIT-2 may represent a novel
negative regulator of proinflammatory responses.
Repeated peripheral administrations of CpG
oligodeoxynucleotides lead to sustained CNS immune
S. Sethi1, I. Wagner2, W. Xiang2, A. Giese2, S. Ebner2
H. Kretzschmar2
Insitute of Medical Microbiology, Microbiology, Homburg
Center for Neuropathology and Prion Research, Prion
Research, Munich, Germany
Bacterial DNA containing CpG motifs activates cells of the
innate immune system. In this study, we examined the effects of
multiple peripheral bacterial DNA-mediated CNS innate
immune stimulation. To study this issue, we repeatedly
peripherally administered synthetic CpG-oligodeoxynucleotides
(CpG-ODN) and assayed effects on CNS-associated TNF-alpha
(TNF ) and C1q mRNA levels. We for the first time accounted
for frequency of CpG-ODN administration and time kinetics of
mRNA expression. We were able show that multiple
intraperitoneal CpG-ODN administrations have a sustainable
effect on immune effectors of the brain and stimulate TNF
mRNA secretion even up to 7 days after the last CpG-ODN
application. This could on the one hand indicate a depot effect
after multiple peripheral CpG-ODN administrations, however, it
could also indicate that the cell producing TNF mRNA remains
activated for the indicated time period. Furthermore, elevated
mRNA levels of C1q were observed, possibly indicating
microglial activation after multiple peripheral bacterial DNA
administrations. In this study, we have correlated frequency of
CpG-ODN administrations with CNS-associated TNF mRNA
levels and show that multiple peripheral administrations of
CpG-ODN lead to a sustained level of a Th1-associated
cytokine in the brain. These findings indicate that the repeated
peripherial administration of CpG oligodeoxynucleotides offer a
therapeutical possibility for CNS-associated infections and
Yersinia enterocolitica induces cell death in splenic dendritic
cells: Evidence for YopP dependent and independent
S. E. Autenrieth1, D. Middendorf1, I. B. Autenrieth1
Eberhard-Karl-University , Institute for Medical Microbiology
und Hygiene, Tuebingen, Germany
The virulence factor YopP of Yersinia enterocolitica (Ye)
induces cell death in mouse bone marrow-derived dendritic cells
(DCs). Here we have investigated whether YopP of Ye might
induce cell death in DCs in vivo. For this purpose, C57BL/6
mice were infected i.v. with Ye wild type (pYV+) or with the
YopP deficient mutant strain (pYV+ yopP). One, two and three
days post infection the number of splenic DCs including
subpopulations as well as the frequency of apoptotic DCs was
analyzed by flow cytometry and immunofluorescence
microscopy. Infection of mice with the pYV+ resulted in a
higher number of CD11c+ cells in the spleen one day post
infection compared to untreated mice. This effect was not
observed in the spleen of mice infected with pYV+ yopP,
indicating that the rapidly increased number of DCs in the
spleen one day after Yersinia infection is mediated by YopP.
Three days post infection the number of CD11c+ cells was three
times lower in the spleen of mice infected with pYV+ compared
to untreated mice. The decrease in CD11c+ cells was associated
with significantly higher numbers of apoptotic and necrotic DCs
three days post infection with the wild type strain pYV+
compared to uninfected mice or to mice infected with the YopP
deficient mutant strain pYV+ yopP as revealed by
immunostaining of active caspase 3, TUNEL reaction and
staining with propidium iodide. However, infection with
pYV+ yopP also resulted in a significant, although lower
increase of apoptotic DCs suggesting that part of DC death after
Yersinia infection in vivo is caused by mechanisms independent
of YopP. Recent published data suggest, that LPS promotes DC
death in bacterial infections in vivo. Furthermore, analysis of
DC subpopulations revealed that Ye induces cell death
predominantly in CD4+CD8 - DCs. Whether and how this
affects priming of T cells required for the control of Ye
infections remains to be shown in future studies. Taken together,
our data demonstrate YopP dependent and YopP independent
induction of DC death in vivo suggesting that YopP, possibly in
concert with LPS, induces cell death in splenic DCs.
Immune modulation mediated by Aggregatibacter
(Actinobacillus) actinomycetemcomitans as a possible
mechanism for the development of periodontitis
A. Schubert1, D. Nickel2, S. Poppert2, H. Bruns2, A. Spahr1
S. Stenger2
University Hospital Ulm, Department of Dentistry
Ulm, Germany
University Hospital Ulm, Institute for Medical Microbiology
und Hygiene, Ulm, Germany
Aggregatibacter (Actinobacillus) actinomycetemcomitans (A.a.)
is a major causative agent of chronic and aggressive
periodontitis. The aim of this study was to investigate the
interaction between A.a. and human peripheral blood
mononuclear cells (PBMC), as the interaction of this pathogen
with the host´s immune system seems to play a major rule in the
development and progression of this disease. To determine the
efficiency of infection, we designed probes specific for A.a. and
performed fluorescence in situ hybridization (FISH). The
majority of the bacteria were in the proximity of or adjacent to
PBMC implying the possibility of immunological interaction.
This finding prompted us to measure cytokine production (TNF,
IFN- ) and bacterial survival using a co-culture system of A.a.
and PBMC. The amounts of cytokines in the co-culture were
measured by Elisa. TNF titers measured after 24 and 72 h were
1300 pg/ml and 540 pg/ml, respectively. IFN- levels were 290
pg/ml after 24 h and 1600 pg/ml after 72 h. Under these
conditions (5% human serum) no viable bacteria were found
after 24 h of incubation. We are currently trying to establish
conditions that permit the survival of both eukaryotic and
prokaryotic cells. Ultimately, we intend to identify immune
deviations in patients with severe periodontitis that account for
the hyper-inflammation characteristic for this disease.
Role of complement in EHEC-induced haemolytic uraemic
syndrome: purified shiga toxin activates complement in
D. Orth1, K. Grif1, A. - B. Khan1, A. Naim1, M. P. Dierich1
L. - B. Zimmerhackl2, R. Würzner1
Innsbruck Med. Univ., Dept. Hygiene, Microbiology & Social
Medicine, Innsbruck, Austria
Innsbruck Med. Univ., Dept. Pediatrics, Innsbruck, Austria
Infections with Enterohaemorrhagic Escherichia coli (EHEC)
represent a major cause for haemolytic uraemic syndrome
(HUS) and acute renal failure in childhood. Shiga toxins (Stx1
and Stx2) are believed to be major virulence factors of EHEC,
contributing to the pathogenesis of HUS.
Besides EHEC-associated HUS, there are hereditary forms of
HUS, which are caused by mutations of complement regulator
proteins, in particular factor H, and not dependent on EHEC
bacteria. In contrast, a direct action of Shiga toxins on renal
cells has been proposed for development of the EHECassociated HUS.
The aim of our study was to investigate whether complement is
also involved in the pathogenesis of diarrhoea associated EHECinduced HUS.
By measuring the Terminal Complement Complex (TCC) using
an ELISA based on a neoepitope on human C9 we could show
that purified Shiga toxin 2 significantly activates complement
when incubated with normal human sera in fluid phase in vitro.
This effect was not seen when heat-inactivated Shiga toxin was
In conclusion, the major EHEC virulence factor Shiga toxin
activates complement in vitro, suggesting that complement also
plays a role in EHEC-induced HUS. Our hypothesis is that
EHEC virulence factors exert some of their destructive action
via complement.
Multiepitope vaccine against extraintestinal pathogenic E.
coli (ExPEC)
A. Wieser1, S. Schubert1
Max-von-Pettenkofer-Institut, Ludwig-Maximilians-University
Munich, Bacteriology, Munich, Germany
ExPEC cause a wide variety of infectious diseases such as sepsis
or urinary tract infections, and are responsible for a significant
mortality and morbidity in humans and animals. As ExPEC
strains become more and more resistant to antibiotics,
preventive measures such as vaccination against these
pathogenic E. coli strains are an achievable goal.
Based on previous findings in genome analyses we selectively
target virulence factors and uropathogen associated proteins. To
identify the relevant immunogenic regions, the unknown three
dimensional structure of these Proteins was simulated using a
combination of Hidden-Markov-Model, and neuronal network
based algorithms. Furthermore MHCI and MHCII epitopes as
well as proteasome cleveage sites were predicted.
Considering these data two recombinant modular proteins were
designed, containing epitope bearing regions separated by linker
sequences unlikely to be presented on MHCI or MHCII
molecules. To express the recombinant vaccine proteins, two
fully synthetic genes have been synthesized using an optimised
codon bias to enhance protein expression in Enterobacteriaceae.
We further evaluated two application routes in the mouse model
for these new multi-epitope vaccine proteins to obtain both, a
high humoral and cellular immune response.
First, we produced the vaccine proteins in vitro using an E. coli
expression system and administered them nasally. Second we
used a novel bacterial antigen delivery system which targets the
vaccine directly into the cytoplasm of mammalian cells in vivo
to enhance the cellular T-cell mediated immune response.
The elicited cellular and humoral immune response was
evaluated using IFN- ELISpot and sub-class specific antibody
ELISA. Immunized mice showed titre increases of specific IgG
in their serum as well as IgA antibodies in vaginal wash and an
increase in cytotoxic T-cells specific for the recombinant
vaccine proteins. In the challenge model of peritonitis a
significant reduction of bacterial load could be achieved in
immunized mice.
Proteins derived from Nocardia farcinica activate human
dendritic cells and induce IL-12 and IL-23 secretion
M. Eisenblätter1, E. Jasny1, A. Buchal1, G. Stübs1
T. Ulrichs1,2, G. Yapici1, R. R. Schumann1, S. Bereswill1
J. Zweigner1, R. Ignatius1
Charité-University Medicine Berlin, Institute for Microbiology
and Hygiene, Berlin, Germany
Max-Planck-Institute for Infection Biology, Berlin, Germany
The gram-positive, partially acid-fast cell wall of the bacillus
Nocardia farcinica is composed of proteins, lipids, lipoproteins,
glycolipoproteins, and peptidoglycan. As dendritic cells (DCs)
are of major importance for the induction of T-cell mediated
immunity, we were interested in the interaction of human DCs
with N. farcinica in vitro as well as in the bacterial components
and cellular mechanisms that might be involved in this
interaction. We investigated the effects of live and killed N.
farcinica as well as lipid and water-soluble non-lipid
preparations of bacterial lysates on human monocyte-derived
DCs in vitro. In contrast to previous findings with DCs infected
with Mycobacterium tuberculosis, DCs exposed to live or killed
N. farcinica in vitro were potently activated, i.e., they developed
a mature phenotype, secreted IL-12 and IL-23, and stimulated
allogeneic T cells in mixed leukocyte reactions. Neither filtered
supernatants of the cultures nor the lipid fraction but only the
water-soluble non-lipid fraction of the bacterial lysate induced
DC maturation. Experiments with Toll-like receptor- (TLR-)
expressing human cell lines demonstrated the involvement of
TLR2 but not TLR4 in the cellular recognition of the
components of the nocardial water-soluble non-lipid fraction.
Similarly, murine bone marrow-derived DCs (bmDCs) of TLR2
knock-out mice secreted less IL-12 than DCs from wild-type or
TLR4 knock-out mice. Thus, water-soluble non-lipid
components of N. farcinica, likely proteins, activate DCs and
TLR2-signaling is involved in the activation. Further
investigations of other TLRs and NOD proteins possibly
contributing to the recognition of nocardial components and
characterization of the activating proteins are ongoing.
IL-6 and maturation govern TLR2 and TLR4 induced TLR
agonist tolerance and cross-tolerance in dendritic cells
J. - S. Frick1, J. Geisel1, F. Kahl1, C. J. Kirschning2, H. Wagner2
I. B. Autenrieth1
Eberhard-Karl-University Tuebingen, Medical Microbiology
and Hygiene, Tuebingen, Germany
Technical University Munich, Institute for Medical
Microbiology, Immunology and Hygiene, Munich, Germany
Stimulation of murine bone-marrow-derived-dendritic-cells
(DC) with lipopolysaccharide (LPS) or the synthetic lipopeptide
(P3CSK4) induces expression of TNF-a in a TLR4- or TLR2dependent fashion, respectively. Pre-treatment of DC with LPS
results in hyporesponiveness to a subsequent LPS challenge,
termed LPS-tolerance and to subsequent P3CSK4 challenge,
termed cross-tolerance. Concordantly, treatment of DC with
P3CSK4 resulted in homo-tolerance towards subsequent P3CSK4
as well as cross-tolerance towards subsequent LPS challenge.
Different mechanisms seemed to account for induction of DC
tolerance. Pre-stimuation with LPS or P3CSK4 at low
concentration induced tolerogenic DC in an IL-6-dependent
fashion and was accompanied neither by activation and
maturation of DC nor by downregulation of TLR2/4 expression.
In contrast, induction of tolerogenic DC by treatment with LPS
or P3CSK4 at high concentrations was independent of IL-6. In
homo-tolerogenic DC degradation of IkB upon challenge was
inhibited, as well as in cross-tolerogenic DC pretreated with
high dose LPS or P3CSK4. In contrary, cross-tolerance in DC
pretreated with low amounts of LPS or P3CSK4 did not correlate
with reduced IkB degradation upon secondary challenge. Our
data indicate that in DC TLR4 and TLR2 stimulation results in
homo- and cross-tolerance and that neither reduction of TLR2 or
TLR4 expression levels, nor reduced NF?kB activtion are
essential for TLR agonist tolerance of DC.
CD8+Foxp3+ T-cells modulate cytotoxic CD8+ T-cell
functions in the intestine
A. M. Westendorf1, J. Bür1,2
German Research Center for Infection Research, Mucosal
Immunity, Brunswich, Germany
Hanover Medical School, Institute of Medical Microbiology
Hanover, Germany
Several studies have observed links between infections and the
development of intestinal autoimmunity. The normal intestine
contains a significant concentration of immune cells in a chronic
state of so-called physiological inflammation, in which the gut is
poised for, but actively restrained from, full immunologic
responses. During the course of infections in the normal host,
full activation of the GALT occurs is rapidly controlled by
regulatory cells. In contrast to antigen specific CD4+ regulatory
T cells in the intestine, the generation and function of
immunomodulatory antigen-specific CD8+ T cells is less well
defined. To dissect the immunologic mechanisms of CD8+ T
cell function in the mucosa, reactivity to a self-antigen
expressed in intestinal epithelium of mice bearing a MHC classI-restricted T-cell-receptor specific for this antigen was studied.
Here, we demonstrate that intestinal self-antigen expression
leads to peripheral induction of antigen-specific CD8+ Foxp3+ T
cells rather than the induction of cytotoxic CD8+ effector T cells
and intestinal pathology. This induction is restricted to the
mesenteric lymphnode and the lamina propria. Antigenexperienced CD8+ T cells in this transgenic mouse model are
characterized by significantly upregulation of CD103, CD83,
GPR83 and granzyme A/B expression, molecules also expressed
on regulatory CD4+ T cell subsets. Despite the fact that naïve
and antigen-experienced CD8+ T cell exhibit the same
proliferative capacity in vitro, CD8+ T cells from this transgenic
mouse model produce much less IFN-gamma and no TNF-alpha
after in vitro stimulation in comparison to naïve CD8+ T cells.
Furthermore, whereas naïve CD8+ T cells further aggravate
CD4+ T cells proliferation in an inhibition assay CD8+ T cells
isolated from this transgenic mouse model slightly reduce
antigen-specific CD4+ T cell proliferation in vitro. In summary,
we demonstrate that self-antigen expression of intestinal
epithelial cells is sufficient to induce CD8+ regulatory T cells
which maintain intestinal homeostasis by down-modulating
effector functions of T cells.
CD4+ T cell immune response induced by bacterial
polysaccharide requires activated IL-6- secreting antigen
presenting cells
S. Meemboor1, E. Flenner1, A. Zingarelli1, M. Bessler1
W. Kalka-Moll1
University of Cologne, Institute for Medical Microbiology
Immunology and Hygiene, Cologne, Germany
Abscess formation associated with intra-abdominal sepsis
causes severe morbidity and are difficult to treat. The presence
of CD4+ T cells is essential for abscess formation. Zwitterionic
polysaccharides (ZPS) from commensal bacteria such as
Staphylococcus aureus represent a novel class of
immunomodulatory bacterial antigens that stimulate CD4+ T
cells in a MHC class II-dependent manner. Sp1 is the capsular
polysaccharide of S. pneumoniae serotype 1 and acts as a ZPS
model antigen. Sp1 possesses a zwitterionic charge motif with
free amino and carboxyl groups and promotes T cell-dependent
intraperitoneal abscesses in an experimental murine model.
Mice are administered Sp1 with sterile cecal content adjuvant
(SCCA) which reflects the spillage of colonic contents that
occurs during the onset of intra-abdominal sepsis in humans and
aids the pro-inflammatory response. SCCA alone has been
proven not to induce abscess formation. In this study we address
the role of the cytokine IL-6 on antigen presenting cells during
Sp1-induced abscess formation. FACS analysis demonstrate that
macrophages are the most prevalent antigen-presenting cells in
intraperitoneal lavage after intraperitoneal Sp1 challenge.
Macrophages activated by Sp1 secrete significant amounts of
IL-6 as shown by intracellular cytokine staining. IL-6 secreting
macrophages are incorporated in the abscess capsule as
immunohistochemical analysis of the Sp1-induced abscesses do
visualize. Migration assays demonstrate a dose-dependent IL-6induced CD4+ T lymphocyte migration. In IL-6-deficient mice
we demonstrate that IL-6 fails to attract CD4+ T cells into the
peritoneal cavity after Sp1 challenge and to hence develop
abscesses. In addition, administration of a neutralizing antibody
specific for IL-6 prevents abscess formation following Sp1
challenge. These data delineate the requirement of activation of
antigen-presenting cells by ZPS and underscore the essential
role of IL-6 in this disease process.
Impaired respiratory burst of human polymorphonuclear
leukocytes after phagocytosis of F. tularensis holarctica and
F. tularensis tularensis strains
W. D. Splettstösser1,2, D. Frangoulidis3, M. Bastian4
P. Kaysser1, E. Seibold1, P. Schuff-Werner4
Bundeswehr Institute of Microbiology, Immunology, Munich
Institute of Medical Microbiology, Virology & Hygiene
Medical Microbiology, Rostock, Germany
Bundeswehr Institute of Microbiology, Munich, Germany
Institute of Clinical Chemistry & Laboratory Medicine
Rostock, Germany
Background: Human polymorphonuclear leukocytes (PMN)
represent the first line of defense against invading
microorganisms. During phagocytosis, PMN produce reactive
oxygen species (ROS) and release cytotoxic granule
components to kill ingested microbes. Only few microorganisms
evade killing by different mechanisms. Francisella tularensis,
the causative agent of tularemia, has been reported to be less
efficiently killed by PMN but the exact mechanisms involved
have so far not been identified.
Methods: Flow cytometric analysis of phagocytosis and
respiratory burst after incubation of PMN with different F.
tularensis strains was performed employing heparinized whole
blood obtained from vaccinated or non-immune donors. The
PhagoTestTM or BurstTestTM (Orpegen Pharma Heidelberg)
were used according to the manufacturer`s instruction, and
bacterial target cells were “live”-stained by different
Results: Regular phagocytosis occurred with all strains even in
the absence of specific antibodies. Strains of the subspecies F.
tularensis tularensis or holarctica did not trigger ROS
production, while F. tularensis subspecies novicida was able to
induce the respiratory burst independently from the opsonization
with normal or specific immune sera. The activation of the
respiratory burst in “none-immune“ PMN depended on the preopsonization with anti-Francisella antibodies.
Conclusion: Immunity to infection with F. tularensis is widely
considered an example of T-cell mediated immune defense,
driven by activated macrophages. However, at least in murine
infection, the critical role of PMN for host defense against
primary infection and their participation in defense against reinfection had been recognized. We demonstrated that
differences in virulence of F. tularensis strains are reflected by
different handling by human PMN. It remains to be elucidated if
ingestion and oxidative killing triggered by Francisella-specific
antibodies are the only ways how PMN can contribute to
immune defense against this intracellular pathogen.
Adenoviral Rab-GFP fusion protein by phagosomal and
endosomal trafficking studies in primary antigen presenting
N. Robinson1, T. Li Stephen1, S. Meemboor1, W. Kalka-Moll1
G. Plum1
University of Cologne, Institute for Medical Microbiology
Immunology and Hygiene, Cologne, Germany
Antigen presentation by major histocompatibility complex
(MHC) class II on antigen presenting cells (APCs) is facilitated
after a sequence of events that starts with a phagocytic process,
followed by phagosomal processing of the antigen,
phagolysosomal degradation of the phagocytosed material and
loading of antigenic peptides onto MHC molecules. A detailed
picture of this process termed phagosome maturation is
beginning to emerge, involving regulators of membrane
trafficking in mammalian cells and phagosomal interactions
with endosomal organelles and the trans-Golgi network. So far,
cell biology studies of antigen presentation can be performed in
primary cells after fixation and permeabilization for staining
with specific antibodies or other endosomal pathway probes.
Investigation in live cells is solely possible in cell lines after
transfection with expression vectors for GFP-tagged endosomal
marker proteins. In an effort to open up ways to elucidate the
phagosomal processing events after antigen uptake in live
primary antigen presenting cells of murine and human origin we
have developed a set of adenoviral Rab-GFP fusion protein
expression vectors that allow observation and tracking
individual phagosomes. Transfection with the early endosomal
marker Rab5-GFP, late endosomal marker Rab7-GFP, and
transferrin receptor recycling marker Rab11-GFP was achieved
in 92% to 93% of mouse dendritic cells, in 89% to 94% of
human macrophages and in 70% to 82% of mouse macrophages.
Rab-GFP fusion proteins were readily expressed in these
primary cells using these highly efficient adenoviral constructs
allowing phagosome and endosome trafficking by fluorescently
labeled fluid phase marker. Our data show that the intracellular
endocytic trafficking events preceeding antigen presentation in
primary APCs can be effectively studied in live cells by using
our adenoviral expression systems.
Analysis of inflammatory mediators in bronchoalveolar
lavage fluid during hospital-acquired pneumonia by a bead
array based multiparameter assay
M. Klotz1, A. Klemmer1, M. Lohoff1
University Hospital Marburg, Institute for Medical
Microbiology, Marburg, Germany
Although BAL has been helpful in the diagnosis and
differentiation of various types of lung diseases including
interstitial lung diseases, malignancies and pulmonary
infections, it is often unclear, whether the presence of bacteria
represents colonization or infection.
During lung diseases cytokines and chemokines are important
mediators of the inflammatory response leading to lung injury.
The proinflammatory cytokines interleukin-1ß and tumor
necrosis factor- are produced by alveolar macrophages. In turn,
they induce the production of a variety of other cytokines and
chemokines by both macrophages and mesenchymal cells
(including fibroblasts, epithelial and endothelial cells), which
recruit more leucocytes to the site of inflammation. Here, we
measured different cytokines and chemokines in BAL from 102
patients with and without clinical symptoms of pneumonia by
flow cytometry using a Cytometric Bead Array (CBA)
multiparameter analysis. Nine different cytokines (IL-2, IL-4,
IL-5, IL-10, TNF , IFN , IL-1ß, IL-6 and IL-12p70) and five
different chemokines (IL-8, CCL5/RANTES, CXCL9/MIG,
CCL2/MCP-1 and CXCL10/IP-10) were analysed. We found a
great heterogeneity in the amount of cytokines. The chemokine
IL-8 was present at high levels in most samples. IL-1ß showed
high levels in BAL from patient with pneumonia and pathogenic
bacteria. Surprisingly, IFN and IL-6 were present at high
amounts in BAL from patients with pneumonia independent of
the presence of bacteria.
These data were correlated with the clinical outcome,
pulmonary infiltration, fever and further laboratory values, such
as leucocytes and procalcitonin. The long-term goal of this study
is to develop an inflammatory-mediator-score-system that helps
to establish a diagnosis of pneumonia.
Bartonella henselae exists as a mosaic of different genetic
variants in the mammalian host
J. Berghoff1, J. Viezens1, M. Arvand1
University Rostock, Institut für Med. Microbiology, Rostock
Bartonella henselae is a fastidious bacterium associated with
chronic infections in humans and cats. The mechanisms
involved in the long-term survival of bartonellae despite
vigorous host immune responses are poorly understood.
Generation of genetic variants is a possible strategy to
circumvent the host specific immune responses. We have
recently demonstrated the coexistence of different genetic
variants within the progeny of three primary B. henselae isolates
from Berlin by PFGE analysis. Aims of the present study were
to determine whether coexistence of different variants is a
common feature of B. henselae isolates worldwide and whether
the genetic variants originally emerged in vivo. Thirty-four
primary isolates from different geographic regions were
analysed by subjecting multiple single colony derived cultures
to PFGE analysis. Up to three genetic variants were detected
within 20 (58.8%) isolates, indicating that most primary isolates
display a mosaic-like structure. The close relatedness of the
genetic variants within an isolate was confirmed by multi-locus
sequence typing (MLST). In contrast to the primary isolates, no
genetic variants were detected within the progeny of 20
experimental clones generated in vitro from 20 primary isolates,
suggesting that the variants were not induced in vitro during the
procedure of PFGE analysis. Hence, the genetic variants within
a primary isolate most likely originally emerged in vivo.
Consideration of the mosaic structure of primary isolates is
essential when interpreting typing studies on B. henselae.
CMV reactivation of patients with septic shock: role of
antiviral immune response
L. von Müller1,2, M. Principi2, A. Klemm2,3, N. Durmus2
M. Schneider3, M. Weiß3, H. Suger-Wiedeck3, T. Mertens2
University of Saarland Hospital, Institute of Medical
Microbiology and Hygiene, Homburg, Germany
University Hospital Ulm, Institute of Virology, Ulm, Germany
University Hospital Ulm, Department of Anesthesiology, Ulm
Cytomegalovirus (CMV) is discussed as a pathogen of emerging
importance in critically ill patients without immunosuppressive
therapy. In this single center prospective study sixty-five
patients with septic shock were recorded. Prospective weekly
monitoring of active CMV infection on the one hand and
immune functions of the other hand were performed using
quantitative pp65-antigenemia, viral isolation, interferon-gamm
release assay for T-Helper1-cell (Th1) function and cytotoxic
Natural Killer cell (NK) assay. Forty-three CMV seropositive
patients with a prolonged ICU stay (<7 days) were included.
Within two weeks fourteen patients (32.6%) developed an active
CMV infection with low pp65-antigenemia (median 3 positive /
5´105 WBCs). Surprisingly, CMV reactivation occurred in
patients with intact CMV specific Th1-cells. Pp65-antigenemia
became undetectable without antiviral therapy after twenty days,
on average. Thereby, the frequency of CMV and superantigen
(SEB) reactive Th1-cells significantly increased in the groups
with active CMV infection but remained low in the group
without active CMV infection. In addition to CMV reactivations
also herpes simplex virus (HSV) reactivations were detected in
sixteen patients in bronchial aspirates which occurred at the
same time in the same patients. Overall, morbidity was not
different between CMV seropositive and seronegative patients;
however, ICU treatment (median 17 vs. 33.5 days) and the need
of mechanical ventilation (median 19 vs. 42.5 days) were
significantly prolonged once active CMV infection occurred.
We conclude that CMV reactivation followed by antiviral
immune response could impair pulmonal function of patients
with septic shock. Early antiviral therapy aimed at preventing
viral associated morbidity of CMV seropositive patients with
septic shock should be evaluated in future clinical as a new
treatment option.
Fig. 1: CMV and SEB reactive Th1-cells
The following scenarios were analyzed. Patients with active
CMV infection (group 1; A and D), patients with negative pp65antigenemia without CMV reactive Th1-cells (group 2; B and E)
and patients with negative pp65-antigenemia with CMV reactive
Th1-cells (group 3; C and F).Statistical analysis was perfomred
using paired t-test , and significance was set at a level of p =
Figure 1:
The control of Anaplasma phagocytophilum in vivo is
dependent on CD4+ T cells, but is independent of typical
Th1 cytokines and a wide spectrum of effector mechanisms
F. von Löwenich1, K. Becher1, Y. Kern1, B. Steiner1
C. Bogdan2
Institute of Medical Microbiology and Hygiene, Freiburg
Institute of Clinical Microbiology, Immunology and Hygiene
Erlangen, Germany
Anaplasma phagocytophilum is a Gram-negative, obligate
intracellular bacterium that is transmitted by ticks and replicates
within neutrophils. In order to define the mechanisms of
immunological control, we studied the course of infection in
transgenic mouse strains with various defects of the immune
system. The mice were systemically infected via i.p. injections
of infected SCID mouse blood. At certain time after infection,
the bacterial load was measured in the blood, spleen and lung by
a quantitative PCR technique. Mice deficient for cytokines and
effector molecules involved in the control of other intracellular
pathogens were unimpaired in eliminating A. phagocytophilum
(IL-12p35-/-, IL-12p35-/-p40-/-, IL-4-/-, IFN-gamma-/-, IFNalphaR-/-, TNF-/-, iNOS-/-, gp91phox-/-, neutrophil elastase-/Cathepsin G-/-, MCP-1-/-, Perforin-/-,Faslpr, Faslgld). SCID and
Rag1-/- mice developed strikingly increased bacterial loads in the
blood compared to wild-type. SCID mice died after about 21 to
30 days of infection, but were rescued by the transfer of T-cells.
Similarly, T-cell deficient nude mice were not able to control
the pathogen, whereas Ig -/- mice (lacking mature B-cells)
cleared the infection. Wild-type mice as well as JHT-mice (no
B-cell-receptor) were protected against a second infection. MHC
II-deficient mice, CD4-depleted mice and Tcrb-/- mice showed a
low level bacterial persistence which indicates that a CD4+-TCell dependent Th1-response plays a role in controlling A.
phagocytophilum in vivo.
Shifts towards pro-inflammatory intestinal bacteria
aggravate acute murine colitis and ileitis via toll-likereceptor signalling
M. M. Heimesaat1, A. Fischer1, B. Siegmund2, A. Batra2
C. Loddenkemper3, O. Liesenfeld1, M. Blaut4, U. B. Göbel1
R. R. Schumann1, S. Bereswill1
Charité-University Medicine Berlin, Microbiology and
Hygiene, Berlin, Germany
Charité-University Medicine Berlin, CBF, Medical Clinic I
Berlin, Germany
Charité-University Medicine Berlin, CBF, Institute for
Pathology, Berlin, Germany
German Institute for Food Research, Gastrointestinale
Microbiology, Potsdam-Rehbruecke (Nuthetal), Germany
Inflammatory bowel diseseases (IBD) are accompanied by shifts
of the gut microflora towards pro-inflammatory bacteria.
Components of gut bacteria are specifically sensed by the innate
immune system via Toll-like-receptors (TLRs) 2 and 4.
Therefore, we assessed gut flora changes and disease
susceptibility in ileitis and colitis by using mice lacking TLR2,
TLR4, or the innate immunity signaling adapter proteins
MyD88 and TRIF.
In DSS-colitis, TLR2-/-, TLR4-/-, and TLR2/4-/- mice displayed
reduced signs of acute colitis, reduced levels of immune
mediators, lower amounts of neutrophils, and less regulatory Tcells in situ as compared to wild-type (wt) animals. During
colitis, E. coli concentrations increased in wt mice but were
significantly lower in TLR2/4-/- animals. Enterococci showed a
tendency to increase in colitis and were also less abundant in
TLR2/4-/- mice. Microflora shifts were confirmed by cultureindependent molecular analyses.
In T. gondii-induced ileitis, E. coli increased by nine orders of
magnitude. Mice impaired in TLR4 or MyD88, but not TRIF or
TLR2 expression, displayed reduced mortality and diminished
immunopathology. Decreased IFN-g- and NO-levels in the
inflamed ileum of TLR4-deficient mice indicated that TLR4signaling aggravates ileitis via local mediator release from
immune cells. E. coli strains isolated from the inflamed ileum
induced TLR4-dependent NF-kB activation and nitric oxide
(NO) production in HEK293 cells and peritoneal macrophages.
In gnotobiotic mice treatment with purified E. coli lipid A and
colonization with live E. coli aggravated ileitis, whereas animals
lacking TLR4 were protected. Treatment with the LPS
Immunohistochemistry and analysis of immune cell populations
at inflamed mucosal sites in situ revealed that the amelioration
of ileitis in TLR-deficient animals was associated with lower
tissue concentrations of neutrophils and regulatory T-cells,
which were both significantly increased in the inflamed ileum.
We conclude that shifts towards pro-inflammatory bacteria
potentiate colitis and ileitis via TLR signaling indicating that the
innate immune system is essential for maintaining the intricate
balance between mucosal homeostasis and intestinal
inflammation. This highlights the innate immune system as a
immunopathology. The application of TLR-antagonists may
represent a novel strategy for amelioration of acute intestinal
inflammation in course of IBD.
Role of dendritic cells in S. pyogenes infection
T. Loof1, M. Rohde2, S. Jung3, E. Medina1
Helmholtz-Center for Infection Research, Mikrobielle
Pathogenicity/Infektion Immunology, Brunswich, Germany
Helmholtz-Center for Infection Research, Mikrobielle
Pathogenicity, Brunswich, Germany
The Weizmann Institute of Science, Immunology, Rehovot
Infections with Streptococcus pyogenes remains a significant
health care problem. The identification of immune components
required for host defenses against S. pyogenes constitutes an
important area of research. Dendritic cells (DCs) are
professional antigen-presenting cells that play a crucial role in
initiation and modulation of specific immune response against
microbes. We investigated the role of DCs during S. pyogenes
infection using an experimental mouse model. S. pyogenes
induces maturation of DCs, indicated by high expression levels
of co-stimulatory molecules such as CD40 or MHC class II and
the release of cytokines such as IL12, IL6, and TNFa in in vitro
experiments. The contribution of DCs to host defenses against
this infection was investigated using CD11c-diphteria toxin
receptor (DTR) transgenic mice, in which DCs can be
transiently depleted in vivo by treatment with low doses of DT.
We show that ablation of DCs led to increased bacterial
dissemination into draining lymph nodes and systemic organs.
Furthermore, ablation of DCs but not of macrophages resulted in
a significant reduction in the levels of IL-12 in the serum of
infected mice. These data suggest that although other cell
populations such as macrophages can also produce IL-12, DCs
are the main producers of this cytokine during in vivo infection.
This is an important finding since IL-12 has been shown to
contribute to host resistance to S. pyogenes infection. The
results of our study are therefore of high relevance to the
understanding of the early events of infection and the onset of
protection against S. pyogenes.
Caspase-1 is essential to control intracellular infection with
Burkholderia pseudomallei
K. Breitbach1, J. Köhler1, P. Wongprompitak1,2, K. Eske1
I. Steinmetz1
University of Greifswald, Friedrich Loeffler Institute of Med.
Microbiology, Greifswald, Germany
Mahidol University, Department of Immunology, Siriraj
Hospital, Bangkok, Thailand
Using murine models of infection, Caspase-1 has been shown to
play an important role in defence against several intracellular
pathogens. However, in some of these models results were
conflicting, since absence of caspase-1 caused increased
susceptibility in vivo but, in contrast, seemed to protect infected
macrophages from cytotoxic effects of the pathogen in vitro.
Recently, it has been shown that the facultative intracellular
gram-negative rod Burkholderia pseudomallei can induce
caspase-1 dependent cell death in macrophages. The present
study was aimed to elucidate the role of caspase-1 during
B.pseudomallei infection in more detail. Our data presented here
shows that cytotoxicity in B.pseudomallei-infected caspase-1-/macrophages was reduced within the first hours after infection,
but strikingly correlated already with an increased intracellular
bacterial burden compared to control cells at this time point. In
contrast, 24 h after infection deficiency of caspase-1 led to
highly increased cell damage of infected cells and a dramatic
increase in bacterial burden. Finally, in agreement with our in
vitro data we found caspase-1-/- mice to be highly susceptible
against B.pseudomallei challenge. Our results suggest that,
although B.pseudomallei induces cell death in macrophages to
some degree via a caspase-1-dependent mechanism in the early
phase of infection, this enzyme is important to control
intracellular bacterial growth in the early and late phase of
infection. This dual role of caspase-1 might explain some
conflicting in vitro and in vivo results on the role of this enzyme
in infection models with other intracellular organisms.
Inhibition of apoptosis conserves neutrophil effector
function and can contribute to inflammation in vivo
T. Frankenberg1, R. Paul2, B. Obermaier2, S. Kirschnek1
H. Häcker3, G. Häcker1, U. Ködel2
Technical University Munich, Institute for Medical
Microbiology, Munich, Germany
Ludwig-Maximilians-University, Department of Neurology
Munich, Germany
St. Jude Children’s Research Hospital, Infectious Disease
Memphis, USA
Neutrophil granulocytes have essential functions during immune
responses against pyogenic bacteria. Following pathogen
clearance, neutrophils have to be removed from sites of
infection. We used a mouse model of pneumococcal meningitis
to test for the role of apoptosis in neutrophils in this termination
of the immune response. In this model, neutrophil function is
known to be crucial for efficient defence against bacterial
infection. Mice overexpressing anti-apoptotic Bcl-2 in the
hematopoietic compartment had more severe symptoms and
higher lethality than wild type littermates. We observed a
similar recruitment of leukocytes into the brain at early time
points during infection but a significantly higher leukocyte
count in the cerebrospinal fluid at later stages. This was
accompagnied by a loss of blood-brain-barrier function,
impaired clearance of bacteria from the blood in Bcl-2transgenic mice and more pronounced histopathological
alterations. The continued presence of neutrophils in these mice
despite clearance of bacteria suggested that the block of
apoptosis caused sustained pleocytosis and contributed to the
severity of the inflammation. To test this, we compared in vitro
apoptosis and effector function of wt and Bcl-2 overexpressing
neutrophils derived from isolated bone-marrow progenitor cells
retrovirally transduced with regulable Hoxb8. In its active state,
Hoxb8 promotes progenitor expansion while upon inactivation it
permits cellular differentiation. This culture system thus
provides the possibility to generate almost unlimited numbers of
‘near-primary’ neutrophils. Differentiated Hoxb8 neutrophils
displayed the typical nuclear morphology, expressed Gr-1, and
were phagocytosis-proficient. No obvious differences in terms
of differentiation and effector functions were observed between
wt and Bcl-2 neutrophils. Upon prolonged culture, the vast
majority of differentiated wt neutrophils underwent apoptosis.
The overexpression of Bcl-2 efficiently protected neutrophils
from cell death and, importantly, these ‘undead’ cells retained at
least some of their effector functions. These data show that
inhibition of apoptosis can prolong neutrophil lifespan and
effector function, which very likely contributes to the more
severe pathology in Streptococcus pneumonia-induced murine
meningitis. This model therefore suggests that granulocyte
apoptosis is an important event for the termination of the innate
immune response.
CD8alpha+ dendritic cells are required for efficient entry of
Listeria monocytogenes into the spleen
M. Neuenhahn1, K. Kerksiek2, M. Nauerth1, M. Suhre1
M. Schiemann1, F. Gebhardt1, C. Stemberger1, K. Panthel3
S. Schröder2, T. Chakraborty4, S. Jung5, H. Hochrein1
H. Rüssmann3, T. Brocker2, D. Busch1
Technical University Munich, Institute for Medical
Microbiology, Immunology and Hygiene, Munich, Germany
Ludwig-Maximilian-University, Institute for Immunology
Munich, Germany
Ludwig-Maximilians-University, Max von Pettenkofer-Institute
Munich, Germany
Justus-Liebig-University, Institute for Medical Microbiology
Giessen, Germany
Weizmann Institute, Department of Immunology, Rehovot
In addition to their bridging function between innate and
adaptive immunity, dendritic cells (DCs) may also contribute to
primary resistance against infection.
Therefore, we analyzed the role of DCs during infection with
Listeria monocytogenes by performing systemic in vivo
depletion of these cells. Surprisingly, we found that CD8alpha+
DCs are crucial for Listeria spreading and proliferation in the
spleen. Efficient and rapid uptake of Listeria by CD8alpha+ DCs
requires the small GTPase Rac1 and is a general characteristic
of this DC subpopulation in filtering particles out of the blood.
Thus, CD8alpha+ DCs appear to play an important role for
efficient bacterial entry into the spleen. Depletion of DC
consequently abrogates subsequent T cell responses, which are
again detectable upon bypassing the dependency of sustained
Listeria infection on the presence of an intact DC compartment.
Based on these important features of early Listeria infection
kinetics, we performed further experiments using Listeria
mutants and bone marrow chimeric mice in order to analyze the
relative contribution of different antigen-presenting cells for the
establishment of protective T-cell responses against intracellular
Role of non-proteasomal proteases in the processing of
Listeria monocytogenes-derived MHC class I-presented
antigenic peptides
S. Grauling-Halama1, U. Bahr1, S. Schenk1, G. Geginat1
University Heidelberg, Faculty for Clin. Medicine Mannheim
Institute for Med. Microbiology and Hygiene, Mannheim
The effective control of the infection of mice with the
facultatively intracellular bacterium Listeria monocytogenes
requires CD8 T cells which recognize bacterial antigenic
peptides presented in the context of host MHC class I
molecules. It is generally accepted that bacterial antigens are
processed by the proteasome, a proteolytic cytoplasmic
multiprotein complex. We observed that presentation of certain
L. monocytogenes-derived CD8 T cell epitopes by infected cells
can not be totally suppressed by inhibitors of the proteasome.
Further analysis revealed that also inhibitors of the cytoplasmic
tripeptidyl protease II suppressed the presentation of L.
monocytogenes-derived CD8 T cell epitopes. Total, synergistic
inhibition of bacteria-derived CD8 T cell epitopes occurred in
the simultaneous presence of inhibitors of the proteasome and
the cytoplasmic tripeptidyl protease II. Our data clearly indicate
that both, the proteasome and other cytoplasmic proteases play a
role in the processing of L. monocytogenes-derived antigenic
Astrocyte gp130-expression is critical for the survival of
murine Toxoplasma encephalitis
U. Helmuth1, M. Deckert2, K. Drögemüller1
M. Sakowicz-Burkiewicz1, D. Reinhold3, D. Gutmann4
W. Müller5, D. Schlüter1
OvG University of Magdeburg, Medical Microbiology
Magdeburg, Germany
University of Cologne, Neuropathology, Cologne, Germany
OvG University of Magdeburg, Immunology, Magdeburg
Washington University School of Medicine, Neurology
St. Louis, USA
Helmholtz Center for Infection Research, Experimental
Immunology, Brunswich, Germany
In response to murine Toxoplasma encephalitis (TE), there is
robust astrocyte activation; however their in vivo function is
largely unknown. To study their role in TE, we generated mice
deficient in astrocytic expression of gp130 (GFAP-Cre
gp130fl/fl), the signal transducing receptor for cytokines of the
IL-6 family. These mice were significantly impaired in control
of T. gondii ultimately dying of a necrotizing TE. While GFAP+
astrocytes of infected gp130fl/fl control mice were activated and
increased in number, which was associated with effective
parasite control, containment of inflammatory lesions and
survival of TE, GFAP-Cre gp130fl/fl mice lossed GFAP+
astrocytes during TE. In vitro, survival of T. gondii-infected,
LPS- and TNF-stimulated astrocytes was also gp130-dependent.
Despite loss of astrocytes, the intracerebal production of
chemokines, cytokines, anti-parasitic effector molecules, and
recruitment of leukocytes was unimpaired in TE of GFAP-Cre
gp130fl/fl mice. These findings together with the observation that
in experimental autoimmune encephalomyelitis astrocyte
survival and control of the intracerebral autoimmune attack was
also gp130-dependent, show that astrocytes are of crucial
importance in TE and EAE and that gp130 is a critical survival
factor of astrocytes.
Innate natural killer cell response in cutaneous and visceral
Leishmaniasis requires toll-like receptor 9-mediated
Interleukin-12 production by myeloid dendritic cells
U. Schleicher1, J. Liese1, S. Kunz1, C. Kurzmann1, C. Bogdan1,2
University Clinic of Freiburg, Medical Microbiology and
Hygiene, Freiburg, Germany
University Clinic of Erlangen, Clinical Microbiology
Immunology and Hygiene, Erlangen, Germany
Natural killer (NK) cells are rapidly activated during the early
immune response to intracellular pathogens including
Leishmania and contribute to the control of the parasites. In
vitro studies suggested that the activation of NK cells during
infections not only results from the balance of stimulatory and
inhibitory signals by NK cell receptors, but depends on the
interaction with plasmacytoid (pDC) or myeloid dendritic cells
(mDC) that sense the pathogen and generate NK cellstimulatory signals. Here, we investigated the cell types, pattern
recognition receptors and cytokines that are required for NK cell
activation during Leishmania infections in vivo.
Plasmacytoid dendritic cells (pDCs) exposed to Leishmania
promastigotes in vitro released both interferon (IFN)-alpha/beta
and interleukin (IL)-12 in a TLR9-dependent manner, although
they did not internalize the parasites. In contrast, myeloid
dendritic cells (mDCs) phagocytosed the parasites and produced
high amounts of IL-12, but not IFN-alpha/beta, in the presence
of TLR9. In vivo, Toll-like receptor (TLR) 9 was indispensable
for NK cell IFN-gamma production and cytotoxicity in both
cutaneous (L. major) and visceral leishmaniasis (L. infantum).
Studies with IL-12- or IFN-alpha/beta receptor (IFNAR)knockout mice revealed that IFNAR-deficiency only slightly
reduced the L. infantum-induced IFN-gamma production by NK
cells, whereas NK cell cytotoxicity and IFN-gamma secretion
was completely abolished in IL-12-deficient mice. The latter
phenotype was also observed in mDC-depleted mice, whereas
NK cell activation in response to L. infantum was maintained
after depletion of pDCs. Intracellular cytokine staining showed
that mDCs represent the source of IL-12 during the early phase
of Leishmania infection. TLR9-deficient mice lacked IL-12
expression by mDCs after infection with L. major or L.
infantum. Thus, NK cell priming in vivo in response to
Leishmania parasites is linked to mDCs, which sense the
pathogen via TLR9 and subsequently deliver IL-12 to stimulate
the NK cells.
Candida albicans and Candida-derived cell wall components
stimulate toll-like receptor and cytokine expression in
human keratinocytes
J. Wagener1, G. Weindl1, P. W. de Groot2, A. de Bör3
M. Weig3, M. Schaller1
Eberhard-Karl-University Tuebingen, Department of
Dermatology, Tuebingen, Germany
University of Amsterdam, Swammerdam Institute of Life
Sciences, Amsterdam, Neterlands
Georg-August-University Goettingen, Department of Medical
Microbiology, Goettingen, Germany
Rapid immune response in Candida infections is mediated in
part by a family of innate recognition molecules known as tolllike receptors (TLRs). TLRs recognise conserved motifs called
pathogen-associated molecular patterns (PAMPs), which
represent broad groups of microbial pathogens or components.
TLR signalling pathways trigger subsequent inflammatory
responses which are crucial for successful host defence against
pathogens. Fungal cell wall components such as beta-glucan and
mannoproteins have been shown to stimulate the innate immune
response in myeloid cells in a TLR-dependent manner,
particularly through TLR2 and TLR4. However, Candida
albicans cell wall components that specifically induce TLR
responses in keratinocytes have not yet been investigated in
In our studies we first analysed the TLR expression of human
keratinocytes after exposure to viable and heat-inactivated C.
albicans yeast cells by quantitative RT-PCR. Stimulation with
viable C. albicans cells showed increased TLR4, TLR9 and
TLR10 mRNA levels in a time and concentration-dependent
manner, associated with increased expression and production of
proinflammatory cytokines, analysed by RT-PCR and ELISA.
In contrast, heat-inactivated C. albicans yeast cells showed only
a weak upregulation of TLR4 and TLR9 mRNA and failed to
induce a cytokine response. In addition, we examined the effect
of different cell wall extractions from C. albicans on TLR gene
expression and found a slight increase of TLR4 and TLR10,
accompanied with an induction of GM-CSF and IL-8 levels
comparable to those observed after treatment with viable cells.
However, different wild type cell wall extractions and cell wall
extractions from C. albicans cell wall mutants showed no major
differences in the TLR expression pattern and cytokine release.
In conclusion, these results indicate that distinct TLRs and
possibly other pattern recognition receptors together trigger
innate immunity in human keratinocytes by recognising
different structures of C. albicans. The similar immune response
to different cell wall extractions suggests that specific ligands
are present in all layers of the fungal cell wall. Furthermore, we
speculate that C. albicans possesses, but as yet unidentified,
TLR ligands which are recognised by TLR9 and TLR10.
Clinical relevance of serum antibody responses elicited by
nasal colonization with Staphylococcus aureus.
S. Holtfreter1, T. T. H. Nguyen1, H. Wertheim2, P. Eichler3
T. T. H. Le4, H. Kusch5, K. Eske1,6, Q.P. Truong4, K. Roschack1
L. Steil4, M. Hecker5, S. Engelmann5, A. van Belkum2
U. Völker4, B. Broeker1
University of Greifswald, Department of Immunology
Greifswald, Germany
University Medical Center Rotterdam, Department of Medical
Microbiology and Infectious Diseases, Rotterdam, Germany
University of Greifswald, Department of Transfusion Medicine,
Greifswald, Germany
University of Greifswald, Department of Functional Genomics
Greifswald, Germany
University of Greifswald, Institute of Microbiology, Greifswald
University of Greifswald, Friedrich-Loeffler-Institute of
Medical Microbiology, Greifswald, Germany
Colonization by Staphylococcus (S.) aureus increases the risk of
nosocomial S. aureus infection. Surprisingly, the outcome of S.
aureus bacteraemia is much better in carriers than in noncarriers. We therefore propose that individuals colonized of with
S. aureus mount an immune response which partially protects
them in the case of infection.
To test this hypothesis, we first used staphylococcal
superantigens (SAg) as indicator antigens because of their high
prevalence and strain variability. Sera from S. aureus carriers
very efficiently inhibited the T cell proliferation induced by the
SAgs of their colonizing strain. In contrast, their neutralizing
serum capacity for secretion products of S. aureus strains with
different SAg genes was much lower and it did not differ from
that of non-carriers. These results show that S. aureus carriers
show a very effective and highly specific neutralizing antibody
response against the SAgs of their commensal strain.
This raises the question, whether colonization with S. aureus per
se is a potent trigger of an adaptive immune response. To
investigate this, we colonized 16 healthy volunteers with the S.
aureus strain 8325-4, which was selected because of its low
virulence, and obtained serum samples before and four weeks
after colonization. The secreted staphylococcal proteins were
separated by two-dimensional gel electrophoresis and
immunoblots with the human sera were produced. Consistent
with our results concerning anti-SAg antibodies, we observed a
large inter-individual variability in the antibody profiles against
S. aureus 8325-4 already before experimental colonization. The
healthy volunteers harboured antibodies directed against a broad
range of extracellular S. aureus proteins, which are likely due to
previous encounters with S. aureus. Only rarely we observed
additional antibody signals or increased signal intensities after
experimental colonization with S. aureus. This shows that short
term nasal colonization with a laboratory strain does not trigger
strong antibody responses to S. aureus. We conclude that the
high antibody titres observed in most healthy individuals, in
particular in S. aureus carriers, require either long lasting
contact with S. aureus or, most likely, minor infections as they
commonly occur with this microorganism, especially with
strains of higher invasive potential.
A molecular mechanism of serum resistance of Borrelia
spielmanii sp. nov.: Binding of immune regulators factor H
and FHL-1 to complement regulator-acquiring surface
P. Herzberger1, C. Siegel1, C. Skerka2, V. Fingerle3
U. C. Schulte-Spechtel3, B. Wilske3, V. Brade1, R. Wallich4
P. F. Zipfel2, P. Kraiczy1
University Hospital , Institute for Medical Microbiology und
Krankenhaushygiene, Frankfurt am Main, Germany
Leibniz-Institute, Department for Infection Biology, Jena
Ludwig-Maximilians-University Munich, Max von PettenkoferInstitute for Medical Microbiology und Hygiene, Munich,
University Heidelberg, Institute for Immunology, Heidelberg
B. spielmanii sp. nov. has recently been identified as a novel
human pathogenic genospecies that cause Lyme disease in
Europe. In order to elucidate immune evasion mechanisms of B.
spielmanii as a means of evading the innate immune system we
have compared the ability of isolates obtained from Lyme
disease patients and tick isolate PC-Eq17 to escape from
complement-mediated bacteriolysis. Applying a growth
inhibition assay, we show that four B. spielmanii isolates,
including PC-Eq17, are serum-resistant whereas a single isolate,
PMew, was more sensitive to complement-mediated lysis. All
isolates activate complement in vitro as demonstrated by
covalent attachment of C3 , however, deposition of later
activation products C6 and C5b-9 was restricted to the
moderately serum-resistant isolate PMew and serum-sensitive
B. garinii isolate G1. Furthermore, serum adsorption
experiments revealed that all B. spielmanii isolates acquire the
host alternative pathway regulators factor H and FHL-1 from
human serum. Both complement regulators retain their factor Imediated C3b inactivation activity when bound to spirochetes.
In addition, two distinct factor H and FHL-1 binding proteins,
BsCRASP-1 and BsCRASP-2, were identified that are
approximately 23 to 25 kDa in size. A further factor H-binding
protein, BsCRASP-3, was exclusively found in the tick isolate
PC-Eq17. In conclusion, this is the first report describing an
immune evasion mechanism utilized by B. spielmanii sp. nov.
and demonstrates capture of human immune regulators to resist
complement-mediated killing. This work was funded by the
Deutsche Forschungsgemeinschaft DFG, Project Kr3383/1-1
and Wa533/7-1.
Immune escape of the Lyme disease spirochete Borrelia
spielmanii sp. nov.: Characterisation of the plasmid-encoded
complement regulator-acquiring surface protein-1 that
binds human immune regulators factor H and FHL-1
P. Herzberger1, C. Siegel1, C. Skerka2, V. Fingerle3
U. C. Schulte-Spechtel3, B. Wilske3, V. Brade1, R. Wallich4
P. F.Zipfel2, P. Kraiczy1
University Hospital, Institute for Medical Microbiology and
Hygiene, Frankfurt am Main, Germany
Leibniz-Institute, Department for Infektion Biology, Jena,
Ludwig-Maximilians-Universität München, Max von
Pettenkofer-Institut für Medizinische Mikrobiologie und
Hygiene, München, Germany
Universität Heidelberg, Institut für Immunologie, Heidelberg
Borrelia spielmanii sp. nov., one of the etiological agents of
Lyme disease found in Europe escapes complement-mediated
killing by recruitment of immune regulators factor H and FHL-1
from human serum. Serum-resistant and intermediate serumresistant isolates express up to three distinct complement
regulator-acquiring surface proteins (CRASPs) that bind to
factor H and/or FHL-1. Here we report identification and
functional characterization of BsCRASP-1, the dominant factor
H and FHL-1 binding protein of B. spielmanii. BsCPASP-1 is a
27.7 kDa outer surface lipoprotein, which after processing is
predicted to be 24.9 kDa. The gene encoding BsCRASP-1 is a
single genetic locus that maps to a linear plasmid of
approximately 55 kb. Ligand affinity blot techniques revealed
that both, native and recombinant BsCRASP-1 from different
isolates, bind to FHL-1 and weaker to factor H. Deletion
mutants of BsCRASP-1 were generated and a high affinity
binding site for factor H and FHL-1 was mapped to a 10 amino
acid residue domain at the C-terminus of BsCRASP-1.
Similarly, the predominant binding site of factor H and FHL-1
was localized to short consensus repeats 5 to 7. Factor H and
FHL-1 maintain their cofactor activity for factor I-mediated C3b
inactivation when bound to full-length BsCRASP-1 but not to a
deletion mutant lacking more than 10 amino acid residues at its
C-terminus. In conclusion, BsCRASP-1 binds the host immune
regulators factor H and FHL-1 with high affinity and is the key
molecule of the spirochetes’ complement resistance. Thus,
BsCRASP-1 most likely contributes to persistence of B.
spielmanii and to pathogenesis of Lyme disease. This work was
funded by the Deutsche Forschungsgemeinschaft DFG, Project
Kr3383/1-1 and Wa533/7-1.
Commensal Bifidobacterium adolescentis prevents
dissemination of Y. enterocolitica infection in vivo by a
TLR2 dependent mechanism
J. - S. Frick1, F. Kahl1, K. Fink1, J. Geisel1, M. Müller1
C .J. Kirschning2, I. B. Autenrieth1
University Tuebingen, Medical Microbiology and Hygiene
Tuebingen, Germany
Technical University Munich, Institute for Medical
Microbiology Immunology and Hygiene, Munich, Germany
An increasing body of evidence suggests that probiotic bacteria
are effective in the treatment of enteric infections although the
molecular basis of this activity remains elusive. In previous
studies we identified a Bifidobacterium adolescentis strain that
suppressed the Yersinia enterocolitica induced inflammatory
host response in vitro. This B. adolescentis strain was
administered to C57BL/6 mice, orally infected with Y.
enterocolitica. Interestingly B. adolescentis fed mice had
significantly lower mean pathogen burden in visceral organs,
decreased intestinal TNF-alpha mRNA expression and loss of
bodyweight upon oral infection with Y. enterocolitica. This
protective effect was abolished in TLR2-/- mice, indicating that
the protective effect of B. adolescentis is mediated via TLR2
dependent signalling. Mice deficient for the DC homing factor
CCR7 were still protected from dissemination of Y.
enterocolitoca infection indicating that rather epithelial cells
than DC contribute to the modulation of Y. enterocolitica
infection by administration of B. adolescentis. This is in line
with the finding that activation and maturation as well as DC
phenotype did not differ between B. adolescentis treated and
only Y. enterocolitica infected mice. Ongoing experiments
investigate whether the protective effect of B. adolescentis can
be related to an increased expression of tight junction proteins
and an enhanced integrity of the intestinal epithelial layer. We
suggest that B. adolescentis increases the resistance of the
intestinal barrier and thereby impedes the translocation of Y.
enetrocolitica and the dissemination of Y. enterocolitica
CD8+ T-cells modulate abscess formation induced by
bacterial polysaccharides
M. Fabri1, A. Zingarelli1, E. Flenner1, M. Bessler1, H. Hafke1
W. Kalka-Moll1
University of Cologne, Microbiology, Cologne, Germany
Polysaccharides have been classically considered T-cellindependent antigens. In contrast, zwitterionic capsular
polysaccharids (ZPS) from bacteria such as Bacteroides fragilis,
Staphylococcus aureus and Streptococcus pneumoniae induce
the formation of intraabdomial abscesses in a CD4+ T cell- and
MHC class II-dependant manner. ZPSs are characterized by
having both positively and negatively charged substitutans on
each repeating unit of a highly repetitive structure. Previous
investigations on ZPS-induced intraabdominal abscesses have
exclusively focused on the role of CD4+ T cells. Herein, we
sought to elucidate the immune response of CD8+ T cells to
ZPSs. We characterize the function of CD8+CD28- T cells in a
experimental model of ZPS-induced abscess formation in vivo
and in vitro. Moreover, we provide evidence that ZPSs modulate
CD8+ T cell activation by a previously unknown mechanism
implying enhanced cross-linking of TCRs on CD8+ T cells.
These data provide substantial new insights in the unique
functions of bacterial ZPSs to induce and regulate T celldependent intra-abdomial abcsess formation.
Panchlamydia PCR for detection and identification of
chlamydia trachomatis, chlamydophilia pneumoniae, and
chlamydophilia psittaci by LightCycler PCR
T. Juretzek1, I. Buder1, W. Bär1
Carl-Thiem-Clinic, Microbiology, Cottbus, Germany
The aim of this study was to evaluate the performance of a
panchlamydia real time PCR for screening of the human
pathogenic Chlamydia, such as Chlamydia trachomatis,
Chlamydophilia psittaci, Chlamydophilia abortus and
Chlamydophilia pneumoniae in clinical specimen. This study
included patients with respiratory affects, lung cancer, neonates
with respiratory affects with possible participatipon of C.
trachomatis or C. pneumoniae, patients from the ophthalmology
clinic, gynaecology, urology as well as pregnant women were
examined. In total3281 Specimens were routinely investigated.
0.96 % (16/1662) of adults with respiratory affects were infected
by C. pneumoniae, 0.3 % (5/1662) with C.psittaci.5.8 %
(34/581) of the pregnants carried C. trachomatis and 0.3 %
(2/581) C.abortus. 2.2 % (8/368) of the neonates were infected
by C .trachomatis 0,27% (1/368) with C pneumoniae. 2.8 %
(18/625) of the children carried C.pneumoniae in the respiratory
tract. 6.6 % (3/45) C. trachomatis were detected in swab of eges.
The melting curve analysis feature of the Lightcycler allowed
identification of C. trachomatis and C. pneumoniae directly,
whereas C. psittaci and C. abortus were confirmed by 16sRNA
gene sequencing. The Panchlamydia PCR detecteds all 4 abovementioned chlamydia within 3 to 4 h. Due to this PanChlamydia
PCR it was possile to reduce the cost for PCR and also rare
found bacteria like C. abortus or C. psittaci were detected.
Prolonged EHEC excretion by a girl and her domestic cat
indicating a possible infection cycle between humans and
U. Busch1, S. Hörmansdorfer1, S. Schranner2, I. Huber1
K. - H. Bogner1, A. Sing1
Bavarian Health and Food Safety Authority, Oberschleissheim
Landratsamt Landshut, Landshut, Germany
Enterohemorrhagic Escherichia coli (EHEC) can cause severe
hemorrhagic colitis characterized by gastrointestinal symptoms
and bloody diarrhea as well as hemolytic uremic syndrome
(HUS). Cattle and small ruminants are the major natural
reservoir of these food-borne pathogens. Besides this foodrelated route of transmission, human infections might develop
after direct contact to cows, goats, sheep and deer. Although
domestic dogs and cats are known as rare EHEC carriers, no
human EHEC infections associated with pet contact have been
reported. Here we present the first case of an EHEC strain of
serotype O145:H- infecting both a child and her domestic cat
over a prolonged period of time.
Bacteriology of biopsy specimens of nasal mucosa and nasal
lavages of patients with Chronic Rhinosinusitis
A. Niederführ1, H. Kirsche1, H. Riechelmann1
N. Wellinghausen2
University Hospital, Otorhinolaryngology, Ulm, Germany
University Hospital, Institute of Medical Microbology and
Hygiene, Ulm, Germany
The pathogenesis of Chronic Rhinosinusitis (CRS) is still
unknown. Aim of this study was to determine the bacteriology
of middle meatal biopsy specimens in comparison to nasal
lavages of patiens with CRS. Special attention was drawn to
Staphylococcus aureus and S. aureus Small Colony Variants
37 CRS patients, including 27 with nasal polyps (CRSNP+), 10
without polyps (CRSNP-) and 11 control patients with septal
deviation without inflammation of the mucosa were
investigated. Detection of the bacteria was done by culture,
including cultures for SCV. Additionally, S. aureus-specific
quantitative LightCycler PCR and Fluorescence in situ
Hybridization (FISH) was done from the biopsy specimens.
Bacteria cultured from biopsy specimens in CRS patients
included mainly staphylococci, streptococci, corynebacteria,
Enterobacteriaceae, Haemophilus spp. and anaerobes. 75 of 123
strains (61%) cultured from biopsies were simultaneously found
in the corresponding lavage. Bacteria cultured exclusively in
biopsy specimens of CRS patients (n=48) included mainly
Propionibacterium spp., Peptostreptococcus spp., E. coli,
Haemophilus spp., and alpha-haemolytic streptococci. S. aureus
was cultured in biopsy specimens in 22% of CRSNP+ patients,
10% CRSNP- patients and 18% of controls. In all patients
simultaneous growth in the nasal lavage was found. 6 of 38
samples investigated were positive in the S. aureus-PCR and in
all of them culture revealed growth of S. aureus. Only 1 of 37
paraffin sections was positive in the FISH. S. aureus SCV were
not detected in any specimen. Altogether, there were no
significant differences in the species spectrum between patients
and controls.
Bacterial culture is the most sensitive method to detect S. aureus
from biopsy specimens and nasal lavages. PCR and FISH do not
increase sensitivity and are, thus, not necessary for routine
workup of specimens. Nasal lavage is a sensitive method to
determine prevalence of S. aureus in patients with CRS. There is
a tendency towards a lower prevalence of S. aureus in CRSNPpatients. S. aureus SCV were not detectable in the 37 CRSpatients investigated. The significance of bacteria cultured
exclusively in biopsies of CRS patients will be further
determined in the ongoing study.
Identification of various CTX-M extended-spectrum lactamases as the leading cause of cephalosporin resistance
of nosocomial Escherichia coli in a German hospital
Y. Pfeifer1, A. Danschke1, H. von Baum2, W. Witte1
Robert-Koch-Institute, Nosocomial Infections, Wernigerode
University of Ulm, Institute of Microbiology and Hygiene
Ulm, Germany
ESBL enzymes of the Ambler class A like SHV and TEM ßlactamases have evolved from plasmid-located enzymes by
mutations resulting in amino acid substitutions and leading to
the expansion of substrate spectrum. Since the late 1990s
diverse ESBL-types of the CTX-M-group occur with increasing
frequency in Enterobacteriaceae in hospital and community
settings worldwide.
For this study 22 isolates of E. coli were collected from a
hospital during a period of eight months in 2006. The strains
were isolated from urine (59%), tracheal secretions (14%),
sputum (9%), wounds (9%) and fecal smears (9%). Phenotypical
susceptibility testing for 3rd and 4th generation cephalosporines
with and without inhibitor protection (microbroth MIC) was
performed. Relevant resistance genes (blaTEM, blaSHV, blaCTX-M)
were amplified by Multiplex-PCR and the amplicons were
sequenced. All isolates were typed by pulse-field gelelectrophoresis (PFGE). Furthermore PCR for phylogenetic
grouping, plasmid analysis and conjugation experiments were
All isolates displayed the ESBL-phenotype. Furthermore the
isolates were resistant to fluoroquinolones and sulfonamides and
most of them were resistant to aminoglycosides and
tetracyclines as well. PCR analysis revealed no “classical”
outbreak situation. Different ESBL genes occurred: One isolate
harbored SHV-5. Among the CTX-M-group ESBL six different
types have been identified. ESBL type CTX-M-15 (n = 12) was
most frequently. One isolate exhibited a CTX-M-type not yet
described (designated as CTX-M-65). The majority of isolates
were attributed to phylogenetic group D. When subjected to
pulse-field gel electrophoresis only eight of 22 isolates exhibited
the same or similar patterns. In one case in which two patients
were hospitalized in the same room clonal dissemination is
likely. The completely different macrorestriction patterns of the
remaining isolates and conjugation experiments are proof for the
wide spread of these ESBL genes and other resistance
determinants by horizontal gene transfer.
Clinical and microbiological characteristics of melioidosis in
Northern Vietnam
T. T. Trung1, D. M. Phuong2, N. Meukow1, K. Breitbach1
N. Q. Tuan3, G. Flunker1, D. D. Khang4, N. X. Quang2
I. Steinmetz1
University of Greifswald, Friedrich Loeffler Institute of Med.
Microbiology, Greifswald, Germany
Bach Mai Hospital, Department of Microbiology, Hanoi
Bach Mai Hospital, Department of Infectious Diseases, Hanoi
Vietnamese Academy of Sciene and Technology, Institute of
Biotechnology, Hanoi, Vietnam
The worldwide distribution of melioidosis, an infectious disease
of the tropics and subtropics caused by the soil bacterium
Burkholderia pseudomallei, is still unknown. In Vietnam, only
very limited data on melioidosis are available for the indigenous
population, although the disease received considerable attention
amongst French colonials and American soldiers. In this
retrospective study, we reviewed clinical and demographic data
of patients with culture-proven melioidosis diagnosed at a single
large referral hospital in Hanoi between November 1997 and
December 2005. There was a striking seasonal accumulation
with a peak of cases admitted during the wet season between
June and September. In 81.6% of melioidosis patients, the
primary diagnosis was septicemia and B.pseudomallei was
isolated from the blood. In 57.5% of septicemic patients, clinical
evidence for the potential source of infection was obtained. 45%
of septicemic patients had clinical signs of respiratory tract
infection. The mortality rate of patients with positive blood
cultures was 42.5%. 48.9% of melioidosis patients had known
risk factors such as e. g. diabetes mellitus or renal disease. The
analysis of the geographical origin of melioidosis patients
showed that melioidosis exists in at least 18 provinces with a
population of more than 30 millions. The characterization of
clinical B.pseudomallei strains isolated between June 2002 and
December 2005 revealed the typical biochemical and antigenic
features of this species. ‘Multi Locus Sequence Typing’, based
on the analysis of 7 house keeping genes, identified a number of
sequence types not previously described. The melioidosis
patients seen at our hospital most likely represent the very tip of
the iceberg of cases in Northern Vietnam. In conclusion, our
study suggests that fatal septicemic melioidosis is widespread
but underdiagnosed in Northern Vietnam. Prospective studies
are needed to unravel the true prevalence of melioidosis and its
clinical presentation in Vietnam.
Emergence of increasing Linezolid-resistance in enterococci
in a post-outbreak situation with Vancomycin-resistant
B. Schulte1, A. Heininger2, I. B. Autenrieth1, C. Wolz1
Institute for Medical Microbiology und Hygiene
University Hospital Tuebingen, Tuebingen, Germany
Klinik für Anaesthesiologie und Intensivmedizin
University Hospital Tuebingen, Tuebingen, Germany
Linezolid is an oxazolidinone antibiotic active against grampositive bacteria approved in Europe in 2001. Here we describe
the emergence of linezolid-resistant enterococci (LRE) in a
university hospital in which linezolid is in use since 2003. In
2004 and in the beginning of 2005 the hospital was affected by
an extensive outbreak of vancomycin-resistant enterococci
(VRE). Linezolid-resistant enterococci emerged during this
period. Due to this outbreak situation, usage of linezolid became
more common also for non-VRE-infections because of good
clinical experiences in treatment. Subsequently, linezolidresistance was not longer limited to VRE. Additionally,
nosocomial spread of linezolid-resistant but vancomycinsusceptible Enterococcus faecium was detected. These strains
now emerged also in patients without prior drug exposure.
Molecular and microscopic epidemiology of intracellular
persisting Staphylococcus aureus in tonsillar epithelial cell
specimens from patients with recurrent tonsillopharyngitis
A. E. Zautner1, M. Krause2, M. Rohde3, A. Podbielski1
Institute for Medical Microbiology, Virologie und Hygiene
Rostock, Germany
Klinik und Poliklinik für Hals-Nasen-Ohrenheilkunde, Rostock
Helmholtz-Center for Infection Research, Brunswich
Treatment failures in acute tonsillopharyngitis and the
occurence of recurrent tonsillopharyngitis have been associated
with the pathogens' formal capability for intracellular
In a prospective study, pre-surgery tonsil swabs and surgically
removed tonsil specimen from 45 / 77 juvenile and adult
patients with chronic recurrent tonsillopharyngitis have been
examined for the presence of extra- and intracellular pathogenic
bacteria. As negative controls served 5 tonsil specimens from
habitual snorers and patients with uvula hyperplasia. The
specimens were conventionally cultured, subjected to antibiotic
protection assays and inspected by fluorescence microscopy
following a selective double immuno stain as well as by high
powered scanning electron microscopy. Staphylococcus aureus,
Haemophilus influenzae and Streptococcus pyogenes were
isolated from 58,2%, 17,2% and 9,8% of all tonsillopharyngitis
patients, respectively. Thus, Staphylococcus aureus was the
predominant pathogenic species in chronic recurrent
tonsillopharyngitis. In accordance with the general antibiotic
resistance pattern of German Staphylococcus aureus strains,
67,6% of tonsil-associated isolates displayed a penicillin
resistance. When comparing the isolates with pulsed field
restriction analysis and different typing methods, a high degree
of diversity could be demontrated among Staphylococcus aureus
and Streptococcus pyogenes strains. Based upon the antibiotic
protection assay results, only three Staphylococcus aureus
strains were exclusively located outside the eukaryotic cells.
The remainder displayed at least partial intracellular location.
The results were confirmed by results from the double immuno
staining assays. Overall, intracellular location and penicillin
resistance of Staphylococcus aureus play obviously a pivotal
role in the etiology and treatment failure of recurrent
Antimicrobial susceptibility of Escherichia coli from wild
S. Günther1, H. Kasper2, S. Schwarz3, J. Jores4, J. Eichberg1
L. H. Wieler1, P. Schierack1
Institute of Microbifology and Epizootics, Berlin, Germany
Bundesamt für Verbraucherschutz und Lebensmittelsicherheit
Berlin, Germany
Institut für Tierzucht, Bundesforschungsanstalt für
Landwirtschaft, Neustadt-Mariensee, Germany
International Lifestock Research Institute, Nairobi, Kenia
While pathogenic as well as commensal Escherichia (E.) coli
from domestic pigs have been studied extensively for
antimicrobial resistance and associated genes, the corresponding
knowledge about E. coli from wild boars is scarce. We therefore
compared E. coli from wild boars with E. coli from clinically
healthy conventionally pigs (piglets and sows) for their
antimicrobial resistance and the resistance genes present.
Initially, 300 isolates from 10 wild boars and 538 isolates from
15 conventional piglets were assigned to different groups by
macrorestriction analysis. Subsequently, representatives of these
groups were tested for antimicrobial susceptibility by
determination of the minimum inhibitory concentration (MIC)
and for resistance genes by PCR (45 E. coli from wild boar / 49
from conventional pigs).
All of the E. coli strains from wild boars were susceptible
against all tested antibiotics. In contrast, 71% of the tested E.
coli strains from healthy domestic piglets were resistant to
spectinomycin, tetracycline or trimethoprim/sulfamethoxacole
though several respective antibiotics were not applied for long
periods to this pig population. Additionally, also MICs of the
susceptible E. coli population were significantly lower in those
E. coli from wild boars indicating a lower effectiveness of the
involved protection mechanisms. None of the E. coli strains
from wild boars carried any of the resistance genes tested. In
contrast, E. coli strains from healthy conventional piglets carried
one to six different resistance genes, with the tetracycline
resistance gene tet(A) and the spectinomycin/streptomycin
resistance gene aadA2 being the most prevalent. Additional
screening of fecal samples from ten conventional sows by real
time PCR proved the existence of two to four different
resistance genes in one DNA sample.
In conclusion, the data confirm that E. coli from wild boars
might lack resistance genes and do not express resistance
phenotypes in contrast to from conventional pigs.
Susceptibility of Staphylococcus saprophyticus subsp.
M. Kaase1, F. Szabados1, T. Sakinc1, S. Friedrich1, B. Kleine1
S. Polywka2, S. Gatermann1
Ruhr-University Bochum, Department of Medical
Microbiology, Bochum, Germany
University Hospital Hamburg-Eppendorf, Institute for Medical
Microbiology, Virologie und Hygiene
Staphylococcus saprophyticus is the second most frequent
pathogen in uncomplicated urinary tract infection of female
outpatients. Only few resistance data exist.
A strain collection of 242 isolates which were phenotypically
saprophyticus were tested for resistance against various
antibiotics by agar diffusion according to CLSI. In addition a
cloverleaf assay was performed for penicillinase detection.
No resistance was found against gentamicin, tobramycin,
kanamycin, nitrofurantoin, rifampicin, linezolid, minocyclin and
ciprofloxacin. Susceptibility rates were 5.4% for fosfomycin
applying the SFM breakpoint, 96.3% for chloramphenicol,
97.5% for sulfamethoxazole-trimethoprim and 92.6% for
tetracyclin. Erythromycin resistance with accompanying
clindaymcin resistance was found in 2.9%, erythromycin
resistance with an inducible MLSB phenotype in 4.5% and
isolated erythromycin resistance in 16.9%. A positive cloverleaf
assay indicating presence of a penicillinase was found in 55%,
an inhibition zone of less than 25 mm for cefoxitin indicating
oxacillin resistance was found in 5.4%. Surprisingly we found
two trehalose positive S. saprophyticus subsp. saprophyticus
strains for which identification was confirmed by sodA
S. saprophyticus subsp. saprophyticus shows susceptibility
against most antibiotics. Resistance against fosfomycin is
detected in almost all isolates as previously reported.
Erythromycin resistance is found in 24.4%. The presence of a
penicillinase is suggested by a positive cloverleaf assay in 55%
of isolates.
Rare cases of nonthyphoidal Salmonella associated
pulmonary infection in immunocompromised patients
R. Schwarz1, J. Matten1
Colonge Merheim Medical Center, Medical Microbiology
Cologne, Germany
Pulmonary infection with nontyphoidal Salmonella is very
uncommon. An increased risk is associated with HIV, diabetes
mellitus, prolonged corticosteroid therapy, alcohol abuse and
neoplastic diseases. Definitive microbiological conformation
from pulmonary specimens is unusual. Here we describe three
cases of nonthypoidal Salmonella pulmonary infections in
febrile patients with lung cancer without any signs of
gastrointestinal symptoms. The organisms were isolated from
the sputum, pleural fluid, blood cultures and the stool. All of the
isolates wereidentified as Salmonella entrica serotype
Typhimurium. After administration of appropriate and adequate
treatment the patients were subsequently discharged from our
hospital without any complications.
Combined Moxifloxacin and Rifampin treatment of
experimentally induced Staphylococcus aureus
periprosthetic osteomyelitis: An animal study
W. Silvis1, J. Schaumburger2, T. Kalteis2, N. Lehn1
University of Ratisbon, Medical Microbiology & Hygiene
Ratisbon, Germany
University of Ratisbon, Orthopaedic Surgery, Bad Abbach
Background: Standard antibiotic therapy forperiprosthetic
osteomyelitisdue to Staphylococcus aureus (SA) is dual therapy
comprising Rifampin (RIF). Previously, we demonstrated
Moxifloxacin (MOX) to be superior to vancomycin (VAN) for
treatment of SAperiprosthetic osteomyelitisin a rat model. MOX
was now compared to dual therapies MOX + RIF and VAN +
RIF and monotherapies VAN and RIF.
Methods:Periprosthetic osteomyelitisof the hind leg femur was
surgically induced in 53 male Wistar rats: 107 colony forming
units (cfu) of methicillin-sensitive SA strain ATCC 29213 were
inoculated into the medullary cavity and a steel implant was
inserted. After 7 days, rats were randomised to intraperitoneal
administration of placebo, MOX 10 mg/kg b.i.d., RIF 20 mg/kg,
VAN 60 mg/kg b.i.d., MOX + RIF or VAN + RIF. After 14
days of treatment, periprosthetic femur, capsular soft tissue of
knee joint and implant were aseptically explanted and cultured
quantitatively. Differences in remaining bacterial burden (cfu
SA/ material) were analysed by ANOVA and Tukey HSD posthoc test.
Results: Treatment results are shown in the table. Significant
differences (p<0.05) to placebo (*) and MOX (#) are indicated.
Conclusion: Dual therapy MOX + RIF was equivalent to VAN
+ RIF and showed higher efficacy than MOX or VAN
monotherapy. M seems a promising new partner for dual
therapy with RIF. Further studies comparing MOX + RIF to
dual therapy betalactams + RIF are under way.
Table 1:
Phenotypic Oxacillin resistance in a Staphylococcus aureus
blood culture isolate lacking mecA
M. Kaase1, A. Anders1, F. Szabados1, S. Friedrich1, T. Sakinc1
B. Kleine1, S. Gatermann1
Ruhr-University Bochum, Department of Medical
Microbiology, Bochum, Germany
We report the case of a 78-year-old male patient from whom
Staphylococcus aureus could be isolated in several blood culture
pairs over two months. About seven weeks after the initial
isolation of methicillin-susceptible S. aureus in six blood culture
pairs we isolated three morphologically clearly distinguishable
S. aureus strains from another blood culture pair.
Of these, isolate I was methicillin-susceptible whereas isolates II
and III showed oxacillin resistance using the VITEK AST-P536
card. Presence of the mecA gene was excluded either by PCR
and by hybridization. Performing agar diffusion a shade of
growth could be detected in the oxacillin inhibition zone which
could be inhibited by placing an amoxicllin/clavulanic acid disk
near the oxacillin disk. Growth on oxacillin screening agar was
detectable for isolates II and III only after 48 h incubation and
the cefoxitin inhibition zone diameter for both isolates was well
above the current threshold. Pulsed field gel electrophoresis
gave identical banding patterns for all three isolates and showed
no relatedness to any currently circulating MRSA clones in our
Therefore we postulate a different mechanism for oxacillin
resistance than expression of PBP2a as in usual MRSA isolates
and suspect that overexpression of penicillinase might be
Evaluation of the new oxoid chromogenic Salmonella
medium mark II (OSCM II) for detecting Salmonella
species, including lactose positive Salmonella in human stool
J. Schmitt1, I. Massow2, J. Stringer3
Oxoid Biotechnik GmbH, Wesel, Germany
Med. VZ Dr.Riegel GmbH, Wiesbaden, Germany
Oxoid Ltd., Basingstoke, United Kingdom
During a clinical study 966 stool samples were examined for the
presence of Salmonella species by direct inoculation on SS
Agar, Rambach Agar and OSCM II. Within 55 positive samples
7 different serotypes were isolated. The sensitivities after 24
hours incubation were 41.8%, 27.3% and 47.3%, respectively,
and increased after 48 hours to 58.2%, 34.5% and 70.9%.
OSCM II is a highly selective medium which includes thenovel
inhibigen concept. So in contrast to the less selective Rambach
medium it could be used as a direct screening medium. In this
study about 50% of the Salmonella positive specimens were
detected after 24 hours and therefore the time to result was
greatly reduced.
Relapse of pneumocystis pneumonia in HIV patients after
beginning of HAART therapy
D. Bandt1, M. Kolditz2, P. Spornraft-Ragaller3
Medical Faculty/Technical University of Dresden, Inst. for
Virology, Dresden, Germany
University Hospital/Technical University of Dresden, Medical
Clinic 1, Dresden, Germany
University Hospital/Technical University of Dresden
Dermatology, Dresden, Germany
Two cases of HIV-infected patients with Pneumocystis jiroveci
pneumonia (PcP) are described. After initially successful
therapy followed by secondary prophylaxis, they suffered from
a relapse of PcP when HAART antiretroviral treatment was
initiated. The sequence of the DHPS gene was analysed. We
found polymorphisms in the sequence, but no known resistenceassociated mutations. Therefore, the relapse was assumed to be
linked to a combination of a recurrence of the infection and an
immune reconstitution syndrome (IRS). The risk factors for a
relapse of PcP and the significance of IRS are discussed.
Membrane permeability in the antibiotic resistance of
Proteus and Providencia
Q.-T. Tran1,2, H. Weingart1, A. Regli2, J.-M. Pagès2
M. Winterhalter1
Jacobs University Bremen, School of Engineering and Science,
Bremen, Germany
Université de la Méditerranée, Laboratoire UMR-MD1
Marseille, France
Proteus and Providencia are pathogenic bacterial agents
involved in hospital-acquired infection. They appear among the
most frequently isolated species in clinic and cause urinary tract
infection. The resistance generally described in these genera is
mainly due to enzymatic mechanisms, predominantly extendedspectrum beta-lactamases of the TEM type. These bacteria
present an intrinsic resistance to imipenem. Moreover, some
clinical isolates of Providencia demonstrate a multidrug
resistance phenotype with a very high level of resistance to
cephalosporins of the last generation such as cefepime and
cefpirome. These antibiotic molecules are known to use porins
as major pathway for cell entry. Our main research focus is on
the characterization of the outer membrane proteins of Proteus
and Providencia for a better understanding of their role in drug
resistance. We observed a negative detection of a special
antigenic site previously reported as internal loop3 marker of
non-specific porins in various Enterobacteriaceae. The
immunoblots using specific antibodies against porins showed
that Proteus and Providencia express OmpF- and OmpCimmunorelated proteins with a modified L3 loop. The porin
genes of Proteus showed about 50% identity with E. coli OmpF,
OmpC and PhoE. The amino acid sequence analysis showed a
high conservation of typical porin structure with an important
modification in the constriction region. These findings suggest a
special structure of the internal loop inside the porin channel of
those bacteria that may be involved in the resistance to
carbapenems and cephalosporins in Proteus and Providencia.
Severe endocarditis due to Streptobacillus moniliformis
W. Geißdörfer1, C. Schlundt2, T. Strecker3, M. Kondruweit3
M. Weyand3, W. G. Daniel2, C. Schörner1
University Erlangen, Institute forr Clinical Microbiology
Erlangen, Germany
University Hospital, Erlangen, Medical Clinic 2 (Cardiology)
Erlangen, Germany
University Hospital Erlangen, Center of Heart Surgery
Erlangen, Germany
Background: Streptobacillus moniliformis is the agent of rat bite
fever. It is found in the oropharynx of rats and can be
transmitted to human by rat bite, contact with rat excreta or
ingestion of contaminated food. The most common
manifestations are arthralgia, fever, and rash. Endocarditis is a
rare complication with only 19 reported cases worldwide, and a
fatality rate of 47 %.
Case: A 29-year-old patient was transferred to our department of
cardiology with the diagnosis of acute endocarditis. On
admission, TEE showed a large vegetation on the aortic valve
and a perivalvular abscess cavity. Aortic valve replacement was
performed immediately, but turned out to be challenging as a
consequence of perivalvular tissue destruction, necessitating a
further surgery for re-fixation of the loosend prosthetic aortic
valve. During the postoperative 7-week stay at our intensive
care unit the patient suffered several complications, i.e.,
postoperative renal and hepatic failure followed by
hemodialysis, dysphagia induced aspiration followed by
transient intubation and ventilation, septic cranial embolism, and
severe pericardial infection due to Candida glabrata. The
antibiotic regimen was adapted several times, and included
penicillin for S. moniliformis endocarditis. The patient was
finally discharged in a satisfactory condition to a rehabilitation
Microbiology: Blood cultures remained negative after 14 days
of incubation. Microscopic examination of the explanted aortic
valve revealed gram-positive/gram-variable bacteria, which
appeared as straight, curved, and filamentous rods. After 48 h
tiny colonies grew on sheep blood agar, showing long
filamentous gram-variable rods. The bacteria were identified as
S. moniliformis by analysis of the 16S rRNA gene sequence.
The identical sequence was amplified directly from the
explanted valve specimen. The S. moniliformis isolate did not
grow on chocolate agar, formed typical cottonballs in
thioglycolate broth supplemented with horse serum, and was
negative for catalase, oxidase, and indole. It was highly
susceptible to penicillins, cephalosporins, carbapenems,
aminoglycosides, quinolones, erythromycin, clindamycin, and
Conclusion: Our case illustrates that endocarditis due to S.
moniliformis is a life-threatening infection. There was no rat
bite, but our patient may have had contact to rat excreta when he
helped his parents with their farm work. In addition, he reported
a cut wound at one hand two weeks before admission, which
may have been the site of infection.
A case of sepsis after dog bite: Rapid identification of
Capnocytophaga canimorsus from blood culture via
pyrosequencing of 16S rRNA
E. I. Kaminski1, I. Seegmüller1, F. J. Meyer2, A. Dalpke1
S. Zimmermann1
University Heidelberg, Hygiene, Heidelberg, Germany
University Heidelberg, Cardiology, Heidelberg, Germany
A 56-year old female was transferred from her primary care
physician to our intensive care unit for fever, pneumonia and
renal failure. She had presented at primary care with a six weeks
history of a productive cough (white sputum) and generalized
weakness, but there had been no history of chest pain, dyspnea
or TB-contacts. She had been treated with levofloxacin. Medical
history revealed that the patient was a bird breeder (parakeets)
with no known allergies. Four days before admission she had
been bitten by a dog in the right hand. Two years earlier she had
been splenectomized for splenic abscess and had undergone
aortic valve replacement because of endocarditis with
Streptococcus oralis. There was no information on
pneumococcal vaccination.
On arrival treatment was switched to imipenem.
Blood cultures were positive and revealed slow growing
fastidious gramnegative rods. Pyrosequencing of the 16S rRNA
variable region 3 (V3) was performed and resulted in the
identification of Capnocytophaga canimorsus within 24 hours
after the positive blood culture signal. Antimicrobial
susceptibility testing happened to be time consuming due to the
slow growing nature of our strain. Imipenem treatment proved
successful and after five weeks of treatment she could be
transferred to an external hospital.
The early determination of C. canimorsus after dog bite in
patients with an increased risk (splenectomy) is essential for
adequate treatment and can be facilitated by pyrosequencing of
16s rRNA.
Helicobacter pylori infection in patients with Coronary
Heart Disease (CHD)
R. Krausse1, M. Hiller1, J. Leiendecker1
University Hospital S.-H, Institute for Infection Medicine, Kiel
Aim: To establish whether Hp is implicated in the development
of CHD.
Methods: Altogether the sera of 365 persons were analysed (186
patients [pat.], mean age 61 y with CHD [65 pat. had a control
angiography after PTCA; 30 with [w] restenosis, 35 without
[w/o] restenosis and 179 control persons, mean age 63 y without
any CHD). The patients were in-patient treatment at the clinic
for Cardiology; the control group was collected by an internist.
The sera were screened by ELISA (IgG/A) (Virion Serion). To
differentiate (Hp type I [VacA or CagA pos] and Hp type II
[VacA and CagA neg] a Western blot (WB) test (Viramed) was
lead through.
There was no significant correlation between the occurrence of
antibodies (Abs) and CHD, neither in the screening nor in the
WB. A small number of individuals (32 pat./18 contr.) who
were only positive for IgG-Abs showed differences in Hp types
(pat.: 46.9% Hp I and 53.1% Hp II; contr.: 100% Hp II).
Patients with restenosis showed significant higher IgA-Abs in
screening. However, these results could not be confirmed in
WB. Several sera were tested positive on both types of Hp, but
without any remarkable difference between the groups.
Conclusion: No correlation was found between the occurrence
of antibodies and CHD. There were no serological differences
between the patients with and without restenosis. An evidence
that Hp is a risk factor for CHD could not be found.
Table 1:
ELISA positive (%)
Patients (186)
Controls (179)
Pat. w
restenosis (30)
Pat. w/o
restenosis (35)
*p = 0,03
Patients (166)
Controls (145)
Pat. w
restenosis (30)
Pat. w/o
restenosis (35)
WB positive (%)
IgG + IgA
IgG + IgA
Hp I
Hp I
Hp I
Hp I +
Moxifloxacin as an adjunctive antibiotic in treatment of
periodontitis is effective in reduction of
periodontopathogenic bacteria
W. Pfister1, K. Wegner1, A. Güntsch2, H. Jentsch3
T. Hoffmann4, S. Eick1
University Hospital Jena, Institute for Medical Microbiology
Jena, Germany
University Hospital of Jena, Department of Conservative
Dentistry, Jena, Germany
University Hospital of Leipzig, Department of Conservative
Dentistry, Leipzig, Germany
University Hospital of Dresden, Department of Conservative
Dentistry, Desden, Germany
Objectives: In vitro studies demonstrated the high effectiveness
of moxifloxacin against periodontopathogenic bacteria. The aim
of this study was to compare the adjunctive use of moxifloxacin
to doxcycline in the treatment of severe chronic periodontitis.
Material and methods: In the randomised prospective study 92
patients underwent a standardized systematic periodontal
therapy. 35 patients got 400 mg moxifloxacin / d, 36 patients
doxycycline (1st day 200 mg, then 100 mg / d) over a time
period of 10 d per os, 21 controls got no antibiotic. Subgingival
plaque samples were collected with paper points in the three
sites with an attachment loss > 5 mm before scaling and root
planing as well as 3, 6 and 12 months later. Aggregatibacter
actinomycetemcomitans (A.a.), Porphyromonas gingivalis
(P.g.), Tannerella forsythia (T.f.) and Treponema denticola
(T.d.) were determined by real-time PCR. The cut-off was about
1,000 bacteria each.
Results: In the moxifloxacin group a significant reduction of the
load of all investigated bacteria was found at each appointment
in comparison to baseline. The number of negative samples
increased from 60% to 83% for A.a., from 20% to 64% for P.g.,
from 26% to 44% for T.f. and from 36% to 60% for T.d 12
months after treatment. In the doxycycline group a significant
decrease of the numbers of P.g. and T.f. was also observed 3, 6
and 12 months after treatment, T.d. was significant lower only 3
months after treatment. The numbers of A.a. did not change at
any time. In controls a significant reduction of the load of
periodontopathogenic bacteria was not detectable.
Conclusions: The adjunctive application of an antibiotic reduces
the load of periodontopathogenic bacteria in periodontitis.
Moxifloxacin seems to be more effective than doxycycline and
might be an alternative drug in treatment of severe cases of
chronic periodontitis.
Effect of antibiotic treatment on development of resistance
in periodontitis
S. Eick1, A. Güntsch2, T. Seltmann2, W. Pfister1
University Hospital of Jena, Institute of Medical Microbiology
Jena, Germany
U2niversity Hospital of Jena, Department of Conservative
Dentistry, Jena, Germany
Objective: In periodontitis usage of antibiotics is common in
treatment. Side effect may be the development of resistance of
bacteria. The purpose of our study was to evaluate the number
of resistant bacteria in subgingival plaque after application of
Methods: 33 patients with severe chronic periodontitis were
treated with scaling and root planing. 16 of them got
additionally moxifloxacin (400 mg/d), 11 doxycycline (200 mg
d1, then 100 mg/d) for 10d. 6 patients served as control. At
baseline, 2 weeks and 3 months after treatment subgingival
plaque was sampled from each two sites (Baseline-PD > 5 mm)
and plated on media containing 1 and 10 mg/l doxycycline and
0.25 and 2.5 mg/l moxifloxacin and without antibiotics. Each
the numbers of resistant bacteria were counted. The MICs of the
two antibiotics against Aggregatibacter actinomycetemcomitans
(A.a.)and Porphyromonas gingivalis (P.g.) strains were
Results: At baseline, in all groups 3.50 % of bacteria showed a
resistance against 1 mg/l and 0.41 % against 10 mg/l
doxycycline. 3.50 % of bacteria grow on media containing 0.25
mg/l and 1 % on media with 2.5 mg/l moxifloxacin.
Immediately after application of doxycycline the percentage of
bacteria increased to 21.5 % exhibiting resistance to 1 mg/l and
to 1.94 % to 10 mg/l doxycycline. The values for moxifloxacin
were 10.66 % against 0.25 mg/l and 2.07 % against 2.5 mg/l. 3
months after treatment we did not find an elevated resistance to
both antibiotics compared to baseline. At all we found 4, in the
doxycycline group 2 P. g. strains with a high resistance against
doxycycline (> 64 mg/l). In A.a. strains exhibiting a resistance
against 6 mg/l Ribo2 gene was found. Without association to the
MICs, 75% of the screened A.a. and P.g. strains had the tetM
gene.In moxifloxacin patients 2 P. g. strains exhibited a
moderate resistance with a MIC of 0.5 mg/l. In these strains, the
Ser-83 Phe mutation in gyrA was not detectable.
The antibiotic therapy tended to a temporary increase in number
of resistance bacteria against the used antibiotic. After
application of doxycycline the development of highly resistant
P. gingivalis strains has to be considered.
PCR method for “serotyping” of Legionella pneumophila
serogroup 1
A. Thürmer1, P. C. Lück1
Technical University of Dresden, Institute of Medical
Microbiology und Hygiene, Dresden, Germany
Legionella pneumophila is commonly detected in environmental
specimens can cause pneumonia (Legionaires’ disease) if
transmitted to susceptible persons. Several PCR assays based on
16SrRNA and virulence-associated genes are available for
detection and subtyping L. pneumophila. However no PCR
methods have been published that can discriminate between
serogroups and the MAb 3-1 (Pontiac) subgroup of L.
pneumophila serogroup 1 the most common serogroup. By
analysing published sequences we developed two PCR methods
specific for serogroup 1. From all serogroup 1 strains (n=23)
including the ATCC type strains specific DNA fragments could
be amplified. In contrast none of the serogroups 2 – 15 strains
(n=27) contained these serogroup 1-specific genes.
Strains associated with illness are often reacting with (MAB 31). This epitope is related to the lag-1 gene that encodes an Oacetyltransferase. Therefore primers specific for the lag-1 were
designed and tested. All MAb 3-1 positive strains contained the
lag-1 gene. In turn 12 of 17 MAb 3-1 negative strains tested
negative in the PCR. However we detected several strains that
did not react with MAb 3-1 and harboured truncated or mutated
lag-1 genes. In summary serogroup 1 strains could differentiated
from strains belonging to other serogroups by using PCR.
However the differentiation of MAb3-1 positive strains and
MAb 3-1 negative strains could not be achieved for all strains.
PCR with primers specific for the serogroup 1 genes can be used
for diagnosis of legionellosis caused by L. pneumophila
serogroup 1 with out need for isolation by culture. These PCR
could be a valuable tool in outbreak investigations and in risk
Major outbreak of wound botulism among injecting drug
users, North Rhine-Westphalia 2005
M. Schröter1, K. Alpers2, U. van Treeck1, C. Frank2
N. Rosenkötter1, R. Schaumann3
Institut for Public Health, NRW, Communicable Diseases
Epidemiology and Hygiene Unit, Muenster, Germany
Robert Koch-Institut, Department for Infectious Disease
Epidemiology, Berlin, Germany
Institute for Medical Microbiology and Epidemiology of
Infectious Diseases University of Leipzig, National Reference
Laboratory for Anaerobes, Leipzig, Germany
Introduction: In developed countries, wound botulism is a rarely
occurring form of the diseases caused by Clostridium botulinum.
In November 2005, several injecting drug users (IDU) with
wound botulism were notified to North Rhine-Westphalia (NW)
health authorities. An outbreak investigation was initiated to
find the source and to prevent the occurrence of more infections.
Patients and Methods: Alerts were issued to public health
departments and emergency physicians, to IDU outreach
organisations and through EU warning systems to find cases.
For a survey, cases were defined as IDUs presenting with acute
onset of flaccid paralysis or cranial nerve palsies between
October and December 2005. As soon as the clinical condition
improved, the patients who gave informed consent were
interviewed during or after their hospital stay. Isolates of C.
botulinum were cultivated from wound swabs and typed by
Results: Altogether, 16 case-patients were notified during the
nine weeks between 13 October and 5 December 2005 living in
eight different districts of NW. Five of the eight interviewed
patients mentioned to have injected a dark brownish form of
heroin recently, although not from an apparent common source.
Likewise, five had injected the drugs intramuscularly or
subcutaneously, and six had observed abscesses and skin
infections prior to onset of botulism symptoms. From wound
swabs of six patients neurotoxin B gene positive C. botulinum
isolates were cultured. Further typing revealed one identical
PFGE pattern for all isolates.
Discussion: The clonality of the C. botulinum isolates and the
results of the epidemiological survey, strongly suggests a
common origin of the outbreak. Although it was impossible to
identify the source, contaminated heroin seems to be most
probable vehicle since other links between the cases (such as
shared needles or dilution materials) could be excluded. The
investigation was hampered by the fact that possession and
usage of heroin is illegal and therefore some IDU were not
willing to cooperate. Early recognition and notification of
potential wound botulism cases is fundamental to allow timely
interventions of Public Health Authorities.
"A forgotten disease?" – Necrobacillosis
A. Benz1, G. Borkenhagen1, C. Kortsik1, E. Siegel2, S. - R. Han2
Katholisches Klinikum Mainz, Klinik für Pneumologie
Beatmungs- und Schlafmedizin, Mainz, Germany
Universitys Hospital Mainz, Medical Microbiology and
Hygiene, Mainz, Germany
Case history: A 24-year old patient was admitted with a high
fever and signs of a severe gastroenteritis accompagnied by
acute tonsillitis. Despite antibiotic therapy his condition
worsened considerably and developed in septic shock syndrome,
associated with pulmonary infiltrates, lung abscesses and pleural
effusions. In several blood cultures, a gram-negative obligate
anaerobe rod could be isolated which PCR-analysis confirmed
as Fusobacterium necrophorum.
Diagnosis: Necrobacillosis / Lemierre'
s Syndrome (caused by
Fusobacterium necrophorum.
Bacterium:Fusobacterium necrophorum is a gram-negative rod
with obligate anaerobic growth.
Diagnosis: Since this condition is rarely observed, it is also
called "the forgotten disease". This life-threatening infection
usually occurs in young healthy adults. The most common
course of this illness is postangial septicaemia or Lemierre'
syndrome: acute oropharyngeal infection with secondary
pulmonary septic lesions. These rare clinical symptoms are very
typical. Upon clinical suspicion, several anaerobe blood cultures
should be obtained. Considering the high mortality rate of 1794
30%, a quick diagnosis with adequate antibiotic therapy is
absolutely vital.
Therapy: Even a specific and immediate antibiotic therapy will
results in a slow clinical recovery response and requires a
protracted treatment regimen. Due to the severeness of the
illness as well as frequent associated metastatic infectious foci, a
4-6 week combination therapy of high-dose penicillin and
metronidazole is recommended.
Clinical Outcome:
Continued adequate antibiotic therapy resulted in complete
recovery and eradication of the severe lung alterations.
First confirmed autochthonous case of human Babesia
microti infection in Europe
A. Hildebrandt1, K. - P. Hunfeld2, M. Baier1, A. Krumbholz1
S. Sachse1, T. Lorenzen3, M. Kiehntopf4, E. Straube1
Institute of Medical Microbiology, Jena, Germany
Institute of Medical Microbiology, Frankfurt am Main
Institute of Transfusion Medicine, Jena, Germany
Institute of Clinical Chemistry and Laboratory Medicine, Jena
Objectives: Babesiosis, caused by infection with Babesia
species, can be acquired either by tick-bite or via transfusion of
infected cellular components. The two most important Babesia
species involved in human babesiosis are Babesia microti and
Babesia divergens. Up to now all reported cases of Babesia
microti in Europe represent imported infections that had been
acquired outside Europe. Here, we report the first confirmed
autochthonous case of human Babesia microti infection in
Europe which occurred in a 42-year-old female patient suffering
from acute myeloid leukaemia. Recently she fell ill with fever
up to 39°C and signs of acute myocardial infarction after the
first cycle of leukaemia specific chemotherapy.
Methods:Suspicion of malaria was ruled out by microscopy and
use of a dip-stick test. In patient material microscopy,
polymerase chain reaction (PCR) for detection of Babesia
species and serologic tests were performed initially and in
follow-up controls. All blood donors to the patient were
investigated for possible Babesia infection by microscopy, PCR
and serological tests.
Results: The diagnosis of Babesia microti infection was finally
established by specific PCR and sequence analysis. Six weeks
after initiation of specific treatment parasites were not longer
detected by light microscopy and PCR. In correlation to this
immunoflourescence assay (IFA) antibody testing revealed a
seroconversion with IgG titers of 1:128. Donors proved negative
for Babesia DNA but serological screening demonstrated
borderline reactivity for Babesia microti (IgG-titer 1:32) in one
out of 44 individuals.
Conclusion: Neither the patient, nor the serological positively
tested blood donor had travelled to North America. Therefore,
the infection has to be considered as autochthonous and most
likely transfusion-transmitted. It is important to note, that
clinical symptoms can occur within several weeks after
transfusion and, as in our case, Babesia infections may arise in
geographic areas where human babesiosis has never been
reported in the past. Further molecular epidemiological and
seroepidemiological studies are urgently needed to get more
information about the distribution and medical relevance of such
pathogens in Europe.
Figure 1:
Emergence of linezolid resistance in a methicillin resistant
Staphylococcus aureus strain
M. Hentschke1, B. Saager2, M. Horstkotte1, S. Scherpe1
H. Kabisch3, R. Grosse3, P. Heisig2, M. Äpfelbacher1
H. Rohde1
University Hospital Hamburg-Eppendorf, Microbiology,
Virology and Hygiene, Hamburg, Germany
University Hamburg, Pharmacognosy Biology and
Microbiology, Hamburg, Germany
University Hospital Hamburg-Eppendorf, Clinik of Pediatric
Hematology and Onkology, Hamburg, Germany
Linezolid is a second line drug for treatment of infections due to
multi-resistant gram-positive bacteria. It acts by binding to the
23S rRNA in the ribosom initiation complex. The fact that the
23s rRNA is encoded by five to six gene copies are present in
the genome of Staphylococcus aureus is believed to result in a
strongly delayed development of resistance by accumulating
point mutations. Indeed, linezolid-resistant strains of S. aureus
have only rarely been detected world-wide. Here we report the
isolation of the first linezolid-resistant methicillin-resistant S.
aureus (MRSA) strain in Germany. It was isolated from a
pediatric, bone-marrow transplantated patient that was treated
with linezolid for prolonged time periods. The strain exhibited
the typical G2576T mutation in three out of five 23S rRNA
alleles, resulting in linezolid resistance (MIC 8 µg/ml). After
discontinuation of linezolid therapy, subsequent MRSA isolates
from the patient displayed linezolid-resistance for at least two
more months. Our report emphasizes the importance to limit the
duration of linezolid therapy to prevent emergence and spread of
resistant bacterial pathogens.
Community acquired MRSA ST8 („USA 300“) has arrived
in Central Europe
W. Witte1, C. Cuny1, B. Strommenger1, U. Nübel1
Robert-Koch-Institute, Wernigerode Branch, Wernigerode
Objective: Characterization of MRSA exhibiting spa sequence
type t008 (congruent with MLST ST8) from community and
nosocomial infections in Germany in order to assess the
emergence and spread of the epidemic community MRSA “USA
300” in Central Europe.
Methods: Multilocus sequence typing (MLST), grouping of
SCCmec elements by means of PCR, PCR demonstration of
lukS-lukF arcA (arginindeiminase, indicator for ACME-island;
msrA (macrolide efflux), mphB (macrolide phosphotransferase)
Results: MRSA exhibiting t008, MLST ST8 which are
exhibiting resistance to oxacillin and to ciprofloxacin but
lacking lukS-lukF, arcA, msrA, mphB are known from
community acquired wound infections and nasal colonization in
the Stuttgart area since 2000. Community acquired MRSA
exhibiting t008, MLST8 and containing lukS-lukF, arcA, msrA,
mphB have been recorded from deep seated infections of skin
and soft tissue in Saarland, Rhineland-Pfalz, North-RhineWestphalia, and lower Saxony as well as in Tyrol (Austria)
since 2004. In two cases spread to other patients occurred after
admission of affected patients to surgical wards. ArcA, msrA,
mphB are characteristic for community MRSA “USA 300” and
were not detected in representatives of epidemic hospital MRSA
and also not in other clonal lineages of lukS-lukF containing
community MRSA.
Conclusion: lukS-lukF containing MRSA carrying other
acquired genes which are characteristic for MRSA “USA 300”
have been recorded from several locations in Germany. For
reliable early warning MRSA exhibiting spa t008 should be
further characterised for containing lukS-lukF, arcA, and mphB.
CTX-M Escherichia coli with reduced susceptibility to
D. Jonas1, A. Buchwald2, K. Biehler1, C. Schneider3, S. Schütt4,
C. Wendt4
University Medical Center Freiburg, Institute of Environmental
Medicine and Hospital Epidemiology, Freiburg, Germany
University Medical Center Freiburg, Depatment of Clinical
Chemistry, Freiburg, Germany
University Medical Center Freiburg, Institute of Medical
Microbiology and Hygiene, Freiburg, Germany
University of Heidelberg, Institute of Hygiene, Heidelberg
Presently, the number of ESBL-positive E. coli, isolated from
patients in hospitals and the community is increasing in many
European countries. Carbapenems are recommended at least for
empirical treatment of severe systemic infections by these
strains. Here we describe the first case in Germany of a patient
with an ESBL-positive E. coli isolate, which lost imipenem
susceptibility under therapy.
The first strain was isolated in November 2006 from a
bronchoalveolar lavage taken from a 33-year old female patient
with recurrent urinary tract infections after renal transplant at
the beginning of the year, and subsequent immunosuppressive
therapy. In the further course, the patient developed a
necrotising HSV-2-hepatitis and was transferred for liver
transplantation to a different university hospital. Here, the
second ESBL E. coli with a reduced susceptibility to imipenem
was noticed in a gall duct specimen.
Genotypic identity of both strains was confirmed by AFLP.
Both isolates were resistant to ceftazidime and cefotaxime with
a decrease of the MIC in the presence of clavulanic acid by
more than 3 dilution steps. However, the 2nd isolate showed an
MIC of 4 µg/ml against imipenem in contrast to 0,25 µg/ml in
the previous isolate. In both isolates the same beta-lactamase
genes encoding for TEM-1, SHV-12 and CTX-M-9 group were
detected. All of them could be mobilised by conjugation into an
E.coli J53 recipient, which became ESBL-positive, but remained
imipenem susceptible. Isoelectric focussing of beta-lactamases
from both strains revealed the same banding patterns, which
could be inhibited by prior overlaying of the gel with clavulanic
acid. Next, outer membrane proteins were prepared from both
strains and controls by ultracentrifugation technique. SDS
PAGE analysis demonstrated the loss of a fragment 38 kDal in
size, which presumably corresponds to the porins OmpF and
OmpC based on their electrophoretic mobility and abundance.
In times of increasing ESBL prevalence we demonstrated the 1st
case of an E. coli strain in Germany losing imipenemsusceptibility with a concomitant loss of outer membrane
proteins. The high prevalence of ESBL-producers might
increase the risk of some of these strains becoming resistant to
carbapenems, which are recommended for empirical therapy.
Scedosporium prolificans in Germany: Patients with deep
infections or long lasting colonisation from 1993 to 2007 and
characterization of the clinical isolates
K. Tintelnot1,2, G. Just-Nübling2, I. Sobottka3, A. Haas4
R. Horré5
Robert-Koch-Institut, Mycology, Berlin, Germany
JWG-University, Center of Internal Medicine, Frankfurt am
Main, Germany
University Hospital Hamburg-Eppendorf, Institute for
Microbiology, Hamburg, Germany
Max-von-Pettenkofer-Institute, Microbiology, Munich
Federal Inst. for Drugs and Medical Devices, Bonn, Germany
Scedosporium prolificans, a mold closely related to
Pseudallescheria boydii, is a rare but emerging pathogen due to
its complete resistance to traditional antimycotic treatment. 20
patients in Germany with deep infection or long lasting
colonization by S. prolificans from 1993 to March 2007 have
been reviewed retrospectively. The mortality rate in all 12
patients with invasive scedosporiosis and a severe underlying
disease (AML, BMT, SOTX, AIDS, long term treatment with
corticosteroids) was 100%. Another 8 patients, 6 of them with
cystic fibrosis, one with chronic sinusitis and one after lung
transplantation, were colonized without development of an
invasive infection. Nevertheless long term colonization is a
contradiction for lung transplantation in patients with Cystic
Fibrosis. All clinical isolates and reference strains from
Belgium, Spain, U.K., Australia and U.S.A. were analyzed by
sequencing of the ITS and parts of the 18S and 28SrDNA
regions and of the translation elongation factor EF1-alpha. No
variability of the ITS gene and flanking regions within the
species S. prolificans was detectable. Interestingly enough
sequencing of the translation elongation factor EF1-alpha
revealed three different genotypes of S. prolificans, which might
be useful for further epidemiological studies.
PCR-DHPLC (polymerase chain reaction - Denaturing high
performance liquid chromatography): A novel method for
rapid monitoring and identification of single and mixed
yeast cultures relevant for the food and beverage industry
M. Hutzler1, O. Goldenberg2
Technical University Munich, Munich, Germany
Transgenomic, Cramlington, United Kingdom
The rapid detection and identification of spoilage yeasts is of
significant interest in quality control in the food and beverage
industries. Spoilage yeasts are able to cause off-flavours,
turbidity, additional CO2 and excess pressure in drinks products.
Rapid, precise detection and identification of spoilage yeasts can
prevent product spoilage and decrease health and economic
risks. The potential of a novel PCR-DHPLC (Polymerase Chain
Chromatography) method was studied to detect the presence of
and identify spoilage yeast populations in single and mixed
yeast cultures related to the food and beverage industries. PCR
was performed with universal primers amplifying the ITS1 and
ITS2 rRNA genes of different yeast species, followed by
DHPLC to separate the mixed PCR products. Applying this
method, 21 out of 25 yeast species tested were clearly separated
from each other, with the remaining four species forming two
clusters. The species investigated included all Saccharomyces
sensu stricto species. The Saccharomyces sensu stricto complex
contains yeasts that are used for several fermentation processes
such as beer, wine, cider, bio-ethanol and indigenous
fermentations. The yeast species of the S. sensu stricto complex
could be clearly separated by PCR-DHPLC using the
aforementioned rRNA genes. It is proposed that this method can
be used to monitor rapidly fermentation processes, controlled
and spontaneous starter cultures as well as yeast-induced food
and beverage spoilage. Additionally a PCR product was
amplified by using a S. cerevisiae specific primer pair situated at
a NTS region and separated by DHPLC to differentiate
industrial Saccharomyces cerevisiae culture strains. These
industrial strains showed different peak patterns. Therefore
PCR-DHPLC can be a useful tool for rapid differentiation at
both species and strain level.
Selective Isolation of Enterobacter sakazakii in infant milk
formula by supplementation of Ferrioxamin E
S. Sanjaq1, M. Fischer1
Central Laboratories Friedrichsdorf, Microbiology Division
Friedrichsdorf, Germany
known but definitely higher than the usual contamination level.
Retrospective analyses of E. sakazakii outbreaks caused by IMF
have shown that in most cases a heavy mishandling of the
prepared formula has been involved.
In a previous study has been demonstrated that Ferrioxamin E
act as a selective iron source for certain groups of bacteria such
as Salmonella and Enterobacter. As a potent growth factor it has
been used in several studies as supplements in standard
enrichment and selection procedures to increase the speed and
sensitivity of detection of members of the genus Salmonella in a
number of food products. Hence, this study was performed to
find the role of iron source on the media for E. sakazakii
detection in infant milk formula by using Ferrioxamin E as an
iron supplement under different treatment conditions.
he application of Ferrioxamin E has been seen increasing the
sensitivity of Salmonella detection especially for heat and dry
stressed strains in respectively treated products. E. sakazakii is a
bacterium of concern especially in powdered infant formula.
This product range runs through a number of heat treatment
steps and is characterized by an extremely low water activity.
Possible E. sakazakii contaminations have to be regarded as
highly heat and dry stressed. First investigations have shown an
increase of the sensitivity for the detection of E. sakazakii in
powdered products.
Improvement and validation of RAPD in comparison to
PFGE analysis of enterobacter sakazakii strains
S. Sanjaq1, A. Lindenstrauß1, M. Fischer1
Central Laboratories Friedrichsdorf, Microbiology Division
Friedrichsdorf, Germany
Enterobacter (E.) sakazakii is an opportunistic pathogen causing
meningitis, septicemia and enterocolitis in premature infants.
Infant formula has been implicated as the source of E. sakazakii
in several clinical outbreaks. The investigation of infection
routes is usually performed by Pulsed field gel electrophoresis
(PFGE). The PFGE is a high sophisticated labor- and costintensive method. Therefore a previously developed randomly
amplified polymorphic DNA (RAPD) protocol was used, to
evaluate a rapid, reproducible and low-cost alternative to PFGE
typing of E. sakazakii strains, which can be used to follow
contamination routes. The RAPD has been evaluated on
repeatability and reproducibility. Especially the discriminatory
potential of the applied method has been investigated for a
number of strains far distinct in source and PFGE profile. The
band pattern of RAPD-PCR reactions represents a genetic
fingerprint that can be used to characterize E. sakazakii strains
and trace contamination routes. Our findings indicate that
RAPD is a useful and reliable tool for typing studies of E.
sakazakii isolates.
Enterobacter (E.) sakazakii is an important opportunistic
pathogen which can cause rare but severe infections in newborn
and preterm infants. In the recent years a number of infections
occurred by infant milk formula (IMF). The contamination level
in IMF is usually very low and a growth of the pathogen in the
IMF is necessary to reach the infection dose, which is not
Studies on the growth kinetics of different bacteria with
impedance technique
S. Zhu1, M. Fischer1
Central Laboratories Friedrichsdorf, Microbiology Division
Friedrichsdorf, Germany
The microbiological risk assessment for food products includes
the storage and handling of those products by the consumers.
The risk associated with the product is partly due to the
microbiological growth conditions provided by the food.
This is especially true for specialised products like clinical
nutrition and infant formula. Since the products are consumed
by very vulnerable consumer groups. The products are
manufactured and distributed in a very high quality state, sterile
or as powdered products where bacterial growth impossible. The
integral risk of specialised nutritional products is very low. The
most critical point of bacterial contamination is the mishandling
by the consumers, which may include the contamination of the
prepared formula and a prolonged storage which allows
bacterial multiplication.
These points have to be included in the risk assessment and the
handling instructions for the concerned products. The individual
risk assessment for certain products is often based on the growth
kinetics of selected model bacteria. However the collection of
data for the growth kinetics is time consuming and labour
The current study shows the surveillance of bacterial growth in
products of specialised nutrition by impedance compared to the
data of conventional microbiology.
The impedance has been detected directly in the product under
investigation inoculated with different bacteria species (E.
sakazakii, B. cereus and Pseudomonas spp), which shows the
growth kinietics in microbiological broths.
The impedance has been detected directly in the product
inoculated with different bacteria species (E. sakazakii, B.
cereus, P. aeruginosa) and compared to the growth kinetics in
microbiological broths for impedance testing.
Detection of sulphite reducing Clostridia species in milk
powder based products
S. Zhu1, M. Fischer1
Central Laboratories Friedrichsdorf, Microbiology Division
Friedrichsdorf, Germany
Clostridia species are a common contamination of milk powder
products. The group includes a number of harmless species but
also pathogenic and opportunistic pathogenic species. Especially
of concern for infants products is C. botulinum which can cause
infant botulism following ingestion. The whole group of
sulphite reducing clostridia (SRC) is used as an index parameter.
However the detection of the SRC group is difficult due to the
inhomogeneous composition of the group. The current ISO
method covers all sulphite reducing bacteria which includes also
Enterobacteriaceae species and some Bacillus species which
grow aerobically. A heat treatment limits the detection to sporeformers, but makes the method also sensitive for a number of
Clostridia species.The current investigations have explored the
sensitivity of four agars (DCA, TSC, ISA, SIA) for 22 Clostridia
species with and without heat treatment.
Sterility test of aseptically filled products with luminescence
M. Fischer1, A. Reidel1
Central Laboratories Friedrichsdorf, Microbiology Division
Friedrichsdorf, Germany
The sterility test of aseptically filled UHT products is a time
consuming and labour intensive work. The products have to be
incubated 10 day and checked for microbiological growth after
that time. The usual procedure is a manual streak on different
agars. There have been a number of alternative methods used
especially for UHT milk. Among others the luminescens is one
of the most promising procedures. The method has been shown
as very reliable for UHT milk and other milk based
products.The current investigation shows results of a validation
trial for products of specialised nutrition. The validation has
been performed for a number of different bacterial species on
several contamination levels. The influence of a number of
flavours on the detection limit has been checked.
Comparative study of the culture method (§64 LFGB 00.0022), PCR and RNA-hybridisation for the detection of
Listeria monocytogenes in foodstuffs.
A. Muelverstedt1, C. Hartke1, H. Dorfmann1
Department of Food Hygiene and Diagnostics, Bad
Langensalza, Germany
For the extensive control of the microbiological risks in the food
production industry, the EU-Commission introduced regulation
(EG) Nr. 2073/2005 on 15. November 2005. This regulation
dictates the investigation of following focal points: Salmonella
spp. and Listeria monocytogenes as safety criteria in foodstuffs
and E. coli as a process hygiene criterion. The detection limit for
L. monocytogenes is ND/25g for consumable products at the
production level, alternatively <102 CFU/25g of consumable
products at retail.
The official method for the detection of Listeria monocytogenes
in foodstuffs (§64 LFGB L 00.00-22) requires the enrichment
and culturing of the bacteria in selective media. Processing
samples in this way may take up to five days for a positive
result. The implementation of a PCR-based method in foodstuff
analysis is one possible method to a dramatic reduction in the
time required to process samples with comparable sensitivity
and specificity to culture methods.
Examination of foodstuffs by molecular biological methods
pose some challenges, especially due to the heterogeneous
protein and fat content of the sample; the addition of colouring
agents and preservatives as well as the potential presence of
concomitant bacterial and non-bacterial agents. This has resulted
in the development and marketing of “alternative” methods for
detection based on immunological or RNA-based methods over
the past years.
In the scope of this investigation five samples (negative by
culture methods) of cheese and sausage, with variable fat and
protein contents, were tested prior to their expiration date. The
samples were spiked with 10, 100, 1 000, 10 000 and 100 000
CFU/g L. monocytogenes and subsequently enriched in either
Peptone Water, ONE-Bouillon, ½-Fraser or Fraser-Bouillon.
The detection of L. monocytogenes was performed by the
reference culture method, PCR and RNA-FastScan®.
Additionally 78 samples were taken out of a routine production
process and compared by all three methods. The results of this
study are discussed below.
Occurrence and properties of Listeria in the food chain
A. Müller1, M. Schmid2, O. Meyer1, F. Meussdörffer1
University Bayreuth, Dept. Microbiology, Bayreuth, Germany
GSF-Research Center for Environment and Health, GmbH
Department Microbs Plant Interactions, Neuherberg
The genus Listeria comprises the six species: L. monocytogenes,
L. ivanovii, L. innocua, L. welshimeri, L. seeligeri and L. grayi.
Of these, only two species, L. monocytogenes and L. ivanovii,
have been shown to be pathogenic for men and animals
respectively. Because of its adaptability and the complex
infection cycle, L. monocytogenes has become a genetic model
organism for intracellular life. Many genes involved in infection
and cell spreading are located at the virulence gene
cluster.Although L. seeligeri too carries homologues of the
virulence genes arranged similarly as in L. monocytogenes, it is
considered apathogenic due to its incapacity to express the
corresponding proteins.
Because of the ability of Listeria to tolerate high salt
concentrations, relatively low pH and to survive and multiply at
refrigeration temperatures, they are ubiquitous present in the
In this study the occurrence of Listeria spp. in the regional food
chain was investigated by microbial analysis of samples from
butcher shops, supermarkets and fecal samples. L. innocua and
L. monocytogenes serogroups 1/2b,3b,7; 1/2a,3a and 4b,4d,4e
were the dominating species in the production facilities, being
particularly present in gullies. Other L. monocytogenes
serogroups as well as L. welshimeri were detected infrequently.
L. seeligeri was detected rather rarely too. However, different
variants could be distinguished with respect to phenotype,
metabolic properties and genetic traits as judged by PCR. Both
variants differed also in their sensitivity towards ß-lactam
antibiotics and the bacteriocin, nisin. Further distinct differences
between variants were detected in the sequences of the iapgenes and the virulence clusters. Moreover, phospholipase
activity was visualized after incubation on chromogenic media
after incubation at 37°C. Accordingly, the protein pattern in
culture filtrates differed depending on cultivation conditions.
Our results indicate that L. seeligeri might be a versatile
organism adapting quite efficiently to its environment.
Characterization of novel toxic mycotoxins in Aspergillus
C. Handrich1, J. Buenger2, G. Westphal1, E. Hallier1
M. Müller1
Georg-August-University, Arbeits- und Sozialmedizin
Goettingen, Germany
Ruhr-University, BGFA, Bochum, Germany
Mycotoxins are secondary metabolites of filamentous fungi, that
can cause various toxic effects including cancer, immune
suppression and teratogenicity. Acute and chronic pulmonary
diseases were reported after inhalation of organic dust
containing toxic moulds and mycotoxins. Exposure to these
airborne contaminants is a major health hazard in waste
treatment facilities. One of the most abundant mould species in
German composting plants is Aspergillus nidulans, which
produces the carcinogenic mycotoxin sterigmatocystin. The
extract of A. nidulans caused serious toxic effects in the A-549
lung cell line, that could not be completely explained by its
content of sterigmatocystin. [Buenger et al. (2004) Toxicology
202, 199]. The presence of additional mycotoxins or other toxic
principles were detected by a structure activity-approach.
Using an HPLC-diode array detector method the components in
the A. nidulans-extract were separated and characterized within
50 minutes/analysis and collected as nine 5-minutes-fractions.
Sterigmatocystin was identified in fraction 8 with a content of
up to 28%. The fractions were tested for cytotoxicity in three
established cell lines (A-549, L-929 and Hep-G2) using the
neutral red assay (NRU). Fraction 9 showed the strongest
cytotoxicity in A-549-pneumocytes. In addition, the
mutagenicity of the 5-minutes-fractions was determined using
the Salmonella typhimurium/mammalian microsome assay
(Ames test) with the strains TA 98 and TA 100. The tests were
performed with and without metabolic activation by a
microsomal mixed function oxidase system (S9 fraction).
Fractions 1-7 did not significantly increase the number of
revertants, while fraction 8 and 9 in low concentrations caused a
significant dose-dependent increase of revertants in both tester
strains after S9 activation. The observed mutagenic effect of
fraction 8 may be predominantly due to sterigmatocystin.
Therefore the single compounds from fraction 9 were collected
and investigated in detail by the NRU and Ames test. The
fraction contains several cytotoxic and mutagenic substances,
which will be further characterized by iontrap mass
spectrometry and NMR spectroscopy.
Occurrence of Aspergillus fumigatus and gliotoxin
formation in meat
H. Schulze1, O. Meyer1, F. Meussdoerffer1
University Bayreuth, Dept. Microbiology, Bayreuth, Germany
The mold Aspergillus fumigatus is ubiquitous in the
environment. However, it is known as causative of aspergillosis
in immunocompromised people and producer of several
mycotoxins, among them the immunosuppressive and cytotoxic
gliotoxin. We have studied the occurrence of Aspergillus
fumigatus in butcher shops. The mold was detected in 46% of
192 air samples from inside butcher premises and their vicinity
as well as in 26% of 73 spice mixtures. Aspergillus fumigatus
was also detected in two out of 330 products tested in densities
above 1,000 cfu/g (fresh weight).
The presence of the gliotoxin gene cluster was demonstrated by
PCR in each of the 13 isolates from different sources examined.
Sequencing parts of the gliJ and gliZ regions of three isolates
(two from spice mixtures, one from sausage) revealed no
differences in these genes between the isolates. This indicates
the potential of environmental Aspergillus fumigatus strains to
form the mycotoxin under appropriate conditions.
We consequently investigated the production of gliotoxin by the
three isolates during growth on different substrates employing
TLC and HPLC. While no gliotoxin was formed on rice, oats or
cheese respectively, the toxin could be detected during growth
of the Aspergillus fumigatus strains on minced meat. This
indicates that gliotoxin might be formed in some foods. In
particular in combination with pathogens known to affect
immunocompromised people this might enhance the hazard to
the consumer.
The weak interaction of LcrV and TLR2 does not contribute
to the virulence of Yersinia pestis
D. Reithmeier-Rost1, J. Hill2, S.J. Elwin2, D. Williamson2
S. Dittmann1, A. Schmid1, G. Wilharm3, A. Sing4
Max von Pettenkofer-Institut, Munich, Germany
DSTL, Porton Down, Porton, United Kingdom
Robert Koch-Institut, Wernigerode, Germany
Bavarian Health and Food Authority, Oberschleissheim
pseudotuberculosis and Yersinia enterocolitica share the
virulence-antigen LcrV. Previously, using reverse genetics we
have proven that LcrV contributes to the virulence of Y.
enterocolitica serotype O:8 by inducing IL-10 via Toll-like
receptor 2 (TLR2). However, both the ability of Y. pestis LcrV
to activate TLR2 and a possible role of TLR2-dependent IL-10
induction by LcrV in Y. pestis are not yet known. To eliminate
interference from additional protein sequences, we produced
LcrVs without affinity tags from Y. pestis and from Y.
enterocolitica O:8 (LcrVO:8). LcrVO:8 was much more potent
in TLR2-activity than Y. pestis LcrV. To analyse the role of
TLR2 in plague, we infected both wild-type and TLR2-/- mice
subcutaneously with Y. pestis GB. While TLR2 -/- mice
exhibited lower blood levels of IL-10 (day 2 post-infection) and
of the pro-inflammatory cytokines TNF-a, IFN-g and MCP-1
(day 4) than wild-type mice, there was no significant difference
in survival. The low TLR2-activity of Y. pestis LcrV and
associated cytokine expression might explain why - in contrast
to Y. enterocolitica O:8 infection - TLR2-deficient mice are not
more resistant than wild-type mice in a bubonic plague model.
The B-dependent yabJ-spoVG locus affects antimicrobial
resistance and capsule formation in Staphylococcus aureus
S. Meier1, B. Schulthess1, C. Görke2, C. Wolz2
B. Berger-Bächi1, M. Bischoff1,3
University of Zurich, Switzerland, Institute of Medical
Microbiology, Zurich, Switzerland
University Hospital Tuebingen, Institute of Medical
Microbiology and Hygiene, Tuebingen, Germany
University of Saarland, Institute of Medical Microbiology and
Hygiene, Hommburg/Saar, Germany
The alternative sigma factor B of Staphylococcus aureus
controls the expression of a variety of genes including virulence
determinants and global regulators and affects the resistance
level formation of methicillin-resistant S. aureus (MRSA) and
glycopeptide intermediate resistant S. aureus (GISA). We report
here that deletion of the B-dependent yabJ-spoVG locus in
MRSA and GISA strains decreased the resistance levels against
oxacillin and teicoplanin, respectively. Moreover, inactivation
of yabJ-spoVG in the capsular polysaccharide type 5 (CP-5)
producing strain Newman strongly impaired CP-5 production.
Trans-complementation of the MRSA and GISA yabJ-spoVG
mutants with yabJ-spoVG under the control of its native
promoter restored the resistance levels to levels seen in the wildtype strains, while neither trans-complementation with yabJ nor
spoVG alone reverted the resistance phenotype. Interestingly,
trans-complementation with a yabJ-spoVG construct harbouring
a frame shift mutation in the 5´-part of the yabJ open reading
frame, yabJ1(Am)-spoVG, allowed to restore the resistance
phenotype, while trans-complementation with a yabJ-spoVG
construct harbouring a frame shift mutation in the 5´-part of the
spoVG open reading frame, yabJ-spoVG1(Am), did not.
Similarly, only yabJ-spoVG and the mutated yabJ1(Am)-spoVG
construct restored CP-5 formation in the
derivative of strain Newman, while neither yabJ, nor spoVG, nor
yabJ-spoVG1(Am) were effective. We therefore hypothesise
that SpoVG is the major factor of the yabJ-spoVG locus that is
of importance for capsule production and resistance formation in
S. aureus and suggest that the yabJ-spoVG mRNA but not YabJ
is necessary for full SpoVG expression and activity.
Functional analysis of Bartonella henselae BadA domains
T. Riess1, P. Kaiser1, D. Linke2, A. Lupas2, A. Schäfer1
V. Kempf1
Eberhard-Karls-Univerity, University Hospital, Institute for
Medical Microbiology and Hygiene, Tuebingen, Germany
Max Planck-Institute for Developmental Biology, Department
for Protein Evolution, Tuebingen, Germany
Bartonella henselae causes cat scratch disease and the
vasculoproliferative disorders bacillary angiomatosis and
peliosis hepatis in humans. One of the best characterized
pathogenicity factors of B. henselae is Bartonella adhesin A
(BadA). BadA belongs to the class of trimeric autotransporter
adhesins and is constructed modularly, consisting of a head
domain, 21 neck-stalk repeats and a membrane anchor. It is
important for, e.g., the adhesion of B. henselae to extracellular
matrix proteins and endothelial cells and for the induction of a
proangiogenic host cell response. As the biological functions of
certain BadA domains are not known so far, we want to
elucidate the role of these domains in B. henselae-infections.
For this purpose, in-frame deletion mutagenesis of badA was
performed followed by a functional analysis of the respective
mutants. First, 21 neck-stalk repeats were deleted leading to the
expression of a truncated BadA. The mutant strain B. henselae
BadA?/Hn23 was analyzed (i) for surface exposure of truncated
BadA, (ii) for autoagglutination, (iii) for adhesion to fibronectin
(Fn) and collagen and (iv) for adhesion to endothelial cells. In
contrast to B. henselae wildtype, B. henselae BadA?/Hn23 did
not bind to Fn indicating a crucial role of the neck-stalk domain
in this process. In all other functional assays, B. henselae BadA/Hn23 showed a similar phenotype as wildtype bacteria. These
results suggest that mainly the head domain is important for the
adhesin function of BadA. Further mutants carrying different
deletions in the head- and neck-stalk domains will be analyzed
in future.
Pseudomonas aeruginosa metabolism and lactate production
in sputum of cystic fibrosis patients
D. Worlitzsch1, T. Bensel1, M. Borneff-Lipp1, G. Doering2
University Hospital Halle-Wittenberg, Institute of Hygiene
Halle, Germany
Eberhard-Karls-Univerity, University Hospital, Institute of
Medical Microbiology and Hygiene, Tuebingen, Germany
In sputum from patients with cystic fibrosis (CF), bacteria such
as Pseudomonas aeruginosa metabolize anaerobically. For
energy generation, P. aeruginosa can use pyruvate which is
produced from glucose via anaerobic glycolysis. Since pyruvate
can be converted to lactate and vice versa, we investigated if P.
aeruginosa may benefit from externally produced lactate.
In 22 CF sputum samples, in aerobically (0 through 24 hrs) and
anaerobically (1 through4 days) grown cultures of P. aeruginosa
and Staphylococcus aureus, and in neutrophils from healthy
donors L-lactate was determined spectrophotometrically, and
total lactate gaschromatographically. After three days of
anaerobic growth P. aeruginosa gene expression was
determined using Affymetrix® microarrays.
In all samples, exclusively L-lactate was found. Lactate in CF
sputum came to 2.9 ± 3.1 mmol/L (range 0.2 to 14.1 mmol/L)
with similar concentrations in sputum colonized with P.
aeruginosa and S. aureus (3.2 ± 3.7 vs. 2.4 ± 1.5 mmol/L,
p=0.30). During anaerobic incubation, neutrophils produced 3.2
mmol/L and S. aureus 2.8 mmol/L. P. aeruginosa did not at all
generate lactate (detection limit: 0.1 mmol/L), neither with nor
without oxygen. P. aeruginosa [1x107 cfu/ml] spiked with 10
mmol/L lactate did not consume lactate. The lactate
dehydrogenase genes were anaerobically downregulated
(PA0927 -11fold, PA2382 -3fold) or unchanged (PA4771 1.2
times). The genes encoding for pyruvate decomposition to
acetyl CoA were upregulated by 208fold (PA3416) and 47fold
(PA3417). We could show that P. aeruginosa does not benefit
from externally produced lactate.
We confirmed the important role of pyruvate metabolism for
anaerobic P. aeruginosa energy generation. Whether lactate
production of PMNs or S. aureus contributes to CF lung
pathophysiology still remains to be investigated.
Supported by the Cystic Fibrosis Foundation, Bethesda,
Maryland, and the Mukoviszidose e.V., Bonn, Germany.
FlpS is essential for the expression of the arginine deiminase
system of Streptococcus suis
M. Fulde1, P. Valentin-Weigand1, R. Goethe1
University of Hanover, Institute for Microbiology, Department
of Infectious Deseases, Hanover, Germany
Streptococcus (S.) suis belongs to the most important agents of
infectious diseases in young pigs. As a zoonotic agent it also
infects humans and causes meningitis and endocarditis. Very
little is known about factors that contribute to pathogenesis in S.
suis. Recently, we identified a new putative virulence factor of
S. suis, the Arginine Deiminase System (ADS), which was
expressed on the cell surface. ADS regulation is mediated by a
multitude of environmental stimuli e.g. arginine, reduced O2
tension, and carbon catabolite repression (CCR).
In this study, we analyzed the impact of oxygen on ADS
expression. Oxygen tension in the medium from bacteria grown
at 37°C without agitation decreased in correlation with the
bacterial growth. Northern and Western blot analysis revealed
an increase in ADS expression in parallel to decreasing oxygen
levels. In contrast, when bacteria were grown with agitation at
250 rpm, the oxygen content in the medium remained at 100%
and no transcriptional induction of the ADS was observed.
In-silico analysis of the 5´-region of the AD operon revealed an
open reading frame (ORF) with homologies to the CRP/FNR
family of transcriptional regulators. Since it is known that
members of this family contribute to oxygen-mediated gene
regulation, we generated an flpS (for FNR-like protein of S.
suis) deficient knock-out strain. Compared to the wild-type
(WT), the mutant strain was not able to induce the ADS under
anaerobic conditions. Furthermore, the mutant strain was
significantly impaired in growth. Complementation of the
mutant strain restored the WT phenotype. Since the ADS is a
catabolic pathway yielding one mole of ATP we hypothesize
that FlpS is essential for S. suis to overcome anaerobic stress
conditions, and therefore, contributing to the biological fitness.
Establishment of a specific test system for quantitatively
detecting extracellular polysaccharides of Streptococcus
E.-M. Decker1, G. Maier1, M. Brecx1, C. von Ohle1
Eberhard-Karls-University, Dental School, Konservative
Dentistry, Tuebingen, Germany
The architecture and properties of (dental) biofilms are
determined by microbial extracellular biopolymers in the form
of a polymeric slime matrix (EPS).
Aim of the study was to develop a specific method for
quantitatively detecting extracellular polysaccharides of Strep.
mutans occurring predominantly in human cariogenic biofilms.
The glucan specific enzyme-linked immunosorbent assay
(ELISA) is based on the sugar specifity of the lectin
concanavalin A labelled with peroxidase (Con A-POD) to
recognize glucose polymers (glucans) synthesized by Strep.
mutans under growth conditions involving sucrose. The
evaluation of the lectin ELISA was realised using three standard
sugars: dextran (pure glucan), xanthan (heterogenous
polysaccharide containing glucose, mannose, glucuronic acid)
and the disaccharide sucrose. The standard sugars were coated
on microtiter plates for creating a calibration curve. After a
wash step the plates were incubated with Con A-POD. The
colour development of the peroxidase reaction was induced by
addition of ABTS and hydrogen peroxide. The optical density
was monitored kinetically every five minutes for one hour by
means of an ELISA reader at 405 nm. Three calibration curves
were created for each standard carbohydrate. The data were
log10 transformed followed by a logarithmic regression for curve
Con A-POD showed the highest sensitivity for the highmolecular standard dextran with a detection limit of 30 ng/ml
within the linear measuring range. The detection limit for
xanthan was 1µg/ml revealed by Con A-POD. Sucrose did not
cause concentration-dependent binding of Con A-POD. The
correlation coefficient was 0.975 for dextran and 0.988 for
By means of the three standard carbohydrates it could be
verified that the lectin ELISA detected glucose or mannose
exclusively as polymers but not as low-molecular glucose.
Based on this finding the new lectin test system may be
qualified to detect extracellular polysaccharides of Strep. mutans
from bacterial and human clinical samples.
Proteome analysis of Burkholderia pseudomallei
biosynthetic pathways mutants
N. Meukow1, B. Voigt2, G. Flunker, M. Hecker2, I. Steinmetz1
University of Greifswald, Friedrich-Loeffler-Institute of Med.
Microbiology, Greifswald, Germany
University of Greifswald, Institute of Microbiology, Greifswald,
The gram-negative saprophyte Burkholderia pseudomallei is the
causative agent of melioidosis, an infectious disease which has
emerged as an important cause of severe community-acquired
infections in certain areas of the tropics.
Burkholderiapseudomallei is capable of intracellular actin-based
motility and has one of the broadest host ranges of any bacterial
pathogen. Presently, there is no vaccine available to protect
against infection with B.pseudomallei. By using transposon
mutagenesis, we isolated mutants which harbour disrupted genes
encoding enzymes involved in the different steps of nucleotide,
amino acid, and fatty acid metabolism. Some of these mutants
were highly attenuated in a mouse model of infection and were
capable of inducing a high degree of protective immunity as live
vaccines against wildtype challenge. Our current studies are
aimed at i) the elucidation of B.pseudomallei biosynthetic
pathways with special importance during infection and their
possible interaction with the expression of virulence factors and
ii) the identification of antigens and predominant mechanisms
B.pseudomallei infection.
Therefore, we compared bacterial supernatants and cytosolic
extracts of B.pseudomallei wildtype and respective mutants by
using two-dimensional (2D) protein gel electrophoresis followed
by a matrix-assisted laser desorption/ionization time of flight
mass spectrometry (MALDI-TOF MS) for protein identification.
A number of differentially expressed protein spots which might
play a role in virulence were identified.
As a first step towards the analysis of B.pseudomalleiantigens,
we performed immunoproteome experiments with sera from
melioidosis patients and identified several immunogenic
Characterization of structural and functional diversity of
the plasmid encoded serine protease EspP in shiga toxinproducing Escherichia coli
J. Brockmeyer1, M. Bielaszewska1, M. L. Bonn1, H. Karch1
University of Muenster, Institute for Hygiene, Muenster
The infection with Shiga toxin-producing Escherichia coli
(STEC) can result in hemorrhagic colitis and the hemolytic
uremic syndrome (HUS), the leading cause of acute renal failure
in children. At an early stage of the disease, laboratory
parameters show a prothrombotic activation of coagulation
factors, platelets and endothelium. We have previously shown,
that the autotransporter EspP is able to cleave coagulation factor
V and thus interferes with the blood coagulation cascade. This
effect may contribute to the pathogenesis of hemorrhagic colitis
and HUS during STEC infection. The aim of this study was to
investigate, whether a structural polymorphism occurs in the
espP gene, and if this is associated with functional variations
such as altered secretion efficiency or differences in proteolytic
activity of the encoded proteins. The espP gene in STEC of 55
serotypes was PCR amplified and investigated for SspI and RsaI
restriction fragment length polymorphism (RFLP) patterns. A
subset of espP genes was sequenced to analyse genetic
differences in detail. Secretion efficiency of EspP was assessed
by western blot analysis using anti-EspP antibody after
purification with affinity chromatography. The proteolytic
activity was investigated by the analysis of cleavage patterns of
EspP against pepsin and pNA-conjugated oligopeptides. Our
data demonstrate that espP shows a significant degree of
heterogeneity among the strains investigated, leading to loss of
either proteolytic activity or transport function for a subset of
strains. Despite this genetic diversity, the functional properties
of EspP remained unchanged in the most virulent strains,
supporting the role of EspP as a putative, serotype-independent,
virulence factor of STEC.
G-Streptococcal IgG-binding molecules have different
impact on opsonization by C1q
D. P. Nitsche-Schmitz1, S. Reißmann1, I. Sastalla1
H. M. Johansson2, I. - M. Frick2, G. S. Chhatwal1
Helmholtz-Center for Infection Research, Mircobial
Pathogenicity, Brunswich, Germany
Lund University, Section for Clinical and Experimental
Infection Medicine, Lund, Denmark
Beta-hemolytic streptococci belonging to Lancefield group C
and G (GCS, GGS) are human pathogens of emerging
epidemiological importance. They cause a similar spectrum of
skin and soft tissue manifestations as group A streptococci,
occasionally resulting in life threatening conditions such as
necrotizing fasciitis. Recent epidemiological data on diseases
caused by GCS and GGS underline that they are an emerging
threat to human health. Among various virulence factors
expressed by GCS and GGS isolates from human infections, Mand M-like proteins are considered important because of their
anti-phagocytic activity. In addition, protein G has been
implicated in the accumulation of IgG on the bacterial surface
through non-immune binding. The function of this interaction,
however, is still not known. Using isogenic mutants, lacking
protein G or the M-like protein FOG, respectively, we could
show that FOG contributes substantially to IgG-binding. A
detailed characterization of the interaction between IgG and
FOG revealed its ability to bind the Fc-region of human IgG and
its binding to the subclasses IgG1, IgG2, and IgG4. FOG was
also found to bind IgG of several animal species. Surface
plasmon resonance measurements indicate a high affinity to
human IgG with a dissociation constant of 2.4 pM. It has long
been speculated about anti-opsonic functions of streptococcal
Fc-binding proteins. The presented data for the first time
provide evidences and, furthermore, indicate functional
differences between protein G and FOG. By obstructing the
interaction between IgG and C1q, protein G prevented
recognition by the classical pathway of the complement system.
In contrast, IgG that was bound to FOG remained capable of
binding C1q, an effect that may have important consequences in
the pathogenesis of GGS infections.
Detection of Yop translocation of Y. enterocolitica in mice
by using a -lactamase reporter system – Indication that the
immune system may modulate Yop translocation in vivo
A. Klein-Günther1, M. Köberle1, I. B. Autenrieth1, E. Bohn1
Eberhard-Karls-University,University Hospital of Tuebingen,
Medical Microbiology and Hygiene, Tuebingen, Germany
Yersinia outer proteins are important virulence factors of
yersiniae which are translocated into host cell via a type three
secretion system. We established a system to detect Yop
translocation mediated by Yersinia enterocolitica in a mouse
infection model. For this purpose a plasmid was introduced
which encodes for a YopE-beta lactamase fusion protein which
is translocated into host cells. Beta-lactamase (Bla) activity
which cleaves the substrate CCF4 can be detected by flow
cytometry indicated by a blue fluorescence signal. Control
experiments revealed that detection of bla-activity depends on
YopE-bla secretion and translocation. In cell culture
experiments bla-activity can be detected up to three hours after
infection. Infection of cultured splenocytes with the reporter
strain demonstrates that Yop translocation occurs without
serious preferences to any cell type of the spleen resulting in a
similar frequency of Bla-activity in B cells, T cells,
macrophages, dendritic cells, granulocytes or NK cells. Two
days after infection of C57BL/6 mice with a high infection dose
about 3% bla-positive cells were detectable in splenocytes. The
composition of bla+ positive cells comprises predominantly B
cells and CD11b+ cells including macrophages, granulocytes
and dendritic cells and only few T cells. The highest frequency
of bla+ cells comprised macrophages, granulocytes and
dendritic cells indicating that those are the cells which
primarilly get actively in contact with bacteria during early
phase of mouse infection. Infection experiments with mice
deficient for important immune defense mechanismus against
Yersinia infection, namely, IFN- Receptor-/-, TNF-Receptor-/-,
or MyD88-/- mice were performed using conditions by which
the bacterial load in the spleen two days after infection was
comparable to those in C57BL/6 congenic mice. The frequency
of bla-positive cells was significantly increased in these
immunodeficient mice which leads to the speculation that
different immune defense mechanisms may be involved to
control either efficiciency of Yop translocation or stability of
translocated proteins during mouse infection. Further
experiments are on the way to prove these hypotheses.
Internalization of the extracellular adherence protein (Eap)
of Staphylococcus aureus by endothelial cells
I. Joost1, L. von Müller1, K. T. Preissner2, M. Herrmann1
University Hospital des Saarlandes, Institute for Medical
Microbiology und Hygiene, Homburg, Germany
University Hospital Giessen und Marburg GmbH, Institut für
Biochemie, Giessen, Germany
Introduction: The extracellular adherence protein (Eap) of S.
aureus belongs to the SERAM family (secretable expanded
repertoire adhesive molecules) and exhibits a broad spectrum of
antiangiogenic functions. While some of these functions can be
explained by Eap interaction with cell adhesion molecules
(ICAM-1), most recently, we have described Eap effects on the
endothelial signalling system suggesting intracellular target(s) of
Eap (Sobke et al., FASEB J 2006). Accordingly, in this study
we have investigated whether or not Eap is endocytosed by
endothelial cells as well as the mechanism of uptake and
intracellular fate of Eap.
Methods: Immunofluorescence microscopy was used to detect
protein uptake by Ea.hy 926 cells and for colocalization studies.
Cell fractions were subjected to Western blots analysis for
detection of Eap in different cell compartments. FACS- analysis
was used to determine uptake kinetics; furthermore, using
pathway-specific uptake inhibitors, putative endocytotic
pathways were elucidated.
Results: Immunofluorescence microscopy showed the uptake of
Eap at concentrations ranging from 1-100 µg/ml. Western Blot
analysis revealed variable amounts of Eap in all tested fractions.
Colocalization studies yielded partial colocalization with the
EEA-1 (early endosomal antigen) but no colocalization with
Lamp-1 (lysosomal associated membrane protein). Uptake of
Eap occurs very fast, with the maximum uptake being observed
at 3 min, yet a substantial amount of Eap remains bound to the
cell surface. Uptake is completely abrogated at 4°C. After 24h
the intracellular amount of Eap is greatly reduced albeit still
detectable. Use of chlorpromazin (CPZ, 30 µM) revealed an
inhibition of Eap-uptake; additional inhibitors are currently
under investigation.
Conclusions: Endothelial cells are able to take up S. aureus Eap
in a time- and concentration-dependent manner. The
temperature dependence and speed of the uptake process
suggests specific uptake mechanism rather than passive
translocation, and chlorpromazine sensitivity indicates
involvement of the clathrin-mediated pathway. Colocalization
studies show a partial overlap with the early endosome but not
with the late endosome. In essence, this demonstration of
specific Eap uptake in concert with its profound effect on
endothelial signalling provides new and exciting aspects on the
role of Eap in S. aureus pathogenicity and a potential use of Eap
in infectious and non-infectious disease.
Identification and characterization of rOrf0, a novel LEEencoded effector protein of enteropathogenic and
enterohemorrhagic Escherichia coli
C. Buss1, D. Müller1, G. Heusipp1, M. A. Schmidt1
Westfaelische-Wilhelms-University Muenster, Institute of
Infectiology-ZMBE, Muenster, Germany
The infection with enteropathogenic Escherichia coli (EPEC),
enterohemorrhagic E. coli (EHEC) results in the induction of
attaching and effacing (A/E) lesions in epithelial cells, which are
characterized by the loss of microvilli, the intimate adherence of
the bacteria to host cells, and the induction of pedestal-like
structures underneath the adherent bacteria due to a
reorganization of the cytoskeleton. The phenotype is associated
with the „Locus of Enterocyte Effacement“ (LEE), a
pathogenicity island encoding a type III secretion system
(TTSS) designed to deliver effector proteins into the host cell.
Whereas the identification of LEE-encoded effector proteins is
thought to be completed, the number of EPEC/EHEC effector
proteins still increases by the identification of additional
prophage or O-island-encoded substrates of the EPEC/EHEC
While sequencing the LEE locus of the clinical isolate ATEC
3431-4/86 we identified rorf0 as an additional LEE-encoded
open reading frame, that encodes a yet undescribed
EPEC/EHEC effector protein. Although absent in the prototype
strains EPEC E2348/69, EHEC O175:H7 Sakai and EHEC
EDL933, PCR analysis revealed that rorf0 is widely distributed
among attaching and effacing pathogens. Furthermore,
sequencing of effector loci indicated, that rorf0 is not
necessarily associated with the LEE PAI, but is also found
encoded on lambdoid prophages. Employing pull-down
experiments, we identified IQGAP1 (IQ motif containing
GTPase activating protein 1), an important scaffolding protein
involved in actin organization during basic cellular processes
like cell migration or adhesion, as a host cell interaction partner
of rOrf0. During infection, both, rOrf0 and IQGAP1 are
recruited to ATEC-induced pedestals, implying an involvement
of both proteins in the ATEC-induced subversion of the host cell
cytoskeleton. Currently, studies are under way to further
characterize the IQGAP1/rOrf0 interaction and its contribution
to the virulence of of EPEC and EHEC strains.
Modulation of the canonical Wnt/ -catenin signalling
pathway in Helicobacter pylori infection
G. Xia1,2, T. Gnad2, U. Lendeckel2, M. Naumann2
Eberhard-Karls-University, University Hospital of Tuebingen
Medical Microbiology and Hygiene, Tuebingen, Germany
Otto-von-Guericke-University Magdeburg, Institute for
Experimentelle Medizin, Magdeburg, Germany
The gastric human pathogen H. pylori settles about fifty percent
of all humans and is implicated in the induction of chronic
gastritis, peptic ulcers and gastric cancer. The mechanisms
involved in these processes are not well understood. The
pathogenesis is strongly dependent on a specialized type IV
secretion system (T4SS), which builds a syringe-like structure
and is encoded by the cag pathogenicity island (PAI). The T4SS
translocates the bacterial effector protein CagA into host cells,
thereby activating c-Met and inducing morphological changes
and cell motility (1). The pathogen further activates a variety of
host factors like the transcription factor NF- B (2) and the
tyrosine kinase receptor EGF (3).
In this study we show that the canonical Wnt/ -catenin
signalling pathway, which is dysregulated in almost every
human cancer, including gastric cancer, is modulated by H.
pylori. Activation of the pathway leads to stabilization of catenin, translocation to the nucleus and subsequent
transcriptional activation of target genes together with
transcription factors of the Lef/Tcf family.
1. Churin, Y. et al. (2003). J Cell Biol 161, 249-255.
2. Foryst-Ludwig, A. and Naumann, M. (2000) J Biol Chem
275, 39779-39785
3. Keats, S. et al. (2001) J Biol Chem 276, 48127-48134.
A small RNA upstream of the ica operon in Staphylococcus
epidermidis is possibly involved in biofilm regulation
M. Eckart1, T. Sakinc2, W. Ziebuhr3
Julius-Maximilians-University, Institut für Molekulare
Infektionsbiologie, Wuerzburg, Germany
Ruhr-University, Medizinische Microbiology, Bochum
s University, School of Biomedical Sciences, Belfast
Northern Ireland
The human pathogen Staphylococcus epidermidis is known for
its ability to form biofilms on implanted medical devices by
synthesis of a polysaccharide intercellular adhesin (PIA). Genes
involved in PIA production are organized in the icaADBC
operon. IcaR is one of the regulators repressing ica-operon
expression. We identified spontaneous mutants of strain 307
which show a reduced biofilm formation in an adherence assay,
carrying an IS256 insertion upstream of the icaR gene. In this
intergenic region, which has a size of about 1.4 kb, we were able
to demonstrate the existence of a RNA transcript of 487 nt. The
5’- and 3’-ends of this RNA could be determined by 5’-3´RACE and primer extension, respectively. Nucleotide sequence
analysis revealed a promoter structure upstream of the
transcription start and a Rho-independent transcription
terminator. But, neither a reasonable ribosomal-binding site nor
a start codon was detectable. Therefore, we regard this transcript
as an untranslated RNA which might be involved in biofilm
regulation. We constructed an IGRica-sRNA deletion mutant by
replacing the IGRica-sRNA gene with an ermB resistance
cassette. The mutant showed the same diminished adherence
phenenotype as the IS256 insertion mutants. To investigate the
influence of the IGRica-sRNA on the expression of the biofilm
genes, quantitative real time PCR experiments were performed.
The relative gene expression of the repressor icaR, the Nacetylglucosaminyl-transferase icaA and the IGRica-sRNA was
measured in the wildtype and the mutants. The obtained data
point to an impact of the transcript in the regulation of PIAmediated biofilm formation in S. epidermidis.
Muropeptide modification - amidation of peptidoglycan Dglutamate does not affect the proinflammatory activity of
Staphylococcus aureus.
D. Kraus1, H. Kalbacher2, J. Buschmann3, B. Berger-Baechi4
F. Goetz3, A. Peschel1
Eberhard-Karl-University, Medical Microbiology and Hygiene
Tuebingen, Germany
Eberhard-Karl-University, Medical and Natural Sciences
Research Center, Tuebingen, Germany
Eberhard-Karl-University, Microbial Genetics, Tuebingen
University of Zurich, Medical Microbiology, Zurich
components, are amidated in Staphylococcus aureus for
unknown reasons. To study whether this modification may
modulate the proinflammatory capacity, cytokine induction by
isogenic S. aureus strains with different amidation levels and by
synthetic amidated/nonamidated muramyldipeptides was
compared. However, amidation did not significantly affect
cytokine induction. This finding contributes to defining
peptidoglycan receptor specificities and indicates that further
rationales for muropeptide amidation have to be considered.
Establishment and optimization of mixed biofilm test
systems with periodontal pathogens
K. Standar1, W. Muenter1, S. Redanz1, B. Kreikemeyer1
A. Podbielski1
University Rostock, Institute for Medical Microbiology
Rostock, Germany
Periodontitis gradually develops in the subgingival sulci when
biofilm-organized physiological bacteria are stepwise
exchanged by pathogenic species. Development of more
elaborate therapies than mechanical removal of biofilms is
hampered by difficulties for in vitro simulation of periodontitisspecific biofilms. In the present study, protocols for the
generation and optimization of mixed species biofilms with both
physiological and pathogenic bacterial species are to be
established as a prerequisite to assess the beneficial effects of
chemical substances, physical measures and probiotic
microflora on such biofilms using functional and transcriptomic
In this study, protocols for two species biofilms under static
culture conditions have been tested. For this purpose,
Streptococcus mitis or S. salivarius were combined with
Aggregatibacter actinomycetemcomitans (A.a.), Fusobacterium
nucleatum (F.n.), Micromonas micros (M.m.), and
Porphyromonas gingivalis (P.g.). As culture media, brain heart
infusion (BHI) and chemical defined medium (CDM) were used
alone or with supplements of artificial saliva and/or human
serum. The bacteria were seeded simultaneously or one day
apart into uncoated polystyrene wells and were cultured for up
to 5 days under anaerobic conditions. Resulting biofilms were
optically quantified by safranin stain and were inspected by
fluorescence or scanning electron microscopy. Initially, more
biofilm was formed in BHI + human serum under anaerobic
conditions for all S. mitis combinations, while all mixtures with
S. salivarius favoured CDM. However, when CDM was
supplemented with 50 mM glucose or sucrose, in many cases
biofilm formation was greatly improved. Transwell assays
showed a positive influence on growth of A.a. by S. mitis and S.
salivarius while there was a reducing effect on biofilm growth
of F.n., P.g. and M.m. Successive seeding did not greatly alter
biofilm amounts if artificial saliva was used. However,
CDM/sucrose medium led to an increase in safranin stain for all
combinations with S. salivarius. Instead, mixtures of S. mitis
with F.n. and P.g. showed a reduction in biofilm amounts when
CDM was supplemented with glucose. In summary, the
influence of both streptococcal species on the remainder in these
mixed biofilms is dependent on the medium composition and
has to be determined for each combination under investigation.
Evidence of a functional two-partner secretion system in
Neisseria meningitidis
C. Schmitt1, D. Turner2, M. Frosch1, O. Kurzai1
University of Wuerzburg, Institute of Hygiene and
Microbiology, Wuerzburg, Germany
University of Nottingham, Division of Microbiology and
Infectious Diseases, Nottingham, United Kingdom
Several large exoproteins in various gram-negative bacteria are
predicted to be secreted via the Two-partner secretion (TPS)
system. However, relatively few of these proteins have been
investigated in detail. The most extensively characterized of
these exoproteins, the filamentous hemagglutinin (FHA) of
Bordetella pertussis, is an important virulence factor of
Bordetella and constitutes the main component of the pertussis
vaccine. In general, FHA and related proteins, termed TpsA for
two-partner-secretion-protein A, are translocated across the
outer membrane via a specific transporter termed TpsB. All
three published meningococcal genomes contain ORFs encoding
putative TpsA (in meningococci termed HrpA) and TpsB
(termed HrpB) proteins as indicated by sequence similarities to
known TPS systems. As we have shown previously, genes
encoding HrpA and HrpB proteins are present in virtually all
clonal complexes. However, a high degree of C-terminal
sequence variation between tpsA genes of different clonal
complexes could be detected. To investigate the expression of
meningococcal hrpA genes and to assess a possible role of the
associated hrpB genes, a polyclonal rabbit antiserum was raised
against the HrpA protein encoded by NMB1779 of strain MC58
(serogroup B, ST-32 complex). For that purpose, NMB1779 was
expressed and purified in E.coli resulting in a protein with a
molecular weight of approximately 210kDa. By Western blot
analysis of whole cell lysates and concentrated supernatants of
strain MC58 and its respective hrpA and hrpB deletion
mutants, we provide evidence, that meningococcal HrpA are
expressed, translocated across the outer membrane by their
cognate transporter HrpB and mainly released into the
environment. During this process, HrpA is proteolytically
processed to a mature 180kDa form. A small percentage of
mature HrpA remains associated with the bacteria and
contributes to the interaction of meningococci with human
epithelial cells.
RNomics: Experimental identification of novel small nonprotein-coding RNAs in Staphylococcus aureus
L. F. Abu-Qatouseh1, J. Seggewiß1, S. V. Chinni2, J. Brosius2
T. S. Rozhdestvensky2, G. Peters1, C. von Eiff1, K. Becker1
University Hospital Muenster, Institute of Medical
Microbiology, Muenster, Germany
University of Muenster, Institute of Experimental Pathology
ZMBE, Muenster, Germany
Background: Only in recent years, the importance of small nonprotein-coding RNAs (npcRNAs) has been fully appreciated.
Most of these RNA transcripts regulate virulence gene
expression in bacterial pathogens in response to host signals.
However, very few npcRNAs have been identified in Grampositive species, including staphylococci. Here, we present for
the first time the experimental identification and characterisation
of small npcRNAs from a clinical strain pair of Staphylococcus
aureus displaying the normal phenotype and the small-colony
variant (SCV) phenotype.
Methods: Total RNA from different growth phases of S. aureus
clinical isolates was extracted and size-fractionated (from 10 to
500 nt). To identify small npcRNAs in isogenic clinical S.
aureus strains exhibiting the normal and the SCV phenotype,
two separate cDNA libraries were constructed. About 300
clones of the cDNA libraries were randomly chosen, sequenced,
and analysed by BLASTN database search against S. aureus N
315 genome. For confirmation of the predicted npcRNA
candidates, Northern blot analyzes were applied.
Results: Thirty four putative candidates for novel small
npcRNAs in S. aureus were identified. Most of these npcRNA
candidates (59%) were located in the intergenic regions of
predicted or hypothetical protein coding genes, in part in
antisense orientation (24%). Interestingly, some of these small
npcRNAs were specific to the S. aureus phenotype and were
differentially expressed.
Conclusion: Beside implications on the understanding of S.
aureus virulence, novel npcRNA candidates in clinical S. aureus
isolates may shed a light on phenotypic variations associated
with S. aureus. Further investigation of the functional aspects of
these novel npcRNAs might provide new strategies in the
control of S. aureus infections.
The broad host range bacteriophage H8 is T5-like and
infects many Salmonella wild-type strains and escherichia
coli through the ferric enterobactin receptor, FepA
W. Rabsch1, L. Ma2, S. M. C. Newton2, M. Schallmey1
J. Laverde-Gomez1, P. E. Klebba2
Robert Koch-Institut, Bereich Wernigerode, Wernigerode
Department of Chemistry and Biochemistry, University of
Oklahoma, Norman, USA
H8 is a bacteriophage of an existing Lalko/Laszlo phage typing
scheme of Salmonella Enteritidis and found by morphology and
genomic structure to closely resemble the virus T5 of the Family
Siphoviridae. The phage infects S. Enteritidis, S. enterica, E.
coli and other Enterobacteriaceae by initial adsorption to the
outer membrane protein FepA. Ferric enterobactin inhibits the
binding of H8 to the E.coli cell surface, other ferric catecholate
receptors (Fiu, Cir, IroN) do not participate in phage adsorption.
H8 penetration into the cell is TonB-dependent, but exbB
mutations in Salmonella and E. coli do not confer resistance to
the bacteriophage. However, exbB and tolQ double mutants are
resistant to H8 infection. Experiments with a collection of
deletion and substitution mutants of E. coli FepA showed that
bacteriophage adsorption and penetration utilizes many of the
same charged residues in the receptor protein that promote the
binding and transport of ferric enterobactin and colicins B and
D. DNA sequence determinations of the complete H8 genome
showed that it is 104,4 kb in size, and extensively homologous
to that of bacteriophage T5. Sequence comparisons (AC171169)
of receptor binding protein rbpH8 showed that relative to the
Oad protein of T5 and the Hrs protein of BF23. Furthermore, the
pore-forming tail proteins of H8 (Tpf), T5 (Pb2), T1 (P33) and 8
(J) are highly homologous along their entire length. This
bacteriophage alone or in combination with other broad host
range phages could play an important role in heterogenous
biofilms control.
Gene expression- and regulation of Staphylococcus aureus
during nasal colonization
M. Burian1, C. Wolz1, C. Goerke1
Eberhard-Karl-University Tuebingen, University of Tuebingen
Institute for Med. Microbiology und Hygiene, Tuebingen
Staphylococcus aureus colonizes the nose of 30% of the human
population. The anterior nares are the primary reservoir from
which S. aureus can cause life-threatening infections. A specific
interaction between bacterial molecules and the host tissue
during nasal colonization has to be assumed. Most genes are
tightly regulated. However, the regulatory circuits leading to
adaptive gene expression during colonisation are largely
unknown. In previous work, we were able to reliably follow
gene expression in vivo by direct transcript analysis
(LightCycler RT-PCR) in specimens contain >104-105 CFU. In
order to analyse nasal colonization in humans (S. aureus
densities <103 CFU/swab) different methods were evaluated to
lower the limit of sensitivity. By optimizing the efficiency of the
RNA isolation and of the RT-PCR employing a two-step
protocol reliable quantification down to 10 copies of specific
transcript was achieved. Additionally, the use of random
hexamer primers during reverse transcription permitted the
quantification of different target genes from a single cDNA
pool. To determine absolute amounts of specific transcripts
RNA standard molecules were constructed for each gene in
question. The expression of the constitutively expressed gene
gyr is used as reference. To study differential gene expression
during colonization nose swabs from five persistent S. aureus
carriers were obtained and isolated RNA subjected to direct
transcript analysis. The in vivo expression of the following
genes is assessed i.) global virulence regulators like agr, sae and
sigB ii.) genes coding for cell wall anchored proteins iii.) genes
coding for secreted proteins and iv.) genes coding for enzymes
necessary for the synthesis of extracellular polysaccharides (ica,
cap). The in vivo results are compared with the transcriptional
pattern of the respective isolates grown in vitro to the
exponential and post exponential growth phase. Furthermore,
transcript levels will be correlated with the expressed surface
antigens detected by immunofluorescence. This approach will
allow us to get insights into the regulatory adaptation of S.
aureus during colonisation of its primary reservoir.
The expression of major virulence genes of S. aureus is
differentially modulated by the histidin kinase SaeS
M. Mainiero1, T. Geiger1, C. Goerke1, C. Wolz1
Eberhard-Karl-University Tuebingen, University of Tuebingen
Institute for Med. Microbiology und Hygiene, Tuebingen
Staphylococcus aureus can produce a wide range of accessory
molecules which mediate specific interactions with the host-cell.
Most of these factors are tightly regulated by global regulatory
loci. Within this network sae appears to be a central downstream regulator which controls the expression of major
virulence genes, e.g., hla (coding for a-hemolysin), hlb (coding
for ß-hemolysin), coa (coding for coagulase), eap (coding for
extracellular adherence protein) or fnbA (coding for fibronectin
binding protein A). The sae operon consists of four ORFs, two
of which code for a two-component system with a histidine
kinase (SaeS) and its corresponding response regulator (SaeR).
The N-terminal sensor domain of SaeS is characterised by two
intramembrane helices separated by a short extracytoplasmatic
domain. Sequence analysis revealed a single AS variation (L to
P) within the first N-terminal intramembrane loop of SaeS from
strain Newman compared to SaeS from other S. aureus strains.
We analysed the effect of this AS exchange on target gene
expression in different strains. The variation results in hyperactivation of the SaeS/R system, most probably due to
constitutive phosphorylation of SaeS and consequently SaeR.
However, the hyper-activation of SaeS results in overexpression of only some of the saeR/S target genes, e.g., fnbA,
coa and eap. Interestingly, hla and hlb expression are unaffected
by the SaeS mutation, indicating that these target genes are
insensitive to SaeS activity. This was further confirmed using
saeS deletion strains. In these strains, hla and hlb was shown to
be activated by SaeR independent of SaeS. Other target genes
such as fnbA, coa and eap are highly dependent on SaeS. We
hypothesise, that SaeR phosphorylation is required only for a
subset of SaeR target genes.
Characterization of a Staphylococcus aureus double mutant
defective in hemin biosynthesis and in the global redox
sensing regulator (Rex)
G. Sander1, S. Strompen1, A. Fischer1, J. Seggewiß1, K. Becker1,
P. McNamara2, G. Peters1, R. A. Proctor2, C. von Eiff1
University Hospital Muenster, Institute of Medical
Microbiology, Muenster, Germany
University of Wisconsin Medical School, Department of
Medical Microbiology and Immunology, Madison, USA
Small-colony variants (SCVs) of Staphylococcus aureus
represent a slow-growing subpopulation with distinctive
phenotypic and pathogenic traits. They are better able to persist
in mammalian cells and are less susceptible to antibiotics than
their isogenic wild-type counterparts. In past studies, sitedirected menadione- or hemin-auxotrophic S. aureus mutants
displaying the SCV phenotype were constructed by interrupting
genes of the menadione (menD) or hemin (hemB) biosynthesis.
Sequence analysis showed that many virulence- and regulator
genes as well as genes of the catalytic network include a
putative binding sequence for a regulator protein called redox
sensing regulator (Rex). This global regulator is predicted to be
involved in regulation of respiratory chain components in
response to oxygen stress. In order to characterize the metabolic
network of SCVs and to figure out whether Rex is involved in
the SCV phenotype, a rex/hemB double mutant was constructed.
The rex mutant was constructed by deleting 20 nucleotides of
the gene including the start codon and leading to a frameshift
using the parent strain S. aureus SH1000. Then, hemB was
interrupted in this mutant by inserting an erythromycin cassette
into the hemB gene. The double mutant exhibited the phenotype
of SCVs and showed the same slow growth rate in Tryptic Soy
Broth as described for the S. aureus hemB mutant. In addition,
the double mutant revealed a reduced susceptibility towards
aminoglycosides and a different protein expression pattern
compared to the wildtype and the rex mutant. In contrast to the
parent strain, the rex mutant and the rex/hemB double mutant
did not produce staphylococcal enterotoxin A, alpha-toxin, SplC
(a serine protease), RNAIII, and SrrA (a response regulator
controlling fermentation enzymes) as shown by Western Blot
analyses and semi-quantitative RT-PCRs.
In conclusion, these results show that Rex might play a role in
regulating virulence- and regulator genes in response to oxygen
stress and might be responsible for some of the biochemical and
physiological characteristics obsverved in SCVs.
Mutations may be involved in the formation of menadioneauxotrophic small-colony variants (SCVs) of Staphylococcus
aureus recovered from clinical specimens
S. Strompen1, T. Cordes1, G. Sander1, A. Fischer1, J. Seggewiß1
R. A. Proctor2, G. Peters1, K. Becker1, C. von Eiff1
University Hospital Muenster, Institute of Medical
Microbiology, Muenster, Germany
University of Wisconsin Medical School, Department of
Medical Microbiology and Immunology, Madison, USA
Small-colony variants (SCVs) of Staphylococcus aureus have
been recovered from patients with chronic, persisting and/or
relapsing infections. These variants are frequently auxotrophic
for menadione or hemin, two compounds required in the
biosynthesis of the electron transport chain components
menaquinone and cytochromes, respectively. Two site-directed
mutants generated by insertionally inactivating (i) the menD
gene using an ermC cassette and (ii) the hemB gene using an
ermB cassette displayed the phenotypic characteristics typical of
clinical SCVs. Main features of both mutants included slow
growth, decreased pigment formation, reduced susceptibility
towards aminoglycosides, low coagulase activity, and reduced
hemolytic activity.
In order to analyze putative genetic events as a cause for the
formation of the SCV phenotype, sequence analyses of the
operons involved in both the menadione (menAFDXB, menCE,
and menG operons) and the hemin biosynthesis (hemAXCDBL
operon) were performed. For this purpose, SCVs recovered from
patients with chronic infections and shown to be auxotrophic for
hemin or menadione were used. In addition, the respective
regions of the clonal identical parent strains (according to
pulsed-field gel electrophoresis) were sequenced.
In two menadione-auxotrophic SCVs, genetic defects were
detected compared to their parent strains. In both cases, the
mutations were located in the menB gene. One mutation was
found to be a deletion of 9 base pairs leading to an alteration of
four amino acids, whereas the other mutation was a single base
pair deletion causing a frame shift and the introduction of a stop
codon after 230 amino acids. Both genetic events are likely to
lead to the production of inactive dihydroxynaphthoate synthase
encoded by menB and, hence, an impaired menadione
biosynthesis. However, no sequence alterations were detected in
two menadione-auxotrophic SCVs and in five heminauxotrophic SCVs compared to their respective parent strains.
Thus, mutations in one of the menadione biosynthesis genes
may be responsible for the SCV phenotype, however, cannot
explain the formation of SCVs in all cases. Subject to further
studies, regulatory mechanisms might be also involved in the
generation of SCVs.
Attachment site tolerance of pathogenicity island
A. Rakin1, U. Antonenka1
Max von Pettenkofer-Institut, Munich, Germany
Recombinases are responsible for the fate of horizontally
acquired pathogenicity islands in the new host. They are
sequence-specific and recognize unique attachment site(s) on
the host chromosome. They utilize Lambda-like site-specific
recombination using the bacterial (attB) and the elementencoded (attP) attachment sites for cointegation with the host
genome. Here we address the specificity of the attB x attP
reaction mediated by recombinases of asn tDNA recognizing
genomic islands.
Four asn tDNA-associated genomic islands, the HighPathogenicity Island (HPI) encoding iron acquisition system in
Yersinia pseudotuberculosis, the “pks island” (Ecoc57N)
encoding a peptide-polyketide genotoxin in uropathogenic
Escherichia coli CFT073, and two islands with unknown
functions, HAI7 and HAI13, from the plant pathogen Erwinia
Enterobacteriaceae. The ability of island-encoded recombinases
to use their own as well as “heterologous” attP sites for
recombination was investigated. The functionality of
recombinases was proven by their ability to promote excisive
recombination of the integrated islands. The attP sites were
recovered from circularized forms of the islands by PCR and
cloned in a Pir-dependent suicide vector. Such plasmids were
used in trans-recombination assay for cross-complementation
with each of four recombination proteins in recA-deficient
background. Only recombinases of two islands, HPI and
Ecoc57N, supported recombination of both cognate and
“heterologous” attP sites with asn tRNA genes. IntHPI mediated
attPEcoc x asn tDNA and IntEcoc - attPHPI x asn tDNA reaction,
but with reduced frequency. In contrast, integrases from HAI7
and HAI13 islands with even higher homology were unable to
substitute one another in recombination. This might be
explained by a higher discrepancy of the attPHAI7 and attPHAI13
attachment sequences.
So, even minor sequence deviations in recombination sites
involved in site-specific recombination could considerably
decrease the ability of the laterally-acquired mobile elements to
cointegrate with the host genome and serve as a restrictive factor
for unlimited acquisition of pathogenicity islands.
Analysis of the Pyp regulatory network controlling the
transcription of hreP and the flp operon in Yersinia
J. Schilling1, K. Wolters1, S. Faelker1, M. A. Schmidt1
G. Heusipp1
University of Muenster, ZMBE, Institute of Infectiology
Muenster, Germany
The coordinated expression of virulence genes in pathogenic
bacteria is tightly regulated. In a previous genetic screen, we
identified three genes (pypA, pypB and pypC; protein involved
in the regulation of Yersinia hreP expression A, B, C), whose
products control the expression of the virulence-associated HreP
protease in the human pathogen Yersinia enterocolitica. PypA is
an inner membrane protein with six or eight membrane domains.
PypB belongs to the family of membrane-localized ToxR-like
transcriptional regulators. PypC is a cytoplasmic protein and
also has a DNA binding domain of typical transcriptional
regulators. In analogy to the ToxR/ToxS/ToxT regulatory
cascade of Vibrio cholerae regulating the expression of the
cholera toxin, PypA, PypB and PypC might constitute a
regulatory network controlling hreP transcription in which
PypA activates PypB or PypC, which then serve as
transcriptional activators of hreP. We characterized the
interactions of the pyp gene products on transcriptional level.
PypB and PypC do not only regulate transcription of hreP, but
also of pypB and pypC. Moreover, pypB and pypC are
autoregulated. However, overproduction of neither Pyp protein
results in increased transcription of the pypA gene. Furthermore,
PypA has no effect on transcription of pypA, pypB or pypC. By
deletion analysis we identified PypB binding regions in the
hreP, pypB, and pypC promoter regions. In addition we could
show that PypB induces the transcription of the flp operon
responsible for type-IV-like pilus production.
Identification of a new regulatory network in Yersinia
K. Wolters1, J. Schilling1, S. Faelker1, M. A. Schmidt1
G. Heusipp1
University of Muenster, ZMBE, Institute of Infectiology
Muenster, Germany
HreP was previously identified as an in vivo expressed protease
important for full virulence of the human pathogen Yersinia
enterocolitica. So far, no in vitro conditions are known that lead
to hreP expression under laboratory conditions. We established
a transposon screen for regulators of hreP transcription in Y.
enterocolitica and identified three genes, termed pypA, pypB,
and pypC (protein involved in regulation of Yersinia hreP
expression A, B, C) which induce hreP transcription after
overproduction. All pyp genes have a low GC% content
reminiscent of horizontal gene transfer. PypA is an inner
membrane protein with 6 or 8 transmembrane regions, while
PypB and PypC have DNA binding domains typical for
transcriptional regulators. Similar to ToxR-like transmembrane
regulators, PypB is located in the cytoplasmic membrane with
an unusually short periplasmic domain. For PypB we could
demonstrate that it directly binds to the hreP promotor region.
Employing various pyp deletion strains we could further show
that induction of hreP transcription after PypC overproduction
depends at least partially on PypA. In Y. enterocolitica PypA is
able to induce hreP transcription independently of PypB and
PypC, while in E. coli PypA overproduction alone is not
sufficient for hreP transcription. In addition to the
characterization of this sophisticated signaling system, we are
now able to analyze the HreP protease after expression in Y.
enterocolitica, which will be helpful in understanding the role of
HreP in pathogenesis of Y. enterocolitica.
Staphylococcus species as potential virulence factors in
bacterial skin infections
F. Layer1, B. Ghebremedhin1, R. Hartig2, W. König1
B. König1
Otto-von-Guericke-University, Institute of Medical
Microbiology, Magdeburg, Germany
Otto-von-Guericke-University, Institute of Immunology
Background: The colonization and infection with staphylococci
is a serious issue in skin disorders especially atopic dermatitis
(AD). Human skin epithelium provides a mechanical barrier to
invading bacteria and also participates in innate immune defence
by producing cationic antimicrobial peptides, e.g. -defensins
(hBD) and the cathelicidin LL-37. The aim of our study was to
determine the potential virulence of staphylococci from AD
patients in bacterial skin infections and to examine the innate
immune response to infection through expression of
antimicrobial peptides.
Methods: Pathogenicity profiles of Staphylococcus spp. isolated
from AD patients were determined by screening genes for
staphylococcal enterotoxins (sea-see, seg-sej), toxic shock
syndrome toxin 1 (tst) and Panton-Valentine leukocidin (lukPV) by PCR. Furthermore we used a model of bacterial skin
infection to test the pathophysiology of these isolates in a human
keratinocyte cell line (HaCaT), including the induction of hBD1, hBD-2, hBD-3, LL-37, IL-6 and IL-8 (real time PCR, ELISA)
and cellular invasion (fluorescence staining, confocal laser
scanning microscopy).
Results: Analyzing of toxin genes revealed various
pathogenicity profiles for S. aureus, although we could not
detect superantigenic exotoxins by coagulase-negative
staphylococci. Exposure of keratinocytes to Staphylococcus spp.
triggers the production of cytokines IL-6 and IL-8 and of defensins (hBD-1 mRNA (2- to 8-fold), hBD-2 mRNA (2- to
270-fold), hBD-3 mRNA (2- to 6-fold)) and the cathelicidin LL37 (2- to 6-fold), whereas the levels of this activity were
different for all clinical isolates. Adherence and invasion to
keratinocytes was shown for clinical isolates of S. schleiferi and
S. aureus, but not for S. hominis, S. epidermidis and S. capitis
Conclusions: In summary, staphylococci from AD patients
showed various pathogenicity profiles, invaded keratinocytes
and triggered the production of cytokines and antimicrobial
peptides. Therefore our results mark them as potential pathogens
in skin infections.
The transcriptional regulator Rgg3 controls fibrinogenbinding of Streptococcus agalactiae and bacterial adherence
to epithelial cells
B. Schaller1, A. Tautzenberger1, B. J. Eikmanns1
D. J. Reinscheid2, F. Borges1
University of Ulm, Institute of Microbiology and Biotechnology
Ulm, Germany
University of applied science, Department of natural Science
Reinbach, Germany
Streptococcus agalactiae is the leading cause of sepsis and
meningitis in human neonates and emerged as an increasing
cause of invasive desease in adults. Beside an important role as
a pathogen, S. agalactiae is also a commensal bacterium of the
human urogenital and gastrointestinal tracts. These different
lifestyles require the presence of regulatory mechanisms in the
bacteria that control the expression of virulence associated
The present work describes the functional characterization of the
putative transcriptional Rgg-like regulator Rgg3 of
Streptococcus agalactiae. Deletion of the rgg3 gene in S.
agalactiae 6313 resulted in an increased adherence of the
mutant to the human lung epithelial cell line A549 and to the
human laryngeal epithelial cell line HEp 2.Futhermore the
ability to bind immobilized human fibrinogen was increased in
the rgg3 deficient mutant. These effects could be restored by the
presence of a plasmid carrying the rgg3 gene in the strain S.
agalactiae 6313
rgg3.Quantitative Real Time-PCR
experiments revealed that Rgg3 does not control the expression
of genes, encoding cell-wall anchored proteins with LPXTG
motif, including the fbsA gene, which encodes the main
fibrinogen receptor in S. agalactiae. Taken together, our
findings suggest an important role of rgg3 in the regulation of
novel genes in S. agalactiae.
Cell wall-deficient (CWD) L-form variants of the food-borne
pathogen Listeria monocytogenes
S. DellEra1, C. Buchrieser2, M. J. Loessner1, M. Schuppler1
ETH Zurich, Institute of Food Science and Nutrition, Zurich
Institute Pasteur, Laboratoire de Génomique des
Microorganismes Pathogènes, Paris, France
L-forms represent cell wall-deficient (CWD) variants of
bacteria. Their main characteristic is the absence of a rigid cell
wall, which is thought to result from an induction process
interfering with cell wall synthesis. Although several reports
exist on morphological, serological and biochemical properties
of L-forms, next to nothing is known about the molecular
background for this unusual and interesting transformation.
We established a stable L-form line from L. monocytogenes
strain ScottA, which was GFP-tagged by single copy insertion
of a modified pPL2 vector into a tRNA gene. L-forms were
obtained on agar plates supplemented with antibiotics. After
serial passage the stable L-forms were cultivated in special soft
agar medium devoid of antibiotics. Complete loss of the rigid
cell wall was confirmed by confocal laser scanning microscopy
and electron microscopy. Division of L-form cells leads to
small, protoplast-like vesicles but also to multi-nucleated macrocells which appear to be surrounded by a single membrane.
Immunological analyses confirmed loss of certain cell wallassociated proteins (e.g. Internalin A), whereas membraneanchored proteins, such as Internalin B, were shown to be still
For transcriptome analysis and gene expression profiling total
RNA was extracted from parental and L-form Scott A cells, and
subjected to whole genome macroarray analysis. The results
indicated to a different expression of numerous important genes
corresponding to differences in morphology and physiology. Lform metabolism seems to be apparently down-regulated,
whereas stress-related genes showed a strong up-regulation.
This observation appears to result from adaptations to the cell
wall-deficient state. GFP-labelled L-form cells were used to
study the pathogenetic potential of L. monocytogenes L-forms
by different methods (fluorescence microscopy, confocal timelapse microscopy, in vitro cell invasion assays). Our
observations suggest that, under specific conditions, Listeria
monocytogenes L-forms (i) can emerge and survive in the
environment; (ii) can multiply and divide actively; (iii) may
survive intracellular after engulfment by macrophages.
Endemic MRSA MLST22 spa-type t310 (MRSA310) carries
the lukF/S-PV genes on a phage highly related to phiPVL
U. Reichholf1, P. C.1, C. Irtenkauf1, A. Patten2, C. Aichinger2
U. Reischl1, N. Lehn1, H. - J. Linde1
University Ratisbon, Inst. Med. Microbiol., Ratisbon
Roche Diagnostics, Penzberg, Germany
Background: MRSA carrying the lukF/S-PV genes for the
Panton Valentine leukocidin (PVL) are emerging pathogens
worldwide. MRSA310 with PVL was first demonstrated in
Eastern Bavaria in 1995, and has since caused community- and
hospital-associated infections. This study investigated the
nucleotide sequence upstream and downstream of lukF/S-PV in
MRSA310 and compared it to published phage sequences.
Methods: MRSA310 was isolated from a 6 year old boy with an
abscess on his shoulder. Typing was performed as described
(; Total genomic DNA was
extracted and sequenced using the Genome Sequencer 20
system (Roche Diagnostics, Penzberg, Germany). For resolution
of ambiguous results appropriate primers were designed for
block cycler PCR and sequencing. Sequence analysis was
performed by BLAST tools ( att sites
were defined according to Kaneko, 1998.
Results: lukF/S-PV genes were detected between putative attL
and attR sites. A total of 41,436 phage related nucleotides (nn)
was identified, flanked by the conserved 29 bp core sequence of
attachment sites. Of all putative open reading frames (ORF), 47
(representing 32,304 nn) showed high homology (>96%) to
phiPVL. Compared to phiPVL, the % nn and % amino acid (aa)
identity and number of nn for selected genes were: Integrase
99/99 (1,206 nn), repressor 99/99 (771), ss-DNA binding
protein 99/100 (471 nn), portal protein 99/100 (1,251 nn), capsid
protein 99/99 (1,248 nn), holin 100/100 (303 bp), amidase 99/99
(1,455 nn), lukS-PV 99/100 (939 nn), lukF-PV 100/100 (978
nn). The putative phage is flanked upstream of the attL site by a
649 bp fragment with 99% identity to ORF SAB1349c from S.
aureus RF122. Downstream of the attR site, the putative phage
is flanked by a 186 bp ORF with 95% identity to ORF 1,380 of
S. aureus USA300.
Conclusion: In MRSA310 the lukF/S-PV genes are contained in
the genome of a phage with high similarity to phiPVL.
Considerable benefit to the host may be conferred by phiPVL or
highly related phage. Functional studies are under way.
Association between biofilm formation, insertion element
IS256, agr group specificity and antibiotic resistance profile
in nosocomial Staphylococcus epidermidis strains
B. Ghebremedhin1, F. Layer1, W. König1, B. König1
OvG University, Clinical Microbiology, Magdeburg, Germany
Background Staphylococcus epidermidis is a normal saprophyte
of the healthy human microflora, but it is also the most common
cause of nosocomial infections associated with catheters and
other medical devices. Isolates from device-associated
infections are known for their pronounced phenotypic and
genetic variability, and in this study we searched for factors that
might contribute to this flexibility.
Materials and Methods 113 Staphylococcus epidermidis isolates
from different sampling sites were analysed for the presence of
IS256 insertion element, agr group specificity, ability of biofilm
formation, presence of icaADBC operon, and antibiotic
resistance. The isolates were collected between June 2005 and
February 2006.
Results The study revealed that, in contrast to those of
commensal strains, the genomes of clinical Staphylococcus
epidermidis strains carry the gene of the insertion sequence
IS256. Detection of IS256 (total n=58; 51%) was found to be
associated with biofilm formation and the presence of the
icaADBC operon (n=46; 41%) as well as with resistance profile
[PEN-OXA-CIP-ERY-CLI] - n=31 of 51 were IS256 positive in the clinical strains. This resistance profile was associated with
the agr group II (n=22 of 43; 51%).
Conclusions We noted that both pathogenic and commensal S.
epidermidis strains can have different as well similar
characteristics. However, the data suggest that IS256 is a
characteristic element in the genome of multiresistant
nosocomial S. epidermidis isolates that might be involved in the
flexibility and adaptation of the genome in clinical isolates. The
formation of biofilm is not significantly associated with the
presence of the insertion sequence IS256 whereas the agr group
II correlated with the presence of the insertion sequence IS256
RhlR expression in Pseudomonas aeruginosa is modulated
by the Pseudomonas quinolone signal (PQS) via PhoBdependent pathways
V. Jensen1, D. Loens1, C. Zaoui1, F. Bredenbruch1
A. Meissner1, G. Dieterich2, R. Munich3, S. Haeussler1
Helmholtz-Center for Infection Research, Chronic
Pseudomonas Infection Research Group, Brunswich
Helmholtz-Center for Infection Research, Department of Cell
Biology, Brunswich, Germany
Technical University Brunswich, Department of Microbiology,
Brunswich, Germany
The expression of virulence determinants in Pseudomonas
aeruginosa is coordinately regulated in response to both the
social environment - commonly referred to as quorum sensing and to environmental cues. In this study we have dissected the
various independent regulation levels for pyocyanin production
which is influenced by the homoserine lactone- and
Pseudomonas quinolone Signal (PQS)-mediated quorum sensing
systems as well as by iron and phosphate availability. We
demonstrated that the phosphate (Pho) regulon is involved in the
transcriptional activation of rhlR and the augmentation of PQS
and pyocyanin production under phosphate limitation. These
results highlight the complexity of secondary metabolite
production in P. aeruginosa via environmental cues and quorum
sensing. Using a bioinformatic approach we have identified 237
putative PHO boxes in the whole P. aeruginosa genome
suggesting an interconnection between environmental cues and
virulence determinants. As a global transcriptional regulator
PhoB is probably not only phosphorylated via its cognate sensor
kinase PhoR but - apart from phosphate starvation - may be
activated under various other environmental conditions, that
warrant further studies.
Distribution of the pks genomic island among
C. Forestier1, E. Carniel2, U. Dobrindt3, T. Oelschlaeger3
J. Hacker3, J. Putze3
University of Auvergne, Clermont-Ferrand, France
Pasteur Institute, Paris, France
Unversity of Wuerzburg, Institute for Molecular Infection
Biology, Wuerzburg, Germany
Escherichia coli is a common commensal of the natural gut
microflora. Healthy individuals are not affected by commensals
but rather have a beneficial effect from those bacteria. Besides
other bacteria like lactobacilli, E. coli strain Nissle 1917 is well
known for its beneficial effects on the gut microflora. There is a
constant communication between commensal/probiotic bacteria
and their hosts but the exact modes of action remain still
E. coli Nissle 1917 lacks virulence factors, is a good colonizer
of the human gut and is a well-studied microorganism. EcN is
therefore used as a model organism to investigate commensal
and probiotic properties.
Recently, a genomic island of approximately 54 kb was
identified in E. coli, which codes for nonribosomal peptide
synthases, polyketide synthases and hybrid nonribosomal
peptide-/polyketide synthases. This pks genomic island is
present in different E. coli strains, e.g. commensal strain E. coli
Nissle 1917, NMEC IHE3034, UPEC CFT073 and commensal
E. coli strains. The synthesized peptide-polyketide induces DNA
double strand breaks in eukaryotic cells, blocks mitosis and
induces megalocytosis. Genomic analysis of the pks genomic
island revealed several indications for horizontal acquisition of
this island, namely an elevated GC-content compared to the core
genome of E. coli strain CFT073, presence of a tRNA-gene
(asnW), presence of a P4-integrase and flanking of the island by
direct repeats. This suggests 1) another source of the pks
genomic island than E. coli and 2) a distinct function of the pks
genomic island as it will most likely not be present in strains
without a selective advantage for the host. To address these
issues we started to investigate the distribution of this genomic
island among members of the Enterobacteriaceae by PCRscreening.
Our results show that the pks genomic island is not exclusively
present in E. coli strains but also in other strains of the family
D-serine deaminase of Staphylococcus saprophyticus;
construction and analysis of an isogenic mutant
T. Sakinc1, N. Michalski1, B. Kleine1, M. Kaase1, F. Szabados1
S. Gatermann1
Ruhr-University of Bochum, Institute for Hygiene and
Microbiology, Bochum, Germany
D-serine is one of the most prevalent amino acids excreted in
mammalian urine at reported levels of 3 to 40 µg/ml, and it can
be found in mammalian blood as well. D-serine catabolism is
biologically important because it is available in some
environments as a readily utilizable nutrient source.
D-serine is bacteriostatic to toxic to many living organisms and
has inhibitory effects for growth.
The dsd locus is comprised of dsdC, a positive transcriptional
regulator, dsdX, a putative D-serine permease and dsdA, the Dserine deaminase (dehydratase) enzyme. These genes are
responsible for the detoxification of D-serine. DsdA converts Dserine to ammonia and pyruvate and D-serine can be used as a
sole carbon and nitrogen source.
We have tested three staphylococci species S. aureus , S.
epidermidis and S. saprophyticus. Only S. saprophyticus
showed growth with D-serine in media. Furthermore, we
amplified a part of the D-serine deaminase gene only from S.
saprophyticus. In addition, we cloned whole D-serine locus and
for further analysis, we constructed an isogenic dsdA knock-out
mutant in S. saprophyticus.
Therefore we conclude that D-serine deaminase is necessary for
S. saprophyticus for survival and growth in urine and presence
of this enzymatic activity may be a general phenomenon in
uropathogenic bacteria.
Detection of SdrI hydrophobicity with BATH - and SAT tests; effects of Ca2+ and EGTA
S. Terjung1, B. Kleine1, S. Gatermann1, T. Sakinc1
Ruhr-University of Bochum, Institute for Hygiene and
Microbiology, Bochum, Germany
A hydrophobic surface might facilitate adherence of bacteria to
the uroepithelium.
The hydrophobic capabilities of S. saprophyticus, a known
cause of urinary tract infections in young woman, have been
examined both by BATH - and SAT -tests.
Both methods indicated that the serine - aspartate repeat protein
of S. saprophyticus, SdrI, is involved in the hydrophobicity of
the bacteria, since the wild - type - S. saprophyticus - strain
7108 and a Sdr I revertant strain were hydrophobic whereas a
SdrI-knock-out-strain was hydrophobic. Additionally, it could
be shown that expression of the SdrI in S. saprophyticus CCM
883 and in S. carnosus clearly increased the hydrophobic
properties of these strains.
When EGTA was added, hydrophobicity was reduced, this
indicates that Calcium binding by the EF - hands present in SdrI
might stabilize the protein or induce a tertiary structure
necessary for this characteristic.
Our data clearly indicate that the collagen ? binding protein SdrI
also conveys hydrophobic surface properties.
However, as S. carnosus expressing the SdrI did not show
collagen binding whereas S. saprophyticus CCM 883 did,
another surface factor must be involved in this property.
Non spa-typable Staphylococcus aureus strains are naturally
occurring protein A mutants
C. Baum1, B. Haslinger-Loeffler1, H. Westh2, K. Boye2
G. Peters1, B. C. Kahl1
University Hospital of Muenster, Institute for Medical
Microbiology, Muenster, Germany
Hvidovre Hospital, Klinisk mikrobiologisk afd., Hvidovre
Background: Protein A (spa) belongs to the covalently bound
cell-wall associated proteins of Staphylococcus aureus with
either 4 or 5 IgG Fc-binding domains, which inhibits
phagocytosis and activates inflammation by activating TNFR1
signaling. Single locus DNA-sequencing of the repeat region of
spa (spa-typing) is used for reliable and discriminatory typing of
the pathogen. Rarely, strains cannot be spa-typed. In this study,
we characterized 5 non spa-typable blood culture isolates on the
molecular level.
Materials and Methods: The entire spa gene of the non spatypable strains was sequenced. Transcriptional analysis of spa
was performed by Northernblot and quantitative RT-PCR.
Protein expression was assessed by Western ligand blot. SpA of
2 strains with mutations and of the standard strain CowanI was
purified using an IgG column. The inflammatory role of SpA
was analyzed by determining the IL-1ß activity of MNCs with
an ELISA test.
Results: Sequence analysis of spa confirmed the presence of the
gene and its repeat region in all strains by using primers
upstream and downstream of the binding sites used for spatyping. It was possible to identify the spa-types of 4 strains, in
which whole IgG Fc-binding domains were deleted while in the
5th strain the spa-type could not be assigned due to deletions at
the beginning of the repeat region resulting in a non-sense
mutation. Transcription of spa was detected in all strains, while
only 4 strains expressed SpA on the protein level. Sufficient
amounts of SpA from CowanI and the 2 strains with SpA
mutations were purified from 2L bacterial cultures, analyzed by
Western ligand blot for specificity and quantified at OD280nm
with one-point calibration. IL-1ß activity of MNCs by
commercial SpA was dose dependent. Further studies are in
progress to analyze the inflammatory roles of purified mutated
Conclusions: Although all strains were not spa-typable using
standard primers, spa was present albeit mutations. The fact that
these strains were isolated from patients with invasive infections
indicates that in spite of these natural occurring spa mutations,
the strains were still highly pathogenic.
Characterization of adherence-mediating protein regions of
Mycoplasma pneumoniae
N. Schurwanz1, R. Dumke1, E. Jacobs1
Technical University of Dresden, Institute of Medical
Microbiology and Hygiene, Dresden, Germany
Mycoplasma pneumoniae (M.p.) is a common agent of
community-acquired human respiratory tract infections such as
atypical pneumonia or tracheobronchitis. Adherence of the
bacterium to the ciliated epithelia cells is a precondition for
colonization and subsequent induction of disease. Cytadherence
process is mediated by a specialised tip-like attachment
organelle. A set of unique surface proteins, including the
adhesin P1 and the adherence-related proteins P30 and P65, is
concentrated in this structure. To prevent infections due to M.p.
by an efficient vaccine, an adherence-inhibiting immune
response directed against adhesion proteins has to be generated.
To prove the role of the P1 protein as major adhesin, specific
antisera against the M.p. wild type strain M129 and a
cytadherence-negative mutant, lacking the P1, were obtained by
immunizing guinea pigs. A quantitative in vitro adhesion
inhibition assay was used to detect the adherence of the wild
type and the mutant to HeLa cells or MRC-5 lung fibroblasts.
Adherence inhibition was investigated by pre-incubating the
wild type with the obtained sera. We could show, that the
adherence inhibitory effect of the mutant antiserum was much
weaker than the influence of the wild type serum. With respect
to the different codon usage in mycoplasmas we expressed
defined regions of the P1, P30 and P65 gene as recombinant
proteins (rp1-15, rp30, rp65). The binding properties of these
peptides to HeLa or MCR-5 cells were quantified using an
ELISA-based assay. 11 of 17 tested recombinant proteins were
shown to bind efficiently to the cells. However, inhibition
studies using specific rp-antisera for pre-incubating the peptides
resulted in a reduced binding of only 6 peptides. Testing the rpspecific antisera in the adhesion inhibition assay seems to be a
promising tool to identify protein regions which are directly
involved in cytadherence and could be of interest to select
candidates for immunization strategies. Besides the inhibition of
M.p. adherence to the respiratory epithelium of the host an
efficient elimination of the pathogen by the cellular immune
response is important to prevent infections due to M.p.
Therefore interactions of human monocytes or guinea pig
alveolar macrophages with mycoplasmas, opsonized with rpspecific antisera, are now investigated.
Role of sigma factors RpoS and RpoE in virulence of avian
pathogenic Escherichia coli
C. Ewers1, S. Günther1, E. - M. Antáo1, A. Bethe1, S. Kiessling1
I. Diehl1, T. Homeier1, G. Li1, S. Glodde1, L. H. Wieler1
Free University of Berlin, Institute for Microbiology and
Epizootics, Berlin, Germany
The alternative sigma factor RpoS is recognized as a global
stationary-phase RNA subunit that controls the expression of a
large number of genes involved in cellular responses to stress.
Another sigma factor, sigma 24 (RpoE), originally identified as
a factor required for survival on exposure to extremely high
temperatures, acts primarily as a repair system for denatured
proteins. While there is extensive knowledge on the role of
sigma factors in Escherichia coli laboratory strains, only a
limited number of studies account for the contribution of sigma
factors to the pathogenicity of wildtype isolates.
We analysed the sequence of the rpoS and rpoE regulons in
avian pathogenic E. coli (APEC), which is the causative agent of
colisepticemia in poultry. Based on virulence features and
sequence types shared with human extraintestinal pathogenic E.
coli, APEC are suggested to be zoonotic. To ascertain the role of
sigma factors RpoS and RpoE in the virulence of APEC, knockout mutants of the encoding genes were generated and employed
in animal experiments and real time PCR assays. Highly
virulent APEC strain IMT5155 (O2:K1:H5), representing one of
the most common phylotype (sequence type 95) of this
pathovar, was used for genetic manipulations.
Real time PCR analysis was performed on 18 virulenceassociated genes, coding for factors involved in adhesion (crlA,
tsh), iron acquisition (fyuA, tonB, iroN, fur, sit, iutA, chuA,
ireA), serum resistance (iss, ompA, colV), and invasion (ibeA,
ibeB, ompA) as well as toxin gene vat. For the the rpoSknockout mutant we observed upregulation particularly in tsh,
iutA and ibeA, while ompA, tonB, fur, chuA, iroN, and vat were
found to be upregulated in the rpoE-knockout mutant by at least
two-fold. Interestingly, the outer membrane gene ompA, which
is known to be associated with invasion of the brain
endothelium by new born meningitis E. coli, is about nine-fold
upregulated in the rpoE-mutant.
In vivo experimental data indicate that the rpoS mutation does
not significantly affect the virulence of APEC strain IMT5155
in 5-week old chickens, as determined by clinical and organ
lesion scores and the bacterial load in chicken organs 24 and
48h post infection; in contrast, a complete loss of virulence was
seen in the rpoE-knockout mutant. The animals did not develop
any clinical symptoms, the organ lesions were negligible, and
bacteria were rapidly cleared from the lung as shown by
calculation of cfu values.
Our data show for the first time the pivotal role of sigma factors
in the virulence and pathogenicity of avian pathogenic strains.
Phage-related mechanisms underlying the differences in
prevalence of Hlb-converting phages and PVL-encoding
phages of Staphylococcus aureus
C. Wirtz1, M. Zink1, C. Wolz1, C. Goerke1
Eberhard-Karl-University of Tuebingen,University Hospital
Institute for Medical Microbiology and Hygiene, Tuebingen
In Staphylococcus aureus virulence factors are often encoded on
bacteriophages. Typical S. aureus phages are the ß-hemoysin
(hlb) converting phage 13, which carries the immune evasion
molecules SAK, CHIPS and SCIN, and the PVL-encoding
phage Sa2mw. While Hlb-converting phages are widely
distributed, PVL-encoding phages seem to be less frequent in S.
aureus. When comparing S. aureus strain collections from
infectious and colonizing situations we could confirm these
differences in prevalence. The aim of our study was to
determine phage-related mechanisms leading to this unequal
distribution in the S. aureus population. Experiments were
performed with lysogens of the prototypic strain 8325-4 to avoid
host-dependent effects. First, we investigated whether 13 and
Sa2mw differ in their ability to be mobilized from the
bacterial chromosome. The amount of extra-chromosomal phage
DNA was detected in the lysogens 8325-4 13 and 83254 Sa2mw, respectively by quantifying the newly formed attP
sites of the phages using Real Time PCR. This method proved
to be more sensitive than the commonly used phage plaque test.
We could show that both phages were inducible with
subinhibitoric concentration of the antibiotics mitomycin and
ciprofloxacin but not by growth at 42°C or under anaerobic
conditions. Overall, 13 showed about 5 times higher rates of
induction compared to Sa2mw. Secondly, possible differences
in the transfer rate of these phages between bacterial strains
were determined in mixed culture experiments employing
selectable resistance labelled phages and recipients. Phage 13
proved to be more transmissible than Sa2mw. Transmission
was in both cases enhanced by adding ciprofloxacin. Next, we
analysed whether both phages are able to form stable lysogens
after transfer into different S. aureus strains. 13 integrated into
its original host 8325-4 with the highest frequency and was also
able to lysogenize an already phage containing strain (83254 13) to a lesser extend. Interestingly, also one clinical isolate
(s64) was sensitive to 13. In contrast, Sa2mw re-integrated
into strain 8325-4 with very low frequencies and the integration
site was often aberrant.
Legionella pneumophila – Phase-variable changes and
delivery of its LPS components in host cells and in broth
K. Reichardt1, E. Jacobs1, J. Helbig1
Technical University of Dresden, Institute of Medical
Microbiology and Hygiene, Dresden, Germany
Modulating effects of LPS components established by L.
pneumophila are under consideration because strains of
serogroup 1 carrying the so-called virulence-associated LPS
epitope, recognized by the monoclonal antibody (MAb 3/1) of
the Dresden Panel, cause most of the community-acquired
legionellosis. Up to now it is unclear, if the LPS equipment of
these strains only enhances the transmission of bacteria in
aerosols or additionally modulates host cells. Therefore, we
tested the MAb 3/1-positive strain Corby for its LPS pattern
with two different MAbs. MAb 3/1 recognizes long and short
chain LPS components whereas the second one, MAb 59/1,
recognizes phase-variable short chain LPS components. By
using ELISA and immunofluorescence, we examined broth
cultures of different growth phases for shed and phase-induced
LPS components. The received results of broth cultures revealed
that LPS components recognized by MAb 3/1 are expressed
continuously onto the bacterial surface and are partially
delivered into the broth. In contrast, LPS components being
positive for MAb 59/1 show an inhomogeneous and phase-
variable expression which increased from 3% of the bacteria in
the exponential up to 34% in post-exponential growth phase.
Concomitant, samples of different host cells infected by strain
Corby were analysed by immunofluorescence microscopy.
Investigations of intracellular life evinced similarities to life in
broth concerning the shedding and the phase-variability.
Accessorily, we demonstrated that shedding of MAb 3/1positive LPS seems to regulate expression of MAb 59/1 epitopes
which increased during the intracellular replication period.
Furthermore, we have noticed that bacteria located in
phagosomes express a LPS pattern which differs from that of
single bacteria. While marginal located bacteria of replicative
phagosomes as well as single bacteria express only MAb 3/1positive LPS, merely bacteria of the inner regions are positive
for MAb 59/1 and attached to each other. Modulating effects of
the LPS were demonstrated by an infection assay with
intracellular delivered MAb 3/1 before infection with
legionellae which shows a reduction of the intracellular
multiplication rate. Our findings substantiate that the LPS
expression is regulated environmentally and therefore the
shedding of LPS is one of the equipment of L. pneumophila to
modulate host cells.
Characterization of an unorthodox sensor kinase in
Pseudomonas aeruginosa
C. Zaoui1, T. Becker1, F. Bredenbruch1, D. Loens1, S. Häußler1
Helmholtz-Center for Infection Research, Chronic
Pseudomonas Infections, Brunswich, Germany
Various classes of antibiotics trigger a Pseudomonas quinolone
signal (PQS) dependent self-induced DNA release in
Pseudomonas aeruginosa. In this study we show that signal
transduction via an unorthodox sensor kinase of a twocomponent system is crucial for this process. The sensor kinase
knock-out mutant did not respond to antibiotic stress with an
enhanced DNA release and the mutation conferred an antibiotic
tolerant phenotype.
Transcriptome analysis of the sensor kinase mutant
supplemented with PQS revealed that most of the genes
belonging to the oxidative stress response -that have previously
been described to be induced in the PAO1 wild type under PQS
supplementation- were either not regulated or down-regulated.
These results point at the involvement of the unorthodox sensor
kinase in the sensing of cellular oxidative stress, and thus its
participation in the activation of the oxidative stress response.
In further studies we aim at identifying the signal(s) upon which
the sensor kinase is activated, and currently perform
phosphorylation assays in presence of various compounds that
might be relevant for the activation of the sensor kinase.
Pathotyping of Pseudomonas aeruginosa isolated from
chronically infected wounds
S. Schmoldt1, I. Özkaya1, M. Götzfried1, J. Heesemann1
M. Hogardt1
Ludwig-Maximilians-University of Munich, Max von
Pettenkofer-Institut, Munich, Germany
Chronic infections with Pseudomonas aeruginosa are
predestinated for the selection of host adapted P. aeruginosa
variants. In this study, we investigated 130 P. aeruginosa
isolates from 73 patients treated<!--[if !supportAnnotations]-->
at the Munich university clinic suffering from chronically
infected wounds with respect to their cell cytotoxicity, mutation
frequencies, antibiotic resistance patterns and several
established virulence-associated traits including the strainspecific endowment with one of the two major type IIIdependent exotoxins ExoS and ExoU.
The exoU- and exoS-prevalence among these strains was found
to be 38.0% and 62.0%, respectively. For comparison, the exoUprevalence among PA isolates from blood cultures and stool
samples was determined and found to be 40% for blood culture
isolates and 35.3% for stool isolates, suggesting an evenly
distribution of exoU-positive strains among gut colonizers,
wound isolates and invasive P. aeruginosa strains.
Hypermutability tests revealed no clear mutator strains among
P. aeruginosa isolates from chronically infected wounds. Most
isolates were positive for the tested virulence-associated traits
(siderophores, pyocyanin, swimming motility, twitching
motility, elastase and protease), while extremely high and low
producers were found. In agreement, a high variability in
cytotoxicity of P. aeruginosa wound isolates against J774
macrophages was measured. Distinct cell cytotoxicity typically
was found for exoU-positive P. aeruginosa isolates. However,
several exoS -/exoU + isolates exhibited only very low
cytotoxicity indicating a defect in ExoU production or secretion,
while several exoS +/exoU - isolates exhibited a remarkable high
cytotoxicity against J774 macrophages.
In conclusion, the genotypic and phenotypic characterization of
P. aeruginosa isolates from chronically infected wounds showed
a broad heterogeneity. The expression and production of the
type III secretion machinery was highly variable and seems to
be a major virulence determinant of PA wound isolates.
D- amino oxidase activity might be a virulence factor for
urinary tract infections in S. saprophyticus
F. Szabados1, N. Guengoer1, A. Orland1, N. Kanageswaran1
B. Kleine1, T. Sakinc1, S. Gatermann1
Ruhr-University Bochum, Institute for Hygiene and
Microbiology, Bochum, Germany
The D-serine deaminase is present in uropathogenic E. coli
strains, in contrast to non- uropathogenic E.coli and reverses the
bactericidal effect of D-serine. In uropathogenic E. coli D-serine
was shown to be an important regulator of uropathogenicity. It
was our hypothesis that D-amino acid metabolism other than Dserine may be involved in uropathogenicity.
To investigate the metabolic pathways of S. saprophyticus, we
to establish a definite artificial urine growth medium for
uropathogenic bacteria. This is the first description of a definite
artificial urine for growth of S. saprophyticus. Definite amino
acid ingredients in physiologic amounts have replaced complex
additives such as tryptic soy broth. For further growth studies
selected L-amino acids were replaced with D-amino acids.
D-alanine inhibits growth of S. aureus and S. epidermidis in
contrast to S. saprophyticus. Interestingly most staphylococci
except S. saprophyticus lack D-amino oxidase genes. The
inhibiting effect of these D-amino acids was increased in
presence of low concentrations of glucose and the effect was
concentration dependent. Although D-alanine is part of the
peptidoglycan of most bacteria, growth of most staphylococci
was inhibited by D-alanine in artificial urine.
S. saprophyticus biofilm formation was impaired in certain Lamino acid compositions, indicating the impact of amino acid
metabolism on biofilm formation under near physiologic
Human urine contains high concentrations of D-amino acids. Dalanine and D-serine have an inhibiting effect on bacterial
growth, indicating a possible host defence mechanism in the
bladder. The bacterial D-amino oxidase may play an important
role in uropathogenic bacteria not only by making new carbon
and nitrogen sources accessible and survival possible n this
harsh environment, but also as a possible regulatory factor for
uropathogenicity in analogy to D-serine deaminase.
Identification of transcriptional regulators associated with
type II secretion systems of Yersinia enterocolitica
B. Sutinoski1, M. A. Schmidt1, G. Heusipp1, K. Wolters1
J. Schilling1
University of Muenster, ZMBE, Institute of Infectiology
Muenster, Germany
The hallmark of an infection with the enteropathogen Yersinia
enterocolitica is the type III secretion of effector proteins
directly into the host cell cytoplasm. While type III secretion is
very well studied, relatively little is known about the role of type
II secretion systems (T2SS) in the pathogenesis of Y.
enterocolitica. In other bacterial pathogens, T2SS have been
shown to secrete proteins necessary for full virulence.
Environmental conditions, growth phase, and quorum sensing
mediate regulation of the expression of T2SS. We show that two
previously described operons encoding T2SS of Y.
enterocolitica, Yst1 and Yst2, are associated with transcriptional
regulators. These regulators, which we termed PypC and PypC2,
share 45% identity on amino acid level. Interestingly, both are
transcribed in one operon with the genes encoding the respective
T2SS. While Yst2 including the PypC regulator is also found in
the other pathogenic Yersinia species Y. pestis and Y.
pseudotuberculosis, Yst1 including PypC2 can only be found in
Y. mollaretii, which is not a human pathogen. The PypC
proteins show similarity to membrane-localized CadC-like
transcriptional activators; however, a transmembrane domain
could not be identified. Interestingly, BLAST searches of
bacterial genomes identified a PypC-like protein with a
transmembrane domain associated with the genes for a T2SS in
Serratia proteamaculans, indicating that the PypC regulators of
Yersinia might have evolved from CadC-like regulators but lost
their membrane localization. In future analyses we will
characterize the regulatory network underlying the expression of
Yst1 and Yst2 as well as aim at identifying putative secretion
Identification of LrhA as a new regulator of Salmonella
typhimurium virulence and motility genes
D. Chikkaballi Anne Gowda1,2, P. Schwerk2, K. Tedin2
Humboldt-University, Institute for Biology, Berlin, Germany
Free University, Institute for Microbiology and Animal
Epidemic, Berlin, Germany
Salmonella enterica serovar Typhimurium is a facultative
intracellular pathogen, capable of invasion and survival in host
cells. Invasion and survival involves numerous virulence
factors, which are tightly regulated by complex environmental
conditions and regulatory pathways.
In previous gene expression studies in our lab, a mutant strain of
Salmonella which is non-invasive and avirulent in mice, showed
severe reductions in expression of nearly all genes previously
shown to be involved in invasion and intracellular survival
despite few changes in expression of global (house-keeping)
regulatory genes. One exception to this general pattern was an
over-expression phenotype for a putative transcriptional
regulator, LrhA.
An lrhA mutant was up to 2-fold more invasive than the
wildtype strain for both epithelial cells and macrophage, but
showed a similar rate of intracellular proliferation. A microarray
analysis of LrhA over-expression revealed severe reductions in
expression of most of the pathogenicity islands, including all the
major regulators of SPI1 gene expression including hilA, hilC,
hilD and rtsA. Transcriptional gene fusions also demonstrated at
least two-fold repression of hilC, hilD and rtsA gene expression
after LrhA over-expression. These results suggest that LrhA
regulation of virulence gene expression could be indirect,
through rtsA regulation. However, LrhA regulates other
pathogenicity islands in addition to SPI1, where RtsA is not
known to play a role in the regulation.
Nucleoid binding proteins have been shown to selectively
silence pathogenicity island genes acquired by horizontal gene
transfer in Salmonella. Over-expression of LrhA resulted in
essentially the same silencing of pathogenicity island genes
without affecting flanking chromosomal regions, suggesting a
similar preference for regulation of virulence genes. Taken
together, these studies suggest LrhA is a potential DNA binding
protein involved in the repression of both motility and virulence
gene expression in Salmonella. Current studies are directed at
determining interactions with other proteins in the regulatory
hierarchy of virulence gene expression in Salmonella.
Characterisation of cell wall hydrolases in Staphylococcus
M. Schlag1, R. Biswas1, S. Zoll2, F. Goetz1
University of Tuebingen, Microbial Genetics, Tuebingen
University of Tuebingen, Institute for Bio-chemistry, Tuebingen
Peptidoglycan (PG) hydrolases or autolysins are a group of
enzymes that catalyse the cleavage of murein at specific sites
during cell separation and growth. Staphylococcus aureus
produces two major PG hydrolases: the major autolysin (AtlA)
and Aaa, an autolysin/adhesin protein. The atl gene product is a
bifunctional protein that consists of an N-terminal N-acetyl Lalanine amidase (Ami) domain and a C-terminal endo-ß-Nacetylglucosaminidase (GL) domain linked by three internal
repeat domains (R1, 2, 3). Proteolytic processing generates the
two extracellular lytic enzymes (62 kDa Ami-R1, 2 and 51 kDa
R3-GL) that can be found in the culture broth of S. aureus. In
the mature protein repeats R1 and R2 are located at the Cterminal portion of the amidase (Ami-R1, 2) and repeat R3 is
located at N-terminal portion of the glucosaminidase (R3-GL).
In our studies, we have purified the Atl amidase, derived from
the related organism Staphylococcus epidermidis. For that we
have expressed the protein in E.coli along with one and two
repeat regions as well as only the catalytic domain linked to
Gluthiatione-S-transferase (GST) in E.coli. We were able to
crystallize the catalytic domain of the AtlE amidase under
different buffer conditions. The X-ray analysis is still in
Furthermore we intend to use purified amidase as a molecular
tool to isolate defined peptidoglycan structures for
immunological studies.
Functional characterization of a new polyketide expressed in
Escherichia coli
S. Homburg1, J. Hacker1, U. Dobrindt1, G. Krumbholz1
University of Wuerzburg, Institute for Molecular Infection
Biology, Wuerzburg, Germany
Polyketides are a remarkable class of natural products with an
enormous range of functional and structural diversity. This
group of secondary metabolites includes numerous important
antibiotics, immunosuppressants, anticholesterolemics, as well
as antitumor, antifungal and antiparasitic agents. Polyketide
synthase genes have been identified from a variety of biological
sources, such as bacteria, fungi, and plants. In the case of E.
coli, comparative genomics led to the discovery of a 54-kilobase
genomic island harbouring genes that show high similarities to
known polyketide synthase and non-ribosomal peptide synthaseencoding genes. E. coli strains possessing this genomic island
(including pathogenic and commensal E. coli) induce a contact
dependent megalocytosis in cultured eukaryotic cells
characterized by an enlargement of the cells and DNA double
strand breaks.
The discovery of this genomic island encoding a putative
polyketide raises the question of function and advantageous
properties for strains expressing the polyketide. To examine the
biological role of the polyketide, its putative function as an
antibiotic or iron uptake system has been studied. Furthermore,
transcription of selected genes of the polyketide determinant
was compared in response to different growth conditions using
reporter gene assays.
To understand the transcriptional organisation of the pks
genomic island, the operon structure was determined by RTPCR. Several genes within the island were shown to be
organized in polycistronic operons. In addition, primer
extension analysis was performed to identify the transcriptional
start points of genes of interest. To allow heterologous
expression of the polyketide, the encoding determinant was
expressed in Pseudomonas putida. Our results provide a basis
for further structural and functional analyses of the novel
Different effects of Helicobacter pylori VacA on human and
murine T cells
S. Prassl1, X. Sewald1, B. Gebert-Vogl1, M. Fabbri2, D. Busch3
P. Sebo4, R. Haas1
Max von Pettenkofer-Institute, Bacteriology, Munich
DIBIT-Scientific Institute San Raffaele, Unit of Leukocyte
Biology, Milan, Italy
Technical University of Munich, Institute for Medical
Microbiology, Immunologie und Hygiene, Munich, Germany
Academy of Sciences of the Czech Republic, Institute of
Microbiology, Prague, Czech Republic
Helicobacter pylori is a bacterial pathogen that colonizes the
stomach of about 50% of the world’s population. In most cases
the initial colonization leads to a chronic infection with the
bacterium which usually lasts for decades or even for life. To
evade the immune system H. pylori expresses a set of virulence
factors, among them the vacuolating cytotoxin (VacA), which
plays an important role in modulating the immune response. The
secreted mature toxin (95 kDa) shows different effects on
different cell types. After binding and uptake into target cells,
VacA causes vacuolation which means the formation of large
acidic vacuoles inside the cell. Another effect of VacA is the
inhibition of T lymphocyte activation by interfering with the Tcell receptor/IL-2 signalling pathway at the level of the
Ca2+/Calmodulin dependent phosphatase calcineurin. IL-2
transcription and therefore the activation and proliferation of Tcells is inhibited. In a human T cell line as well as in primary
human T cells a VacA dependent inhibition of T cell activation
as well as vacuolation of the cells can clearly be shown. In
contrast, murine T cells do not respond in the same way if
exposed to the toxin. A weaker binding compared to the human
T cells can be observed but there is no vacuolation and no
inhibition of IL-2 production of the murine T cells.
The human integrin subunit 2 is necessary for uptake of the
toxin into the cell. Stable transfected murine T cells expressing
the human Leukocyte Function Associated Antigen-1 (LFA-1;
CD11a/CD18) or Mac1 (CD11b/CD18) gain sensitivity towards
induction of vacuolation by VacA. Interestingly, the inhibition
of IL-2 production can not be observed. Other intracellular
components which differ in humans and mice may be involved.
As a consequence, the suitability of the mouse model for H.
pylori virulence studies has to be questioned.
Role of capsular polysaccharides in decreased susceptibility
to vancomycin in Staphylococcus aureus
A. Jansen1, C. Szekat1, C. Wolz2, C. Goerke2, G. Bierbaum1
University of Bonn, Institute for Medical Microbiology
Immunology and Parasitology, Bonn, Germany
Eberhard-Karl-University , Institute for Medical Microbiology
and Hygiene, Tuebingen, Germany
Intermediate vancomycin resistance in Staphylococcus aureus
(CLSI: MIC=4-8 µg/ml) has been assigned to an increased cell
wall thickness as a consequence of an activated cell wall
biosynthesis and decreased autolysis. The mechanism of
resistance was shown to be based on an enhanced amount of
free d-Ala-d-Ala termini in the cell wall acting as false target
sites. This way, the diffusion of the vancomycin molecules
through the cell wall is impeded. In order to investigate the
genetic basis of this resistance mechanism, the expression
profiles of two vancomycin intermediately resistant S. aureus
(VISA) strains were compared with a clonally related
susceptible control strain employing microarrays. Surprisingly,
the genes involved in type 5 capsule biosynthesis figured
foremost in the microarray experiments. Thus, the role of the
microcapsule in decreased susceptibility against vancomycin
was investigated further. To this end, capsule production was
determined in different growth phases of selected VISA and
vancomycin susceptible S. aureus (VSSA) strains by
immunofluorescent labelling and transcript quantification using
real time PCR. Additionally, vancomycin susceptibility levels of
capsule defective mutants of a VSSA and a VISA strain were
analysed by MIC determinations, cultivation on vancomycin
gradient agar and population analysis. As could be shown by the
transcription levels of the essential gene capE, capsule
expression of VISA strains SA137/93A, SA137/93G and Mu50
was increased in comparison with the susceptible S. aureus
strains Reynolds, Newman and SA1450/94 in exponential as
well as in stationary growth phase. The capsule defective mutant
of VISA strain SA137/93G showed higher susceptibility to
vancomycin, while the knock-out had no effect on susceptibility
in the highly sensitive strain Reynolds. In conclusion, an
increased production of capsular polysaccharides seems to
contribute to intermediate vancomycin resistance in
Staphylococcus aureus.
Signalling activity of staphylococcal cell wall components
J. Buschmann1, R. Biswas1, M. Nega1, T. Volz2
T. Biedermann2, F. Goetz1
Eberhard-Karl-University , Microbial Genetic, Tuebingen
Eberhard-Karl-University , Universitys-Hautklinik, Tuebingen
One of the first lines of defence is the innate immune system.
This type of immune response is stimulated by the recognition
of conserved components of microorganisms called pathogenassociated molecular patterns (PAMPs). These PAMPs consist
of molecules not found in the host, including bacterial cell wall
components such as peptidoglycan (PG). Recognition of
PAMPs by specific proteins called pattern recognition
molecules (PRMs) activates inflammatory signaling pathways.
The gram-positive bacterium Staphylococcus aureus is a major
pathogen responsible for a variety of diseases. We are
investigating the role of the staphylococcal cell wall components
in signaling in human cell-lines and murine dendritic cells.
Defined PG-structures of S. aureus are purfied by HPLC and the
use of PG-hydrolytic enzymes. We want to find out which
staphylococcal envelope structures have the main function in
host signaling and how this structures are interacting.
The effect of moxifloxacin on post-stationary phase growing
senescent Staphylococcus aureus population
I. Chatterjee1, R. Rajbhandari1, M. Herrmann1
University of Saarland, Department of Medical Microbiology
Homburg/Saar, Germany
Introduction: Staphylococcus aureus infections can be difficult
to treat due to their remarkable capability to persist in the host.
Persistence and/or the evolution of resistance may be related to
several complex regulatory and metabolic networks, which
modifies transcription in response to environmental stress. As a
result, S. aureus has shown resistance to many commonly used
antibiotics. To understand how S. aureus persists during
antibiotic-therapy and eventually emerge resistant, we decided
to characterize the effect of a broad spectrum fluoroquinolone,
moxifloxacin (MXF), on stationary phase senescent S. aureus
and previously characterized clpC and sigB mutants (Chatterjee
et al., 2005).
Methods: Long-term growth and survival of S. aureus
DSM20231 (WT) and its isogenic mutants (both MIC = 0.03
mg/ml) were analyzed up to 5 days in presence of sub-inhibitory
concentrations of MXF (half the MIC), added at the beginning
(at 0 h) of the survival assay. Real-time RT-PCR was used to
examine the gene expression pattern of select S. aureus genes in
presence of MXF. Additionally, assays for glucose and acetate
concentration were performed.
Results: Presence of MXF (0.5 x MIC; added at beginning of
growth assay) was found to induce the transcription of cidC
(encoding pyruvate oxidase) in the WT strain during poststationary phase (74 h). Moreover, an up-regulation of asp23
(SigmaB target gene) and a sigmaB-dependent HSP, clpL were
also recorded in the WT, in presence of MXF. Additionally, the
WT (+ MXF) also accumulated higher concentration of acetate
as compared to the WT (- MXF). Consequently, the
staphylococcal cells (+ MXF) were found to enter the death
phase (³ 48 h) earlier as compared to the staphylococcal
population (-MXF). In contrast to these phenotypes, both the
clpC and the sigB mutants lacked an up-regulation of the asp23,
cidC and clpL transcript levels and an induction of enhanced
death-phase entry, in presence of MXF.
Summary: Our observations suggest an effect of subinhibitory
MXF concentration on global S. aureus regulation with the
consequence of an early induction of the death phase during
post-stationary phases. Moreover, the induction of early death
phase by MXF was evident in S. aureus only in presence of both
ClpC and SigB (+ ClpC + SigB dependent manner).
Collectively, our observation may explains the effect of MXF in
treating and eradicating staphylococcal population in chronic
infections such as endovascular & bone diseases.
Characterization of the type III secretion system chaperone
SycO of Yersinia enterocolitica
S. Dittmann1, A. Schmid1, G. Wilharm2, J. Heesemann1
University of Munich, Max von Pettenkofer-Institute, Munich
Robert Koch-Institute, Wernigerode, Germany
Y.pestis, Y.pseudotuberculosis, and Y.enterocolitica are the three
human pathogenic species within the genus Yersinia. These
Gram-negative bacteria share a type III secretion system (T3SS)
which allows them to translocate effector proteins into the host
cell. These so-called Yops (Yersinia outer proteins) inhibit the
immune response and enables them to survive and proliferate in
the host.
It has been shown that most of these effectors build a complex
with their specific Yop chaperone (Syc) in the bacterial cytosol
and are at least partially folded before secretion.
We succeeded in purifying recombinantly expressed SycO in its
outright form by ammonium sulfate precipitation followed by
size exclusion chromatography to near homogeneity. Further,
we confirmed dimerization by cross-linking experiments and
found a new interaction partner. In addition we present evidence
that this chaperone, like SycH, is integrated into the Yop
regulatory network.
Cross-talk between the type three secretion system and
metabolism in Yersinia enterocolitica
A. Schmid1, W. Neumayer1, G. Wilharm2, J. Heesemann1
University of Munich, Max von Pettenkofer-Institute, Munich
Robert-Koch-Institute, Wernigerode, Germany
Pathogenic Yersinia spp. (Y. pestis, Y. pseudotuberculosis and Y.
enterocolitica) share a protein transport machinery, called type
three secretion system (T3SS), with other gram-negative
pathogenic bacteria. This machinery is encoded on the pYV
plasmid and enables the bacteria to circumvent the innate
immune system by injecting so-called Yop (Yersinia outer
protein) effectors directly into the cytosol of host cells. YscM1
of Y. enterocolitica (LcrQ in Y. pseudotuberculosis and Y.
pestis) and the paraloguos YscM2 (missing inY.
pseudotuberculosis and Y. pestis) with 57% identity to
YscM1/LcrQ have been reported to be functionally equivalent
negative regulators of the T3SS. Using recombinant GSTYscM1 and GST-YscM2 in a pulldown assay to determine new
interaction partners we found an approximately 95 kDa protein
associating with both YscM1 and YscM2. This protein was
chromosomally encoded
spectrometry. We were able to confirm binding of recombinant
PEPC to recombinant YscM1 and YscM2 by native gel
electrophoresis. The enzymatic activity of the PEPC in the
presence of YscM1 or YscM2 was determined by coupling the
PEPC reaction to an NADH oxidating reaction. We could show
that in the presence of increasing amounts of YscM1 PEPC
activity decreases gradually. In contrast, increasing amounts of
YscM2 had no effect. In addition we could demonstrate that
YscM1 acts synergetically to the known PEPC inhibitors citrate
and aspartate and antagonistic to the PEPC activator acetyl
coenzyme A. As the PEPC is required for cells growing on
glucose as sole carbon source we performed growth experiments
with the Y. enterocolitica wildtype overproducing YscM1 or
YscM2 in M9 minimal medium. We could demonstrate that the
overproduction of YscM1 leads to an attenuation in growth.
Ex-vivo detection of extracellular adherence protein (Eap) of
S. aureus in wounds
I. Joost1, D. Blass1, M. Burian2, C. Görke2, M. Herrmann1
University Hospital des Saarlandes, Institute for Medical
Microbiology und Hygiene, Homburg, Germany
University Hospital Tuebingen, Institute for Medical
Microbiology und Hygiene, Tuebingen, Germany
Comparison of the interaction between THP-1 cells and cell
wall components of Mycobacterium avium ssp.
paratuberculosis and Mycobacterium smegmatis
E. Borrmann1, H. Köhler1, B. Burkert1, A. Hinsching1
B. Rohrmann1, C. Muselmann1
Federal Institute for Animal Health, Molecular Pathogenesis
Jena, Germany
Introduction: S. aureus is one of the most important pathogens
found in wounds. Previously, we have observed that the S.
aureus Eap exerts a strong antiangiogenic effect
(Athanasopoulos et al., Blood 2005) through inhibition of
MAPK signalling (Sobke et al., FASEB J 2006), resulting in
delayed wound closure. Accordingly, it is possible that Eap
plays a major role in delayed healing of S. aureus infected
wounds. However, while it is shown that all clinical S. aureus
strains contain eap, quantitative expression of eap under
authentic conditions in the wound has not yet been studied.
Accordingly, we wish to determine in situ eap expression
directly obtained from human specimen.
Methods: To establish ex-vivo analysis of eap expression, nasal
swabs from volunteers were obtained and snap-frozen in liquid
nitrogen. RNA preparations were performed from cultured S.
aureus as well as from direct swab specimen at RNAse free
conditions. c-DNA was generated by reverse transcription.
Sequence modified RNA standards were engineered for the
house-keeping gene gyrB and two different standards for eap,
reflecting two genetic distinct subtypes. eap expression levels
were compared to gyrB by qRT-PCR. For control, expression
was also analyzed in non-S.aureus species
Results: We confirmed that eap expression differs between
different growth phases, i.e. during the exponential phase 8-20
fold elevated eap transcript levels were detected compared to
gyrB transcripts, whereas during stationary growth the level the
amount of eap transcripts remains substantially below the gyrB.
First data obtained with colonization specimen suggest that the
amount of eap expression in situ corresponds to that of the in
vitro condition with 2-16 fold elevated eap transcripts compared
to gyrB transcripts. Other bacterial organisms (putatively
encountered in wounds) revelead no detection of RNA.
Conclusions: Here we present the successful establishment of a
protocol determining in situ eap expression in S. aureus. The
application of the protocol for authentic wound infection is
currently in progress in our laboratory. This protocol provides
the basis to determine the expression of eap in situ, and allows
for important evidence to the pathomechanistic consequences of
S. aureus wound infection. Such evidence may open new
preventive or therapeutic aspects directed against S. aureus Eap
in acute or chronic wound or soft tissue infection.
Paratuberculosis (John’s disease) is a chronic granulomatous
enteritis in ruminants caused by Mycobacterium avium ssp.
paratuberculosis (MAP).The crucial process in the pathogenesis
of this disease is the internalization of MAP by intestinal
macrophages, survival and intracellular multiplication. Although
the zoonotic potential of MAP has controversially been
discussed in several reports, an aetiological association between
MAP and Morbus Crohn could not be proved unequivocally
until now.
A unique pattern of all mycobacteria is the structure and
composition of the cell envelope consisting of the plasma
membrane and the wall. The specific cell wall structure is
believed to play an important role in the successful persistence
of MAP in macrophages. Lipoarabinomannan (LAM), one of
the biologically active cell wall components, is a
multiglycosylated form of the mannosyl phophatidyl-inositol. It
is known that LAM’s from diverse mycobacterial species differ
in the modification of terminal arabinose residues. These
modifications influence the macrophage response and so the
effect on the immune system.
The interaction between the human monocyte cell line THP-1
and LAM isolated from MAP as well as from M. smegmatis was
investigated. Both LAM’s were produced after cultivation of
both MAP and M. smegmatis using a Triton X-114 phase
separation technique (Severn et al., 1997). In combination with
nuclease and protease digestion and subsequent purification by
ultracentrifugation the isolation procedure delivered LAM’s
from bacterial cells without contaminating protein or DNA. The
purity of the products was tested by electrophoresis.
For the determination of the impact of these molecules on
modulating the macrophage response the production of mRNA
of the cytokines TNF-alpha, IL-1 and IL-10 were determined by
PCR. The biological active proteins IL-1 and IL-10 were
measured using ELISA and TNF-alpha using a cell test.
First results show that both mycobacterial species differ in their
effect on THP-1 cells.
Severn, W.B., Jones, A.M., Kittelberger, R., de Lisle, G.W.,
Atkinson, P.H.: Improved procedure for the isolation and
purification of Lipoarabinomannan from Mycobacterium bovis
strain AN5. J. Microbiol.Methods, 28 (1997) 123-130.
PQS production promotes tolerance towards SDS
M. Müsken1, T. Becker1, S. Häußler1
Helmholtz-Center for Infection Research, Chronic
Pseudomonas Infections, Brunswich, Germany
In Pseudomonas aeruginosa, diverse virulence determinants and
the formation of biofilms are regulated via the action of a
hierarchical cell-cell communication system which integrates
two classes of signal molecules, the N-acylhomoserine lactones
and the 4-quinolones. While the homoserine lactone molecules
diffuse freely between the cells, the Pseudomonas Quinolone
Signal, 2-heptyl-3-hydroxy-4-quinolone (PQS), is packaged into
membrane vesicles that serve to traffic this molecule within a
population (Mashburn and Whiteley, Nature, 2005). As PQS
directly promotes its package into the membrane vesicles and
PQS non-producing strains do not form membrane vesicles, we
hypothesized that a membrane-targeting antimicrobial agent
may exhibit an increased efficiency against PQS non-producers.
This might account for the previously observed effect that
biofilms of PQS negative strains are more sensitive to the
detergent sodium dodecyl sulfate (SDS) (Allesen-Holm et al.,
Mol. Micro., 2006). We investigated the influence of 0.2 % SDS
to planktonic cultures. By measuring the optical density and
determining bacterial viability by counting the colony forming
units (CFU), we provide evidence for a higher tolerance of the
wild-type towards SDS as compared to a PQS non-producing
mutant. Our results indicate that a PQS-dependent formation of
membrane vesicles may be an essential envelope stress
response. Moreover, since it has recently been shown that the
exogenous addition of rhamnolipids or SDS mediate central
hollowing of bacterial microcolonies and promote bacterial
dispersal from biofilms, a PQS differential within the
microcolony forming subpopulations might play a central role in
the dynamics of biofilm development and dispersal processes.
Characteristics of site-specific modified Legionella
pneumophila Pad mutants in the protozoan host
Acanthamoeba castellanii
F. Reschke1,2, L. Igel1,2, E. Kuhlisch2, P. C. Lueck1
Technical University of Dresden, Medical Microbiology and
Hygiene, Dresden, Germany
Technical University of Dresden, Medical Statistic and
Informatics, Dresden, Germany
Legionella pneumophila is a gram-negative bacterium
commonly isolated from lakes, streams, potable water supplies
and cooling towers. In these aquatic settings L. pneumophila
cells act as a facultative intracellular parasite, able to invade and
replicate within free-living amoebae. Thus, these protozoa may
serve as important reservoirs for the bacteria in an aquatic
environment. L. pneumophila can be transmitted from there by
aerosolization and may lead to Legionnnaire`s disease, a severe
form of atypical pneumonia or the milder form named Pontiac
L. pneumophila expresses the 16kDa protein Pad
(L.pneumophila amoebal adhesin) which is involved in the
uptake of the bacterium in the amoebal host, Accanthamoeba.
It is a Legionella pneumophila-specific fitness factor exposed at
the surface of the bacterial cell with a length of 136 amino acids.
A monoclonal antibody (MAb 61/1) and a polyclonal antiserum against the protein can block the uptake of Legionella
within amoeba. Epitope mapping by peptid-spot-synthesis
showed a binding site at aminoacid11-20 (C1) for MAb 61-1.
Site-directed mutation in the putative epitope C1 with pLIHis11, pLI-Thr12, pLI-Leu13, pLI-Ile14, pLI-Phe15, pLI-Ile16,
pLI-Phe17, pLI-Gly18, pLIGln19, pLI-Pro20 result in different
binding ability of Mab 61-1 in comparison with the wildtype
(Corby) and the pad knockout mutant (Cp7).
In cell-culture-assays Acanthamoeba castellanii was infected
with the wildtype, the knocked-out mutant (CP7) and the sitespecific modified mutants with a cell to bacteria ratio of 1:100.
Immunfluorescence microscopy and culture assay showed that
the uptake of the mutants and the binding ability of Mab 61/1
relate to each other. However, the intracellular replication of the
bacterial clones was not affected.
We suppose that the C1-epitope of the Pad-protein is involved in
the uptake of L. pneumophila by Acanthamoeba castellanii but
not to the intracellular replication.
Metabolic pathways and intracellular persistence associated
with thymidine-dependent small colony variants of
Staphylococcus aureus
J. Zander1, S. Besier1, S. Saum2, F. Dehghani3, S. Loitsch4
V. Brade1, T. A. Wichelhaus1
University of Frankfurt, Institute for Medical Microbiology and
Hygiene, Frankfurt am Main, Germany
University of Frankfurt, Institute of Molecular Biosciences
Frankfurt am Main, Germany
University of Frankfurt, Institute of Anatomy II, Frankfurt am
Main, Germany
University of Frankfurt, Department of Medicine II, Frankfurt
am Main, Germany
Inactivation and regulation of a new Na+/H+-dicarboxylate
symporter operon of Pseudomonas aeruginosa found to be
up-regulated during chronic cystic fibrosis lung infection.
C. Henke1, J. Heesemann1, M. Hogardt1
Max von Pettenkofer-Institute, Bacteriology, Munich, Germany
P. aeruginosa is the major pathogen during chronic infection of
the cystic fibrosis (CF) lung. It has been suggested that the
persistence of P. aeruginosa in the CF lung is associated with its
growth as biofilm-like macrocolonies in the microaerophilic and
viscous CF mucus. Usually, CF respiratory secretions contain
high concentrations of mucin, lipids, DNA and amino acids.
Comparison of the proteoms and transcriptomes of sequential P.
aeruginosa mutator and non-mutator CF isolates revealed a
significant up-regulation of the genes PA0119, PA0120 &
PA0121 whose predicted products are associated with the amino
acid/dicarboxylate uptake and biofilm formation which
suggested a major contribution of amino acid availability to the
successful survival of P. aeruginosa in the CF lung.
The PA0119 gene encodes a protein with high identity to a
membrane associated C4-dicarboxylate Na+/H+ symporter DctA
of Rhizobium meliloti. We showed that PA0119 is organized in
an operon with two putative GntR-like transcriptonal regulators:
PA0120 and PA0121. To determine the regulation of these
genes under varying conditions the upstream region of PA0119
was cloned into a lacZ-reporter plasmid. Highest reporter
activity was observed during early to late stationary growth
phase and when P. aeruginosa was grown on fumarate, aketoglutaric acid, aspartate, succinate or glutamate. Inactivation
of PA0120 resulted in an increased reporter activity, suggesting
a negative regulation on PA0119. PA0119 reporter assays using
P. aeruginosa rpoS-mutant, rhlI- and rhlR-mutants yielded
decreased promotor activity as compared to wildtype PA01
indicating a RpoS & Quorum Sensing dependent regulation of
the PA0119-PA0121 operon. These data indicate that the
PA0119-PA0121 dicarboxylate transporter operon probably
contribute to the growth and survival of P. aeruginosa in the
nutritional rich environment of the CF lung.
Functional studies on the IL-8 degrading surface protease of
Streptococcus pyogenes
S. J. Kaur1, S. R. Talay1, R. Graham1, R. Frank1, E. Hanski2
G. S. Chhatwal1
Helmholtz-Center for Infection Research, Microbial
Pathogenicity, Brunswich, Germany
The Hebrew University-Hadassah Medical School, Institute of
Microbiology, Jerusalem, Israel
Group A Streptococcus (GAS) is an important human pathogen
which causes various diseases including pharyngitis, meningitis,
necrotising fasciitis and streptococcal toxic shock syndrome.
Popularly known as ‘flesh eating bacteria’ GAS can cause
diseases ranging from minor to life threatening. Proteins
secreted by Streptococcus pyogenes are potential vaccine
candidates against this bacteria. The GAS surface protein called
‘SpyCEP’ (Streptococcus cell envelope protease) has proven to
be a potential protective antigen against GAS infection. SpyCEP,
also known as ScpC can degrade an important chemotactic
factor, IL-8 and thus retard neutrophil influx at the site of
infection during necrotising fasciitis. To elucidate the properties
of this protease, recombinant expression of ScpC was performed
and purified protein was used for identifying and testing
chromogenic substrates for ScpC. Contrary to what has been
proposed, ScpC did not cleave known trypsin substrates. A
chromogenic substrate including the cleavage site of IL-8 was
synthesised and tested with ScpC. In vitro adherence and
invasion assays using human epithelial cells and a scpC knock
out mutant of S. pyogenes suggests a role of ScpC in invasion of
host cell.
Analysis of the adaptation of adhesins of Staphylococcus
aureus during chronic airway infection of cystic fibrosis
M. Knauer1, S. Deiwick1, K. Wardecki1, B. Kahl1
University Clinics Muenster, Medical Microbiology, Muenster
Background: During persistent airway infection of cystic
fibrosis (CF) patients Staphylococcus aureus isolates can be
affected by various mutations of regulator and virulence genes.
To accomplish colonization, S. aureus possesses a number of
adhesive genes, which all possess regions with different
numbers of repeats.
Aims: To assess the degree of genotypic adaptation, 77 S.
aureus isolates of two patients (6.9 and 26.1 years old), who
were chronically infected by S. aureus for 4.3 and 11.8 years,
were further investigated.
Methods: Single or multiplex PCR were performed for the
different adhesins: fnbA, fnbB, clfA, clfB, cna sdrD, sdrC, sdrE.
Molecular typing was performed by pulsed-field gel
electrophoresis (PFGE) and determination of agr-specificity
Results: By PFGE, the first patient harboured 6 and the second 3
different S. aureus strains with subtypes due to fragment pattern
differences. The strains of the first patient belonged to agr group
I (4 strains), III and IV (1 strain each) and the strains of the
second patient to agr group I, II and IV. By PCR, all strains
were positive for fnbA, fnbB, clfA, clfB, sdrC and 62/77 positive
for cna. There were different sizes of amplicons in some strains
and their subtypes for cna, fnbA, fnbB, clfA and clfB. 17 strains
were negative for sdrD and 6 for sdrE.
Conclusions: By PCR analysis, strains were identified, which
showed differences in amplicon sizes of special adhesins
indicating changes in the repeat area assumingly due to
adaptation of the microorganism to the hostile environment
during longterm persistence.
Expression and cell wall anchoring of Pls is required for
reduced adherence and cellular invasiveness of
Staphylococcus aureus
M. Hussain1, K. M. Juuti2, G. Peters1, P. I. Kuusela3, B. Sinha4
University of Muenster, Medical Microbiology, Muenster
University of Helsinki, General Microbiology, Helsinki
University of Helsinki, Bacteriology and Immunology, Helsinki
University of Wuerzburg, Hygiene and Microbiology
Wuerzburg, Germany
Background: The surface protein Pls (plasmin-sensitive) of
methicillin resistant Staphylococcus aureus (MRSA) reduces
adhesion to immobilized host proteins and host cell invasion by
an unknown mechanism, requiring pls gene expression. Here,
we tested the effect of Pls expression in two different MSSA
Methods: The pls–negative MSSA isolates Cowan and 6850
were transformed with plasmids carrying WT pls (pPLS4), pls
LPDTG (lacking the sortase motif; pPLS5) and pls SD
(lacking the SD repeat region; pPLS6). Transformants were then
tested for adherence to immobilized fibrinogen (Fg) and
fibronectin (Fn) and, invasion of 293 and EA.hy 926 cells (flow
cytometric assay), and compared to the respective WT strains.
Gene expression was monitored by real time RT-PCR.
Results: In Pls-expressing strains (pPLS4), adhesion to
immobilized Fg and Fn was reduced by ~ 80% and ~ 75%,
respectively. Invasion of 293 cells and EA.hy 926 cells was
reduced by up to 85% compared to WT strains. Surprisingly, Pls
expression upregulated fnbA and spa transcription, but
downregulated clfA and hla transcription. Competition
experiments using purified Pls protein (25 µg/mL) did not affect
invasiveness, as opposed to purified FnBPA (25 µg/mL;
reduction to ~ 10% of WT). Invasiveness of strains 6850
(pPLS5; no LPDTG sortase motif) and 6850 (pPLS6; no SD
repeat region) was only moderately reduced (to ~ 80% and ~
85% of WT, respectively).
Conclusions: Expression of Pls appears to reduce adherence and
invasiveness independently of an MRSA/SCCmec background.
Cell wall anchoring of Pls appears to be required for reduced
invasiveness, which occurs despite upregulation of fnbA
transcription. Thus, Pls may modulate invasion by steric
hindrance, rather than by competitive binding or altered adhesin
abrogated suggesting a profound impact of LTA on
physicochemical properties of bacterial surfaces.
A Staphylococcus aureus mutant with strongly reduced
lipoteichoic acid (LTA) content: LTA governs bacterial
surface properties and autolysin activity
I. Fedtke1, D. Mader1, G. Nicholson2, K. Henseler1, F. Goetz3
U. Zaehringer4, A. Peschel1
Eberhard-Karl-University , Medical Microbiology and Hygiene
Tuebingen, Germany
Eberhard-Karl-University , Organic Chemistry, Tuebingen
Eberhard-Karl-University , Microbial Genetics, Tuebingen
Research Center Borstel-Center for Medicine and Biosciences
Immunochemistry and Biochemical Microbiology, Borstel
The epidermal cell differentiation inhibitor B (EDIN-B) of
Staphylococcus aureus belongs to a family of exotoxins that
ADP-ribosylate and thereby inactivate Rho GTPases. The gene
coding for EDIN-B (edinB) is located on the etd-pathogenicity
island which consists of 7 ORFs including the coding sequence
for the exfoliative toxin D (etd) and a putative endopeptidase
(orf2). We found that patients suffering from blood stream
infections due to edinB-positive S. aureus exhibited anti-EDINB antibodies. Thus, edinB is expressed in vivo and might play
an important role in S. aureus infections. Therefore we
investigated the prevalence and organisation of the etdpathogenicity island in clonal independent S. aureus blood
culture isolates. Eight of the 121 (6,6 %) clinical isolates were
tested positive for the etd-pathogenicity island. By sequence
analysis of the etd-pathogenicity island in these and number of
additional edinB-positive S. aureus strains three genotypes
according to the presence or absence of specific genes as
compared to S. aureus TY114 can be differentiated, with
genotype I being the most prevalent.The regulatory principles of
edinB expression on the transcriptional and translational level
were assessed in S. aureus RN6390, Newman and their
respective agr and sarA deletion mutants. The influence of these
regulator elements on edinB expression was analysed in
exponential and in stationary growth phase by semi-quatitative
transcription analysis, demonstrating that sarA is an essential
positive regulator of edinB expression, while no direct effects of
agr on edinB transcription were detected. In apparent contrast to
the results of transcription analysis, EDIN-B amounts in
supernatants of RN6390agr were higher than in the wild type.
However, this discrepancy was abolished by the addition of
a2macroglobulin to the growth medium, indicating that agr
indirectly, via its positive effect on exoprotease expression, has
as a negative impact on EDIN-B protein levels. These findings
underline the tight interplay of regulatory components and their
influence on potential pathogenicity factors. Future studies will
concentrate on the functional analysis of exoproteins encoded on
the etd-pathogenicity island.
Staphylococcus aureus, a leading cause of community- and
hospital-acquired infection, produces two subclasses of teichoic
acids (TA). These are polyanionic molecules found in the cell
envelope of Gram-positive bacteria only. While wall TA (WTA)
is covalently linked to peptidoglycan and dispensable in S.
aureus, lipo-TA (LTA) is anchored via a glycolipid in the outer
leaflet of the cytoplasmic membrane and was recently identified
as essential. LTA and WTA seem to contribute to the virulence
potential of this major pathogen, but many studies are based on
mutants with structural alterations in both, LTA and WTA, and
do not allow to define LTA-specific effects. Deletions of the
gene responsible for the biosynthesis of the glycolipid used as
LTA anchoring structure resulted in the lack of glycolipids and
LTA is anchored via diacylglycerol. Interestingly, in S. aureus
SA113 the LTA content was reduced to 13% (both in culture
supernatants and cell-associated) and although LTA is essential
the residual LTA allowed the mutant to grow normally
indicating that this amount is sufficient at least under laboratory
conditions. The same mutation, however, led in S. aureus
RN4220 supernatants to two to threefold increased LTA
amounts. Characterization of these mutants offered for the first
time the opportunity to discriminate effects due to reduced LTA
content or lack of glygolipids. Our data reveal that LTA may be
less important in protein attachment than previously thought
while LTA controls autolysin activity and has a profound impact
on the viability in late stationary phase. Moreover the ability of
the LTA mutants to form biofilms on plastic was completely
The etd-pathogenicity island in Staphylococcus aureus:
Organisation and regulation
G. Franke1, H. Rohde1, A. Böckenholt1, C. Wolz2, M. Sugai3
M. Äpfelbacher1
University Hospital of Hamburg-Eppendorf, Microbiology
Virologie und Hygiene, Hamburg, Germany
Eberhard-Karl-University , Institute for Medical Microbiology
und Hygiene, Tuebingen, Germany
Hiroshima University Graduate School of Biomedical Sciences
Department of Bacteriology, Hiroshima, Japan
Characterization of virulence gene expression in different
Staphylococcus aureus mastitis isolates
C. Wolf1, I. Burghardt1, C. von Eiff2, S. Holtfreter3, B. Bröker3
P. Rainard4, W. Petzl5, M. Hecker1, S. Engelmann1
Institut für Microbiology, Greifswald, Germany
Institute for Medical Microbiology, University Hospital
Muenster, Germany
Institute for Immunology and Transfusionmedicine, Greifswald
Institut National de la Recherche Agronomique, Laboratoire de
Pathologie Infectieuse et Immunologie, Nouzilly, France
Klinik für Wiederkäuer mit Ambulanz und Bestandsbetreuung
der LMU Munich, Oberschleissheim, Germany
Staphylococcus aureus is a Gram-positive pathogen and
regarded as an important pathogenic bacterium of bovine and
human mastitis which is characterized by inflammation of
mammary glands. This bacterium is noted for its diversity in the
equipment with virulence factors which are supposed to
contribute to the pathogenesis. Extracellular as well as surface
associated proteins represent a reservoir of virulence factors.
Thus, the comprehensive analysis of these proteins of S. aureus
may reveal whether there are virulence factors that might be
specific for mastitis isolates. Moreover, proteins involved in the
particular interaction of S. aureus with the respective host
(human or cattle) are of great interest.
In the present study, 23 S. aureus isolates of either human (7
isolates) or bovine (16 isolates) origin were analyzed. For
genetic and epidemiological studies, we utilized the well
established Multi-Locus-Sequence-Typing (MLST) technique.
Moreover, the agr type and the presence of some virulence
genes (e.g. superantigens, eta, etd) were determined by using
Multiplex PCR. The proteomics approach is a useful tool for
analyses of virulence gene expression in different isolates. The
comparison of the extracellular protein patterns revealed a high
heterogeneity between human and bovine strains as well as
within strains from the same origin. Altogether 9 extracellular
proteins were found in at least 80 % of the bovine isolates e.g.
autolysin (Atl), alpha-hemolysin precursor (Hla), aureolysin
(Aur), SspA and SspB. 17 extracellular proteins could be
detected in less than 20 % of the bovine isolates, including
Leukotoxin E subunit (LukE), three staphylococcal enterotoxins
(SEC2, SEC-bov and SEL) and the toxic shock syndrome toxin
Investigation of the structure and transfer of the Yersinia
high-pathogenicity island in Klebsiella pneumoniae
O. Benedek1,2, S. Schubert1
Max-von-Pettenkofer-Institute, Ludwig-MaximiliansUniversity, Munich, Bacteriology, Munich, Germany
University of Pécs, Institute of Medical Microbiology and
Immunology, Pécs, Hungary
The structure and function of the Yersinia-High Pathogenicity
Island (HPI) is well-characterised in human pathogenic
Yersiniae and in extraintestinal E. coli. In spite of the fact that
the island is widely distributed in further members of the family
Enterobacteriaceae, its genetic organisation and transfer
mechanisms have not yet been studied in details. A recent
discovery of a unique type HPI in E. coli ECOR31 resembling
integrative-conjugative elements (ICEEc1) indicates the possible
role of conjugative transfer in dissemination.
From a collection of HPI-positive enteric bacteria isolated from
extra-intestinal infections we identified five distinct K.
pneumoniae strains bearing an unusually structured, ICE-like
HPI. Although these Klebsiella-HPIs share common features,
they are not identical since they suffered different truncations
and deletions. Furthermore, our sequencing data reveal that their
most downstream region is not only completely different from
the one of ICEEc1, but there is an individual variation among the
distinct K. pneumoniae strains. Moreover, we were able to
detect an episomal interspecies transfer of the ICE-like
Klebsiella-HPIs to E. coli and Citrobacter sp. utilizing
Klebsiella wild type plasmids. These plasmid-borne ICE-like
HPIs, however, appear to be rather unstable in its new hosts.
ClpV, a novel Hsp100 member of pathogenic proteobacteria
G. Boenemann1, A. Mogk1
University of Heidelberg, ZMBH, Molecular Biology
Heidelberg, Germany
AAA+ proteins are ATPases that are associated with diverse
cellular activities. These ring-forming ATPases are able to
transform chemical energy into biological activity. One of their
subfamilies are Hsp100/Clp heat shock proteins, which are key
players in the protein quality control network of cells and
function in the degradation and refolding of misfolded or
aggregated proteins. Recently, a new class of Hsp100/Clp
proteins was identified and termed as ClpV proteins as they are
present in many pathogenic bacteria (V for virulent), but are
absent in their non-pathogenic relatives. Although these proteins
show a similar domain organization than ClpB proteins, they
lack disaggregation activity and cluster in a separate
phylogenetic tree with remarkable distance to ClpB. Strikingly,
clpV is organized in a conserved gene cluster in bacteria that
live in intimate contact with eukaryotic cells. The genes of this
cluster are hardly characterised, but seem to encode a potential
secretion system. Diverse studies within the last few years could
show that mutational inactivation of different genes of this
cluster affect host selectivity, cell invasion and cytotoxicity.
Particularly, the analysis of corresponding genes in Vibrio
cholerae, termed as the virulence associated secretion (vas)
genes showed their importance in virulence towards the
macrophages. This new type 6 secretion system involves
extracellular translocation of proteins that lack N-terminal
hydrophobic leader sequences and Hcp and VgrG were
determined as substrates.
In our own studies we show that clpV in Vibrio cholerae is
essential for Hcp secretion. Currently we are analyzing the role
of clpV in more detail, beside identifying its interaction partners
to better understand the molecular mechanism of this new
secretion system.
Effects of D-Serine on growth of Staphylococci under
minimal medium
T. Sakinc1, S. Friedrich1, N. Michalski1, B. Kleine1
F. Szabados1, S. Gatermann1
Ruhr-University Bochum, Institute for Hygiene and
Microbiology, Bochum, Germany
D-Serine catabolism is biologically important because D-serine
is available in some environments as a readily utilizable nutrient
source, but it can also have inhibitory effects on growth. Despite
the fact that D-serine is toxic to many living organisms, it is one
of the most prevalent amino acids excreted in mammalian urine
at reported levels of 3 to 115 µg/ml, and it can be found in
mammalian blood as well.
We are interested in the mechanisms for uptake of D-serin in
staphylococci. For that, we generate a defined minimal medium
without yeast extract for growing staphylococci. Therefore we
used the protocol from Pattee et al.,1975 as basic and stepwise
reduce the concentration or left components out. We could
reduce known medium to ten amino acids and included only 2
mg/ml glucose and a defined salt solution, minerals and
Growth assays were done with 10 strains each of S.
saprophyticus, S. aureus and S. epidermidis in defined minimal
medium with or without D-serine. In addition, we also tested the
growth conditions in PY-media.
Our experiments suggest that only S. saprophyticus could show
growth in both media. For S. epidermidis and S. aureus D-serine
is an inhibitory factor or even toxic.
Characterisation of the S. saprophyticus surface associated
lipase: Biochemical properties and catalytic triad
B. Kleine1, S. Friedrich1, F. Szabados1, S. Gatermann1
T. Sakinc1
Ruhr-University Bochum, Department of Medical
Microbiology, Bochum, Germany
S. saprophyticus, an important cause of urinary tract infection
especially in young women, expresses the surface-associated
lipase Ssp. We recently cloned Ssp and described its activity
depending on pH and concentration of calcium. Here we
finished our characterisation regarding to substrate preference,
dependence on temperature as well as its stability in front of
different pH and temperature. In a spectrophotometrical assay
with p-nitrophenyl esters Ssp prefers short?chain substrates in
homology to other staphylococcal lipases. The temperature
optimum is around 30°C with a long stability at this temperature
but even after 5 min. at 100°C we could recover about 80%
activity. Ssp is stable at physiological pH-values in the urine up
to 9 but loses its activity fast at pH-values above 10. First in
vivo experiments regarding the virulence of Ssp reveal a
significant difference between the wild-type strain 7108 and the
ssp knock-out mutant. To determine if the lipolytic activity of
Ssp or just the high amount of proteins responsible for this we
used PCR-based site-directed mutagenesis. We replace the
serine482 in the putative catalytic triad Ser-His-Asp of Ssp with
cysteine, Asp 673 with serine and His712 with proline. With
these experiments we can validate the position of the catalytic
site with analysing its activity and probably the role of the
lipolytic activity of Ssp in the infection process.
Virulence-associated genes of the flagellar regulon of L.
T. Schulz1, S. Jacobi1, C. Albert-Weissenberger1, K. Heuner1
University of Wuerzburg, Institute for Molecular, Infection
Biology, Germany
Legionella pneumophila is the causative agent of Legionnaires´
disease, an atypical pneumonia. After inhalation of infectious
aerosol L. pneumophila is able to replicate within alveolar
macrophages of the human lung. L. pneumophila is commonly
found in aquatic habitats and replicates inside of protozoa.
The expression of virulence factors is genetically linked to the
expression of the flagellum (motility) in L. pneumophila. We
showed that the flagellin (FlaA) and the alternative sigma factor
FliA are involved in the invasion and replication of Legionella
in host cells. We identified most of the yet known regulators and
genes of the flagellar regulon. FliA is also involved in the
expression of virulence traits of L. pneumophila. In preliminary
studies we identified and analysed genes of the FliA regulon. In
addition, we started to analyse the expression of fliA.
Unlike the wild type strain, the flaA mutant of L. pneumophila is
able to proliferate in bone-marrow derived mouse macrophages.
It seems as if FlaA is recognized by the cytosolic Naip5 receptor
of the macrophages. We now try to identify the amino acids of
FlaA involved in this process.
Contribution of the lipase Ssp and the SdrI of
Staphylococcus saprophyticus to virulence in experimental
urinary tract infections of mice
S. Gatermann1, K. Kline2, M. Ingersoll2, S. J. Hultgren2
T. Sakinc1
University of Bochum, Med. Microbiology, Bochum, Germany
Washington University School of Medicine, Molecular
Microbiology, St. Louis, USA
Staphylococcus saprophyticus causes urinary tract infections
especially in young women without predisposing medical
conditions. Previously we have described that its urease
contributes to virulence in experimental infections of rats. In
addition, S. saprophyticus expresses a number of surface
proteins such as a surface-associated lipase (Ssp) and an SDrepeat protein (SdrI) that binds collagen.
In this work we used specific isogenic mutants deficient in
expression of the lipase or the SdrI in experimental infections of
mice to show that both surface-associated proteins are involved
in the pathogenesis of urinary tract infections caused by S.
Ten mice were infected in each group and cfu of S.
saprophyticus were determined at 2 and 7 days post infection.
At 2 days the ssp and sdrI mutants showed significantly fewer
bacteria in bladders and kidneys than the wild-type strain. After
7 days only the colony counts in the kidneys differed
significantly. In addition, IL-1 and IL-12, markers for
macrophage activation, were significantly reduced in kidneys
infected with the mutants, indicating that both antigens
contribute to macrophage activation. The ssp mutant did not
show reduced recruitment of macrophages into kidneys whereas
macrophages were less prevalent in kidneys infected with the
sdrI mutant. Therefore SdrI might be involved in macrophage
recruitment and activation whereas Ssp might be involved in
activation only.
Our data indicate that both the lipase (Ssp) and the collagenbinding SdrI of S. saprophyticus contribute to pathogenesis of
experimental urinary tract infections. Our data also provide the
first experimental evidence that a lipase plays a role in infection
and that this enzyme may also contribute to pathogenesis of
urinary tract infections.
Lipopolysaccharide shed by Legionella pneumophila arrest
phagolysosomal maturation in host cells
E. M. Seeger1, M. Thuma1, E. Jacobs1, J. Helbig1
Technical University of Dresden, Medical Faculty, Institute
Medical Microbiology und Hygiene, Dresden, Germany
Legionella pneumophila, the causative agent of Legionnaires’
diseases, multiplies in amoeba and its equipment for bypassing
the lysosomal pathway is also very strong in monocytic host
cells. L. pneumophila gains ability to arrest phagolysosome
fusion when cultured in broth to transmissive (infective) growth
phase. Several gene loci of L. pneumophila were confirmed to
be involved in host cell modulation, but there exist no certain
evidences for LPS components. To elucidate the role of LPS in
this mechanism, we investigate its influence inside the natural
host cell Acanthamoebae castellanii, but also inside U937 cells,
human monocytes, and A/J mouse macrophages. For this, we
have cultured L. pneumophila strain Corby in broth to
replicative (not infective) or transmissive growth phase. Shed
LPS enriched outer membrane vesicles (OMV) and molecular
LPS, respectively, were separated by size filtration and coated
on latex beads carrying the LPS-specific monoclonal antibody
MAb 3/1. After staining of host cell lysosomes by FluoresceinDextran, these beads were added for phagocytosis. Built
lysosomal or non-lysosomal phagosomes were counted. As
proved in an other study with A/J mouse macrophages using
purified OMV, which contain beside LPS also proteins involved
in pathogenicity, in our study all other host cells used were
evaded significantly the lysosomal degradation up to five hours.
Furthermore, we could substantiate for the first time that
arresting the phagolysosomal maturation can be induced by LPS
shed in molecular form during the transmissive growth phase
characterized by expression of virulence traits. Most
interestingly, LPS shed during the replicative growth phase has
the same impact. Therefore, it can be concluded, that
intraphagosomal shed LPS contributes to modulation of host
cells during the early intracellular life of legionellae.
Identification of Staphylococcus aureus ser/thr kinase
substrates by phosphoproteome analysis
S. Donat1, S. Fuchs2, S. Engelmann2, S. Rakette3, T. Stehle3
K. Ohlsen1
University of Wuezburg, Infection Biology, Wuerzburg
University of Greifswald, Microbiology, Greifswald, Germany
Eberhard-Karl-University , Structure Biology, Tuebingen
Signal transduction pathways are essential for the regulation of
gene expression in both prokaryotes and eukaryotes. Signal
transduction in prokaryotes was considered to occur primarily
by histidine protein kinases that activate transcription by the
phosphorylation of cognate response regulators (two component
system). In contrast, signal transduction in eukaryotes occurs via
phosphorylation on serine, threonine, and tyrosine residues.
Until recently, it has been believed that this type of
phosphorylation is restricted to eukaryotes. However, whole
genome sequencing revealed that kinases using this type of
phosphorylation are more widespread than previously thought.
Almost all prokaryotes also encode so called ESTPKs
(eukaryotic-type ser/thr protein kinases). Especially in the
genome of bacteria with complex live cycles like
Cyanobacterium several putative ESTPKs have been identified.
Compared to their eukaryotic counterparts, the signal to which
ESTPKs respond, the mechanism of signal transduction, and
their substrates remain poorly understood. Staphylococcus
aureus also encodes an ESTPK that shows homology to PknB
of Mycobacterium tuberculosis.
The aim of our work is to characterize this ESTPK and to find in
vivo substrates of the PknB kinase. Therefore we constructed a
deletion mutant of pknB in S. aureus strain 8325. We performed
a phosphoproteome approach, in which we compared via 2DGE
the set of phosphorylated proteins of the wild type with that of
the isogenic mutant. Here we found around six proteins, which
are less or no more phosphorylated in the ?pknB mutant, for
example trigger factor, translation elongation factor, and
proteins involved in the central metabolic pathways.
Further, we investigated the phosphorylation ability of PknB.
Therefore, we constructed a plasmid for the overexpression of
the catalytic domain in E. coli. The purified protein is a
functional protein kinase and was able to phosphorylate a model
substrate, MBP. The kinase activity was also tested in an
autophosphorylation assay. The phosphorylation depends in
both cases on divalent cations. PknB1-274 showes a clear
preference for Mn2+ versus Mg2+ ions.
Discovery of a generalized O-linked glycosylation pathway
in Neisseria gonorrhoeae
W. Egge-Jacobsen1, S. Vik1, F.E. Aas1, J. H. Anonsen1
M. Koomey1
University of Oslo, Kristine Bonnevies House, Department of
Molecular Biosciences, Mass Spectrometry & Proteomics, Oslo
Type IV pili of Neisseria gonorrhoeae (Ng), filamentous surface
protein structures critical to the colonization of their human
host, are known to undergo differential posttranslational
modifications. By top down and bottom up mass spectrometry
we could show that PilE is modified with phosphoethanolamine
(PE) and phosphocholine (PC) at serine 68 (S68) and 156
(S156), respectively.
Furthermore PilE is glycosylated with an disaccharide
composed of an acetylated hexose residue linked to a proximal
2,4-diacetamido-2,4,6-trideoxyhexose sugar
(AcetylHexDATDH) at S63.
By immunoblotting of whole cell extracts from Ng with rabbit
antibodies raised against wild-type glycosylated PilE we
discovered that many proteins in addition to PilE are carrying
the exact same glyco moiety, suggesting a general O-linked
glycosylation parthway in Ng. 2D gel electrophoresis of Ng
membrane fractions in combination with immunoblotting
allowed us to identify 8 further glycoproteins by mass
fingerprinting. Based on relatedness to known proteins, they are
homologs of proteins known to have a potential role in redox
potential and oxidative stress respond, and should be localized
to the cytoplasmic membrane.
All 8 proteins have a four AA long sequence in common and for
three of those proteins the AspN or trypsin derived peptides
carrying the glyco modification have already been identified, by
monitoring the specific oxonium ion signal of 433 m/z formed
by in-source fragmentation and CID. However, the exact
position of the modification could only be determined by
performing ETD (Electron Transfer Dissociation), identifying
serine residues within or close to the four AA long sequon as Oglycan attachment site.
Therefore in addition to the two already described generalized
protein glycosylation systems (which like eukaryotic systems
target a broad array of substrates) in Campylobacter jejuni (Nlinked system which targets at least 21 proteins in the periplasm)
and in Mycobacterium tuberculosis (O-mannosylation system
for exported proteins) we here describe a third system in Ng.
Nutritional factors and structural components determining
Streptococcus pyogenes biofilm formation and development
C. Lembke1, A. Podbielski1, B. Kreikemeyer1
University of Rostock, Institute for Medical Microbiology
Rostock, Germany
Streptococcus pyogenes (group A Streptococcus, GAS)
persistently colonizes the throat or skin of its human hosts.
Currently, this behavior is explained by the bacterial capability
to internalize into epithelial cells and to stay there protected
from various human defense mechanisms. We have recently
shown that GAS can form monospecies biofilms. Biofilms
shield their inhabitants from forces of the human defense system
and/or antibiotic therapies. This trait could be an alternative
reason for the long term persistence of GAS.
Little is known about structure and components of GAS
biofilms. Here we show that GAS biofilm formation is strictly a
strain-specific capacity and does not correlate with GAS
serotypes. Scanning electron (SE) and confocal laser scan
microscopy (CLSM) studies revealed variations in structure and
number of cell layers of various GAS strains. Thus, we
examined components of the extracellular polymeric substance
(EPS) of the GAS biofilms by applying sets of
chromatographical / spectrometrical techniques. First results
identified rhamnose and ribose polysaccharides as EPS
Additionally, we added specific nutrient components, enzymes
and antimicrobial substances to different GAS biofilm
developmental stages to investigate effects on the biofilm
structure. We could demonstrate destruct effects of selected
carbohydrates, artifical saliva, or human serum supplements of
the growth media.
DNase treatment demonstrated that extracellular DNA is
essential for initial biofilm establishment. GAS biofilm cells
proofed to be less sensitive than planctonic cells when exposed
to chloramphenicol, kanamycin or tetracycline.
Finally, we employed defined GAS mutants to monitor gene
effects on biofilm forming capacity. By using dpp and emm
gene mutants we revealed that streptococcal dipeptide permease
is necessary for initial biofilm development, whereas the M
protein does not contribute to early developmental stages.
Moreover, biofilm formation is subject to antagonistic
regulation processes when studying corresponding Mga and
RofA global regulator mutants.
In summary, our study will uncover the role of GAS biofilm
formation for bacterial persistence in human hosts and may open
up new avenues for therapeutical approaches to eliminate
persistent GAS.
PavB is a novel multifunctional adhesin of Streptococcus
pneumoniae contributing to colonization
M. Rothe1, D. Somplatzki1, I. Jensch1,2, S. Ebert3
S. Bergmann2,1, R. Nau3, S. Hammerschmidt2,1
University of Wuerzburg, Research Center for Infectious
Diseases, Wuerzburg, Germany
University of Munich, Max von Pettenkofer Insitut, Munich
University of Goettingen, Department of Neurology
Goettingen, Germany
The genomic analysis of Streptococcus pneumoniae identified a
putative surface-exposed multidomain protein. This protein
(SP0082 in TIGR4 and SPR0075 in S.p. R6) contains
N?terminally a leader peptide and C-terminally a LPXTG-motif,
which anchors the protein in a sortase-dependent manner to the
cell wall. The major part of the protein is composed of
conserved repetitive sequences, each of approximately 150
amino acids, which have been suggested to interact with
immobilized fibronectin. The repeats were named streptococcal
surface repeats (SSURE) and the protein is termed
pneumococcal adherence and virulence protein B (PavB). Our
molecular analysis of 23 genes encoding PavB in different
pneumococcal serotypes indicated that the molecular mass of
PavB varies among pneumococcal strains. Strikingly, the
identified numbers of SSURE differ from the annotated numbers
and are responsible for the differences in the molecular mass of
PavB. The repeat domain has no recognizable homology with
any other known protein. Binding experiments with His6?tagged
SSURE peptides (2 or 5 SSURE) indicated a specific interaction
of these peptides with fibronectin, plasminogen, and
thrombospondin-1. Moreover, mice infection experiments
indicated that PavB contributes to pneumococcal colonization.
In an experimental meningitis model no differences were
observed for the wild?type and the pavB?deficient mutant. In
contrast, in a mouse sepsis and pneumonia model the
pavB?deficient mutant was significantly less virulent compared
to the isogenic wild-type. Moreover, the pavB-deficient mutant
showed a delay in transmigration to the lung as shown by
determination of bacterial vegetation and imaging the
dissemination by use of the IVIS Imaging System 100
(Xenogen). In conclusion, PavB is a multifunctional
pneumococcal adhesin which is involved in the first stage of
infections, namely, colonization of the airways.
Nasal colonization, the major source of Staphylococcus
aureus infections, is a multifactorial process involving
surface protein and wall teichoic acid-mediated interactions
T. Kohler1, E. Kulauzovic1, C. Weidenmaier1, A. Peschel1
University Hospital Tuebingen, Medical Microbiology and
Hygiene, Tuebingen, Germany
Most of the severe bacterial infections originate from the
endogenous microflora of human body surfaces. However, the
molecular basis of colonization, e.g. of the human nose by
Staphylococcus aureus has remained incompletely understood.
Several surface-exposed proteins and wall teichoic acid (WTA)
polymers have previously been implicated in S. aureus
attachment to nasal epithelial cells. Here we dissect the role of
these molecules in colonization using S. aureus srtA (sortase A)
and tagO mutants deficient in surface protein and WTA display,
respectively. The two mutants were affected in interaction with
different types of epithelial ligands (keratin vs. scavenger
receptor-like molecules) and in binding to nasal cells. Moreover,
both mutants exhibited nasal colonization defects in a cotton rat
model, albeit at different colonization stages. These data
indicate that S. aureus nasal colonization involves several types
of receptor/ligand interaction, which may be useful targets for
new preventive and therapeutic strategies.
Characterization of the promoter activities of the regulatory
sae operon in Staphylococcus aureus
T. Geiger1, M. Mainiero1, C. Goerke1, C. Wolz1
University Hospital Tuebingen, Medical Microbiology and
Hygiene, Tuebingen, Germany
Staphylococcus aureus possess several regulatory systems
which allow the organism to use physiological parameters as
signals for specific gene expression. The two component system
SaeR/S is considerably involved in the activation of major
virulence factors. However, little is known about the signals
leading to saeR/S activation. There are two additional open
reading frames (ORF3 and ORF4) located upstream of saeR/S.
Altogether four overlapping transcripts, from three different
transcription starting points are expressed, indicating three
putative promoters (P1, P2 and P3). We aimed to characterize
these three putative promoters using a -galactosidase reporter
system. Putative promoter regions were cloned in front of a
promoterless lacZ gene, located in an integration vector which
integrates into the lipase gene of S. aureus. The promoter assay
reveals that the first sae promoter (P1) upstream of ORF4 is the
main sae promoter with the highest activity. No promoter
activity could be detected for the putative sae promoter (P2)
localized in front of ORF3. Results obtained by RACE and
Northern hybridization using constructs in which the upstream
region of saeR/S were partially deleted confirmed that the most
abundant transcript starting in front of ORF4 is generated by
mRNA processing. The putative sae promoter P3 upstream of
saeR/S is a weak secondary promoter. Further investigations
revealed a cis-enhancer element located upstream of P3
indicating an unknown activator binding site for the P3
promoter. Comparison of promoter activities between wild type
and sae-mutants demonstrate that P1 is strongly auto-activated
by sae, the P3 promoter is constitutively expressed independent
of sae and the P3 promoter including the enhancer element is
auto-repressed by sae. Interactions with other regulators show
that the P1 promoter is inhibited by the alternative sigma factor
B. Additionally, P1 is inhibited by low pH and high NaCl
concentrations (1M) but activated by subinhibitory
concentrations of hydrogen peroxide. In summary, we could
show that there are only two active promoters in the sae operon.
The main promoter P1 is strongly autoregulated and reactive to
external signals such as H2O2, pH and NaCl.
Molecular analysis of the thymidine-auxotrophic small
colony variant phenotype of Staphylococcus aureus
S. Besier1, A. Ludwig1, K. Ohlsen2, V. Brade1
T. A. Wichelhaus1
University Hospital of Frankfurt am Main, Institute of Medical
Microbiology and Infection Control, Frankfurt am Main
University of Wuerzburg, Institute for Molecular Infection
Biology, Wuerzburg, Germany
Thymidine-auxotrophic small colony variants (SCVs) of
Staphylococcus aureus can be frequently isolated from the
chronically infected airways of patients suffering from cystic
fibrosis. To date, little is known regarding the molecular basis
underlying the formation of this special phenotype, but the
auxotrophism for thymidine suggests a perturbed pyrimidine
pathway with an impaired dTMP synthesis.
The formation of dTMP from dUMP with concomitant
conversion of 5, 10-methylenetetrahydrofolate to dihydrofolate
is catalyzed by the thymidylate synthase. Sequence analysis of
the thymidylate synthase-encoding thyA gene of six clinical
thymidine-auxotrophic S. aureus SCVs revealed that all isolates
had mutations within this gene. In five isolates the function of
the thymidylate synthase was definitely impaired: three of them
showed a truncation of the thyA coding sequence by nonsense or
frame-shift mutations, in one further isolate the active site of the
enzyme was affected by an internal 12-bp deletion, and another
isolate had a 173 bp-deletion spanning the 5’-terminal region of
thyA and the preceding DNA sequence. The sixth isolate showed
two amino acid substitutions within the thyA gene product. To
confirm the importance of impaired thymidylate synthase
synthesis or activity for the formation of the thymidineauxotrophic SCV phenotype, we constructed a thyA knock-out
mutant of a wild-type S. aureus strain. This mutant showed all
characteristics of clinical SCVs, such as slow growth, decreased
pigment formation, reduced hemolytic activity, auxotrophism
for thymidine, resistance to trimethoprim/sulfamethoxazol, and
reduced plasma coagulase activity. Complementation of the
thyA knock-out mutant with intact thyA in trans nearly restored
the normal phenotype.
In conclusion, these data confirm at the molecular level that
impaired thymidylate synthase function is causative for the
formation of the thymidine-auxotrophic SCV phenotype in S.
S. saprophyticus strain ATCC 15305 invades the urothelial
cell line 5637
F. Szabados1, B. Kleine1, A. Anders1, M. Kaase1, T. Sakinc1
I. Schmitz2, S. Gatermann1
Ruhr-University Bochum, Institute for Hygiene and
Microbiology, Bochum, Germany
Ruhr-University Bergmannsheil, Institute for Pathology
Bochum, Germany
Uropathogenic E. coli (UPEC) and S. saprophyticus may cause
acute and chronic urinary tract infections. Certain UPEC strains,
for example UTI89, are known to invade cells from urothelial
cell lines. This invasion is initiated in UPEC via pilus adhesion
and is described as an important pathogenicity factor of acute
and chronic infections. Within a cell, the bacteria are usually
protected from anti-infective therapy and the bacteria can
maintain the infection from there. S. saprophyticus shows strong
adhesion to urothelial and Hep2 cells, we therefore investigated
the ability of S. saprophyticus for invasion into the urothelial
cell line 5637 using a FACS-based method to detect
internalization. The bacteria were stained with FITC and were
incubated for one hour with the cells, gentamicin and
lysostaphin was added for one hour. The cells were washed and
treated with trypsin to remove adhering bacteria and to detach
the eukaryotic cells. For further discrimination, a gating strategy
was used, because bacteria were smaller in size and FITC
stained. We could show invasion by S. saprophyticus strain
ATCC 15305 in this assay, compared to S. carnosus strain
TM300 and S. epidermidis strain ATCC 12228 where no
invasion was documented. S. saprophyticus ATCC 15305 was
detected in a significant higher amount above the 3-times
detection threshold of non-invasive controls of S. carnosus
TM300 and S. epidermidis ATCC 122228. The internalization
of S. saprophyticus was less compared with S. aureus Cowan I
strain. These results were validated in a classical gentamicin
protection assay, in histological stains and transmission electron
In these experiments, we found strong evidence for invasion of
uropathogenic S. saprophyticus ATCC 15305 in a validated
FACS invasion assay. To bear analogy to UPEC strains these
results suggest a new mechanism for pathogenicity of
coagulase-negative S. saprophyticus ATCC 15305 in urinary
tract infections in addition to other suspected or proven
pathogenicity factors.
Development of antibody-based therapy targeting
immunodominant antigens of Staphylococcus aureus
K. Ohlsen1, U. Lorenz2, T. Schäfer1, B. Lorenz1, C. Erck3
J. Wehland3, A. Thiede2, J. Hacker1
University of Wuezburg, Infection Biology, Wuerzburg
University of Wuezrburg, Surgery, Wuerzburg, Germany
Helmholtz-Center for Infection Research, Cell Biology
Brunswich, Germany
The use of antibodies for the treatment of staphylococcal
infections is currently under investigation in academics and
industry. We developed a murine monoclonal antibody targeting
the immunodominant antigen IsaA and applied the antibody in
two animal infection models. The study revealed that
application of an anti-IsaA MAB lowers the infection burden
both in acatheter-related infection model and in a model of soft
tissue infection.In vitro studies suggest an enhancement
ofantibody-mediated phagocytosis of S. aureus by neutrophils in
the presence of IsaA-binding antibodies. Moreover, the antibody
may interfere with the function of IsaAthatshows homology to
lytic transglycosylases which areinvolved in the completion of
the nascent cell wall of S. aureus. Overall, the results strongly
support the idea that implementation of antibodies against IsaA
can serve as an alternative strategy to combat infections caused
by S. aureus.
Characterization of a murine Staphylococcus aureus
pneumonia model
J. Koehler1, K. Breitbach1, S. Vogelgesang2, K. Rogasch3
M. Hecker3, S. Engelmann3, I. Steinmetz1
University of Greifswald, Friedrich Loeffler Institute of Med.
Microbiology, Greifswald, Germany
University of Greifswald, Institute of Pathology, Greifswald
University of Greifswald, Institute of Microbiology, Greifswald
Staphylococcus aureus is a major pathogen of hospital-acquired
pneumonia. Additionally, there are reports that the prevalence of
severe clinical courses of community-acquired S.aureus
pneumonia, often due to multiresistant strains, is increasing.
Currently, there is little knowledge on S.aureus virulence factors
which are important in causing lower respiratory tract infection.
Moreover, the host factors which are crucial in the defense of
S.aureus lung infection are largely unknown. In this study, we
developed a murine model of S.aureus pneumonia and
compared different strains of mice. Intranasal infection of
BALB/c mice with S.aureus Newman led to severe
bronchopneumonia with a massive influx of neutrophils as
demonstrated by lung histology and bronchoalveolar lavage.
BALB/c mice were more susceptible compared to C57Bl/6
mice, resulting in a significantly higher mortality after infection.
Depletion of neutrophils prior to infection showed that these
cells are essential for the early control of lung infection in both
BALB/c and C57Bl/6 mice. Intranasal infection of BALB/c
mice with a S.aureus Newman saeS mutant resulted in no
mortality, indicating that the two-component system SaeRS
which regulates the expression of a number of virulence factors
is important in pulmonary infection. Moreover, the saeS mutant
showed an impaired ability to persist in the lung of BALB/c
mice after infection with a sublethal dose, when compared to the
wildtype. In conclusion, we have established an experimental
system which should enable us to further analyze host defense
mechanisms as well as S.aureus virulence factors in pulmonary
Global transcriptomic and proteomic analysis of a
Staphylococcus aureus clpC mutant: implications for
physiology, metabolism and survival during late stationary
I. Chatterjee1, C. F. Batzilla2, S. Schmitt3, S. Engelmann4
M. Hecker4, W. Ziebuhr2, K. T. Preissner3, G. A. Somerville5
M. Herrmann1
University of Saarland, Department of Medical Microbiology
and Hygiene, Homburg/Saar, Germany
University of Wuerzburg, Institute for Molecular Infectious
Biology, Wuerzburg, Germany
University of Giessen, Institute for Biochemistry, Giessen
University of Greifswald, Institute for Microbiology and
Molecular Biology, Greifswald, Germany
University of Nebraska, Department of Veterinary and
Biomedical Sciences, Lincoln, Nebraska, USA
Introduction: Clp ATPases are molecular chaperones
influencing cell physiology functions including an aconitasemediated profound effect on post-stationary growth, acetate
catabolism and entry into death phase (Chatterjee et al., 2005,
2007). To get a comprehensive picture of the role of ClpC on
physiology, metabolism and late-stationary phase survival in S.
aureus, a clpC mutant strain was constructed in strain S.
aureusDSM20231 and was used to study the global regulation
during post-stationary phase.
Methods: DNA microarray analysis was used to determine
differential gene expression patterns of the WT and its clpC
mutant during late stationary phases of growth. Cytoplasmic
protein extracts were obtained from WT and clpC mutant, and
were subjected to high resolution 2-D protein electrophoresis
followed by MALDI-TOF MS. As the clpC mutant lacked TCA
cycle activity, we performed intracellular redox assay to
determine the redox status of both strains between 24-96 h.
Results: DNA microarray analysis of the S. aureus clpC mutant
and its isogenic WT denoted differentially expressed genes
involved in different functions. Importantly, genes involved
with physiology and metabolism (eg. qoxABCD, sdhAB, odhA),
transcriptional regulation (eg. codY, gapR and glnR), ribosomal
proteins and other genes (eg. srrA, rsbU) were all up-regulated
in late-stationary phase surviving clpC mutant. Interestingly,
proteins induced in the clpC mutant or in the WT also indicated
towards the modifications of global staphylococcal physiology
involving energy metabolism and stress regulation. Increased
protein levels in the clpC mutant were IlvA (member of isobranched chain fatty acids biosynthesis family), AhpCF, ArcA,
SA1272 (alanine biosynthesis), and LacD. In WT, among others
OdhB and GudB were up-regulated. As our clpC mutant was
seen to lack TCA cycle activity, we wanted to see if the lack of
ClpC could also alter the redox status during post-stationary
phase of growth. Accordingly, redox assay was performed and
as anticipated, the intracellular concentration of NADH and
NAD+ were markedly reduced in the S. aureus clpC mutant.
Conclusion: The results of this ongoing study further confirm
that the ClpC ATPase is involved in a broad spectrum of
staphylococcal energy metabolism and stress regulation
functions and that ClpC may be a key element for
staphylococcal physiology, particularly during prolonged times
of bacterial life such as encountered in sessile and/or persistent
staphylococcal populations.
Essential roles of environmental iron and iron-mediated
regulation in Helicobacter pylori resistance against oxidative
stress, metronidazole and gastric acid
S. Bereswill1, M. Kist2
Charité-Universitysmedicine Berlin, Microbiology and
Hygiene, Berlin, Germany
University Hospital Freiburg, Medical Microbiology and
Hygiene, Freiburg, Germany
During persistent gastric infection and chronic inflammation
Helicobacter pylori is continuously exposed to drastic changes
in the environmental iron concentration and to toxic oxygen
radicals produced by immune cells. Because radical formation is
catalysed by iron and H. pylori controls the cellular iron
concentration via repression of iron uptake by the Fur protein,
we investigated roles of iron and Fur in resistance against
oxidative stress, metronidazole and acid by growth of H. pylori
wildtype (wt) strains and fur mutants in media supplemented
with iron, urea and oxidative agents. Oxidative stress was
generated with peroxide, paraquat and sodium-nitroprusside
(SNP). The latter release superoxide anions and nitric-oxide
(NO) upon resolution in growth media. The results indicated
that after preloading with 0.5 mM iron, Fur was essential for
resistance to superoxide, NO, peroxide and metronidazole, as
indicated by significant growth reduction, as compared to the wt
strain. In the H. pylori wt strain 26695 addition iron to the
growth medium did strongly inhibit urease-mediated acidresistance. At pH5 the addition of 0.5 mM iron reduced the
urea-dependent growth to about 50%. The iron-mediated
reduction of urease-dependent acid-resistance was more
pronounced in the corresponding fur mutant, which showed no
growth in the presence of iron and urea at pH5. These findings
confirm the important role of iron-mediated homeostasis in
gastric adaptation of H. pylori and help to explain the complete
colonization defect of H. pylori fur mutants in the Mongolian
gerbil-based infection model.
The molecular mechanism of c-Src and Abl tyrosine kinase
activation by the Helicobacter pylori type IV secretion
system encoded in the cag pathogenicity island
D. Zabler1, N. Tegtmeyer1, S. Brandt1, I. Tammer1, S. Gieseler1
W. König1, M. Rohde2, S. Backert1
Otto-von-Guericke-University, Medical Microbiology
Magdeburg, Germany
Helmholtz-Center for Infection Research, Microbial
Pathogenesis, Brunswich, Germany
The pathogenesis of Helicobacter pylori-associated diseases
depends on a specialized type IV secretion system in the cag
(cytotoxin-associated genes) pathogenicity island which encodes
a pilus structure for the injection of the CagA effector protein
into target cells. Within the infected AGS gastric epithelial cells,
CagA becomes phosphorylated on tyrosine residues and initiates
actin cytoskeletal rearrangements and cell scattering. We have
demonstrated recently that injected CagA can be phosphorylated
in vivo and in vitro by Src and Abl tyrosine kinases both of
which are crucial mediators of H. pylori-induced phenotypical
outcome. However, the molecular mechanism by which H.
pylori activates Src and Abl are unknown. Here, we show that
H. pylori activates a integrin>focal adhesion kinase
(FAK)>Src>Abl signaling pathway. Upon activation, FAK is
autophosphorylated at tyrosine residue Y-397 in an integrindependent manner which then serves as a high-affinity binding
site for the SH2 domain of Src resulting in an active FAK-Src
signaling complex. Incubation of AGS gastric epithelial cells
with wild-type H. pylori but not type IV secretion mutants led to
the transient activation of FAK, Src and Abl, as indicated by the
accumulation of phosphorylated FAK-PY-397, Src-PY-418 and
Abl-PY-412, respectively. Furthermore, the subsequent
activation of the three tyrosine kinases correlated with the timedependent occurence of phosphorylated CagA in infected cells,
supporting the notion that integrin activation by H. pylori and
subsequently the activation of FAK, Src and Abl rapidly results
in tyrosine phosphorylation of injected CagA. These findings
have important implications on the role of integrins in H. pyloriinduced infections. Taken together, our results suggest that H.
pylori has evolved a mechanism to use at least three of several
functionally redundant tyrosine kinases which play an important
role in the pathogenesis of this bacterium.
Helicobacter hepaticus HHGI1 is a pathogenicity island
associated with colitis in IL-10-/- and RAG2-/- mice
T. Sterzenbach1, Z. Ge2, J. Schulze1, B. Brenneke1, M. Whary2
B. Rickman2, A. Rogers2, Z. Shen2, N. Taylor2, C. Josenhans1
S. Sürbaum1
Hanover Medical University, Institute for Medical
Microbiology, Hanover, Germany
Massachusetts Institute for Technology, Division of
Comparative Medicine, Cambridge MA, USA
Helicobacter hepaticus belongs to the enterohepatic group of
Helicobacters which persistently colonize the intestinal tract of
humans and various animals. H. hepaticus lives in the upper
bowel and hepatobiliary tract of animals where it can lead to
hepatitis and liver cancer. In several strains of
immunocompromised mice an infection with H. hepaticus
causes colitis, colon carcinoma, and chronic inflammation of the
intestinal tract, which resembles inflammatory bowel disease in
humans, and therefore is used as a model for the study of these
diseases in its natural host. Little is known until now about the
factors of H. hepaticus responsible for its colitogenic properties.
The only pathogenicity factor which has been studied in detail is
the cytoletal distending toxin CDT. The complete genome
sequence of H. hepaticus revealed a putative pathogenicity
island named HHGI1 coding for a type IV secretion system and
several other virulence-associated genes. Type IV secretion
systems play an important role for many bacterial species
regarding their pathogenesis and host interaction. The HHGI1
island is missing in some natural isolates of H. hepaticus, and
we could recently show that HHGI1-deficient strains lead to a
weaker degree of hepatitis than HHGI1-containing strains in
A/JCr mice. In our present study, we wanted to verify that the
HHGI1 island is involved in the development of colitis in
different models of immunocompromised mice.
Results: A mutant lacking a major part of the HHGI1 island
(HH_PAIdel1-mutant) and mutants in several genes encoding
components of the predicted type IV secretion system were
constructed by allelic exchange mutagenesis. The HH_PAIdel1mutant led to a significantly reduced degree of typhlocolitis and
hyperplasia in IL-10-/- C57BL/129 mice, accompanied by
reduced expression of IFN- and TNF- and lower levels of
TH1-associated IgG2c antibodies. In a short term infection
model in IL-10-/- BALB/c mice, it could be confirmed that caeca
and colonic cells of HH_PAIdel1-infected mice secrete lower
amounts of IFN- and IL-12p40 than mice infected with the
wild type, accompanied by reduced levels of IgG2a antibodies.
In RAG2-/- 129S6/Sv mice which do not possess functional Tand B-cells, therefore lacking an adaptive immune system, the
HH_PAIdel1 mutant also induced a reduced degree of colitis in
the caecum. Our data confirm that the HHGI1 island is an
important factor in pathogenicity and host immune modulation.
Investigation of in vivo gene regulation of Escherichia coli
Nissle 1917
D. S. Schmidt1, S. Nicolaisen2, A. Bleich3, A. Smoczek3
H. Blöcker4, I. Deyneko4, M. Hartmann5, F. Gunzer6
Technical University of Dresden, Faculty of Medicine Carl
Gustav Carus, Institute for Medical Microbiology and Hygiene,
Dresden, Germany
Hanover Medical School, Medical Microbiology and Hospital
Epidemiology, Hanover, Germany
Hanover Medical School, Institute for Laboratory Animal
Science and Central Animal Facility, Hanover, Germany
Helmholtz-Center for Infection Research, Department of
Genome Analysis, Brunswich, Germany
Medical Microbiology and Hospital Epidemiology, Hanover
Medical School, Hanover, Germany
Technical University of Dresden, Institute for Medical
Microbiology and Hygiene, Faculty of Medicine Carl-GustavCarus, Dresden, Germany
Escherichia coli strain Nissle 1917 (EcN) is a fecal isolate
which is used as a probiotic and comprises a therapeutic
alternative for the treatment of inflammatory bowel diseases.
Clinical trials comparing EcN to standard medication used for
therapy of ulcerative colitis revealed an equal effectiveness of
the probiotic in maintaining remission, associated with less or
no side-effects. EcN is a good colonizer in the gut of humans
and animals. Specific characteristics of the strain are the
production of microcins or a semirough LPS, for example.
However, the molecular mechanisms which are responsible for
the beneficial behaviour of EcN during passage and colonization
are not very well understood.
Investigation of in vivo gene regulation in EcN will provide
important information for a better understanding of the strains
probiotic traits. Therefore, we constructed a promoter trap
library. Mechanically sheared fragments of EcN genomic DNA
were randomly cloned in front of a promoterless gfp on a
plasmid and transformed into EcN. Plasmids containing
functional promoters would lead to GFP expression in these
clones. To identify in vivo active promoters, the library was fed
to Balb/c mice followed by FACS sorting of green fluorescent
bacteria in the feces. Sequencing of the cloned promoter
fragments and a BLAST search against the EcN genome as well
as a computer based analysis of the promoter regions revealed
genes with potential in vivo activity. Selected genes from a
collection of in vivo regulated ones were chosen as candidates
for further analysis.
Regulation of virulence factor expression by DNA
methylation in Yersinia enterocolitica
S. Faelker1, J. Schilling1, M. A. Schmidt1, G. Heusipp1
University of Muenster, ZMBE, Institute of Infectiology
Muenster, Germany
In -proteobacteria, methylation of DNA at GATC sequences by
the DNA adenine methyltransferase (Dam) regulates various
physiological processes. In addition, differential methylation of
DNA influences the binding of transcription factors, thereby
affecting transcriptional regulation. DNA methylation by Dam
interferes with the coordinated expression of virulence functions
in an increasing number of pathogens. While analyzing the
effect of Dam on virulence of the human pathogen Yersinia
enterocolitica, we observed various altered phenotypes,
including type III secretion of Yop effector proteins under nonpermissive conditions and increased invasion into eukaryotic
cells. We could previously show that the effect of Dam on type
III secretion is mediated posttranscriptionally via the
degradation of the regulatory protein LcrG by the ClpP protease,
but the molecular mechanism behind increased invasion
remained unknown. As invasion and motility are coordinately
regulated in Y. enterocolitica, we analyzed the motility of a Dam
overproducing (DamOP) strain and found it to be highly motile.
In DamOP strains, the operon encoding the master regulator of
flagella biosynthesis, flhDC, is upregulated. We show that the
increased invasion is not due to enhanced expression of known
and putative Y. enterocolitica invasion and adhesion factors like
Invasin, YadA, Ail, Myf fibrils, Pil or Flp pili. This indicates
either that additional so far not identified invasion factors are
upregulated following DamOP or that invasion factors are easier
accessible to cellular receptors. Indeed, we show that DamOP
results in an increased amount of rough lipopolysaccharide
(LPS) molecules lacking O antigen side chains, implying that a
reduced steric hindrance by LPS might contribute to the
increased invasion of a Y. enterocolitica DamOP strain. Our data
indicate that Dam targets regulatory processes modulating the
composition and function of the bacterial surface.
Salmonella typhimurium exploits inflammation to compete
with the intestinal microbiota
B. Stecher1, R. Robbiani1, A. Walker2, A. Westendorf3
M. Barthel1, M. Kremer4, A. J. Macpherson5, C. von Mehring6
W. - D. Hardt1
ETH Zurich, Institute of Microbiology, Zurich, Switzerland
Sanger Center, Wellcome Trust, Genome Campus, Cambridge
United Kingdom
Helmholtz-Center for Infection Research, Department of
Mucosal Immunity, Brunswich, Germany
Ludwig-Maximilian-University of Munich, Pathology, Munich
Mcmaster University, Hamilton, Canada
University of Zurich, Molecular Biology, Zurich, Switzerland
Most mucosal surfaces of the mammalian body are colonized by
microbial communities (microbiota). A high density of
commensal microbiota inhabits the intestine and shields from
infection (‘colonization resistance’). The virulence strategies
allowing enteropathogenic bacteria to successfully compete with
the microbiota and overcome colonization resistance are poorly
understood. Here, we investigated manipulation of the intestinal
microbiota by the enteropathogenic bacterium Salmonella
Typhimurium in a mouse colitis model: We found that
inflammatory host responses induced by S. Typhimurium
changed microbiota composition and suppressed its growth. In
contrast to wild type S. Typhimurium, an avirulent invGsseDmutant failing to trigger colitis was outcompeted by the
microbiota. This competitive defect was reverted if
inflammation was provided ‘in trans’ by mixed infection with
wild type S. Typhimurium or in mice (IL10-/-, Villin-HA+CL4TCR) suffering from inflammatory bowel disease. Thus,
inflammation is necessary and sufficient for overcoming
colonization resistance. This reveals a new concept in infectious
disease: In contrast to current thinking, inflammation is not
always detrimental for the pathogen. Triggering the hosts
immune defense can shift the balance between the protective
microbiota and the pathogen in favour of the pathogen.
Identification of intracellular signalling cascades mediating
Salmonella invasion into epithelial cells
B. Misselwitz1, S. Dilling1, R. Sacher2, B. Snijder2
L. Pelkmans2, W. - D. Hardt1
ETH Zurich, Microbiology, Zurich, Switzerland
ETH Zurich, Institute of Molecular Systems Biology, Zurich,
Salmonella spp. are a major cause of food born diseases. Central
to Salmonella pathogenicity is its ability to invade gut epithelial
cells. To this end, Salmonella spp. inject virulence factors (so
called effectors) into the cytoplasm of the host’s cells. The most
important effectors responsible for Salmonella invasion are
SopE, SopE2, SopB and SipA. These effectors can either
directly or indirectly activate actin polymerization, causing an
uptake of the bacteria by the host. Even though several
intracellular targets of these effectors have been identified, the
complexity of signaling mechanisms leading to Salmonella
uptake has not been addressed.
To investigate the invasion mechanism we have established a
new microscopy based Salmonella invasion assay that enables
us to perform high throughput experiments. Moreover, using
Salmonella single effector mutants expressing only one of the
important Salmonella effectors we can test invasion mediated by
only one protein without interference of the others. We are
currently testing a 50 kinase library and plan to proceed to
testing a genome wide library. With our experiments we will
identify cellular signaling cascades important for an efficient
invasion of Salmonella.
Functional characterisation of the non-fimbrial adhesin SiiE
C. Wagner1, M. Hensel1
University Hospital Erlangen, Institute of Microbiology
Erlangenm, Germany
The complex pathogenesis of infections with Salmonella
enterica is a result of different interactions between pathogen
and host cells, where so-called pathogenicity islands (PAI) play
a major role in the infectious processes. Recently it has been
shown that Salmonella pathogenicity island 4 (SPI4) has an
important, but so far unknown function in adhesion to, and
invasion of polarised, microvilli-forming epithelial cells. SPI4
genes code for the components of a type I secretion system
(T1SS), its substrate protein SiiE, which serves as a nonfimbrial adhesin to mediate the adhesion and two accessory
proteins, SiiA and SiiB, whose function is not defined so far.
SiiE is the largest protein of the proteome of Salmonella with a
molecular weight of about 600 kDa. SiiE has a predicted domain
structure, consisting of a N-terminal part which forms ß-sheets
and coiled-coil domains, followed by 53 highly repetitive Igdomains with an insertion of about 60 residues between the last
two Ig-domains and a C-terminal part without characteristic
structural elements. To understand the binding process of SPI4dependent adhesion, it is important to characterise SiiE and to
dissect the functions of the various SiiE domains more precisely.
Proteins secreted by T1SS are commonly dependent on a Cterminal signal sequence. This led us to investigate the Cterminal part for the ability to mediate the secretion of SiiE. By
means of truncations of the C-terminus of SiiE, it could be
shown that the signal sequence for the recognition and secretion
of SiiE by the T1SS is located maximal 9 residues apart from
the C-terminus of SiiE and that the ultimate residues of the
signal sequence were essential for the secretion, as the Cterminal truncations of SiiE proteins abolished the secretion.
Furthermore it could be shown that the secretion was dependent
on SPI4-encoded T1SS, as a siiF mutant was not able to secrete
full-length SiiE. Besides bearing the signal sequence, another
function of the C-terminal part has been suggested: the
recognition and binding of a putative host cell receptor, which is
currently under investigation. Regarding other domains of SiiE,
the N-terminus is supposed to anchor the protein to the bacterial
cell, and the numerous Ig-domains might be essential to
elongate the adhesin to protrude above LPS-length and to ensure
a certain distance.
Serotype-dependent escape of Yersinia enterocolitica YopE
from degradation by the ubiquitin-proteasome pathway
M. Hentschke1, K. Truelzsch2, J. Heesemann2
M. Äpfelbacher1, K. Ruckdeschel1
University Hospital Eppendorf, Institute for Medical
Microbiology, Hamburg, Germany
Max von Pettenkofer-Institute, Munich, Germany
Pathogenic Yersinia spp. engage a type III protein secretion
system that translocates several Yersinia outer proteins (Yops)
into the host cell to modify host immune responses. The
degradation of injected bacterial virulence proteins through the
ubiquitin-proteasome pathway is one strategy of the infected
host cell to resist the bacterial attack. The cytotoxin YopE is a
known target protein of this proteolytic system.We
investigatedYopE protein species belonging to different
enteropathogenic Y. enterocolitica serogroups towards
ubiquitination and proteasomal degradation. Our data indicate
that YopE from the highly pathogenic Y. enterocolitica serotype
O8 is subjected to proteasomal destabilization, whereas YopE
from the serogroups O3 and O9 evades degradation. The
accumulation of YopE from the serotypes O3 and O9is
accompanied by an enhanced cytotoxic effect. Using Yersinia
strains that specifically produce YopE from either Y.
enterocolitica O8 or O9 we found that solely YopE from
serogroup O8 is modified by polyubiquitination. We determined
twoN-terminal lysinesin serogroup O8 YopE not present in
serogroup O9 YopE that serve as polyubiquitin acceptor sites.
Insertion of either lysine in serotype O9 YopE enabled its
ubiquitination and destabilization. These results define a
serotype-dependent difference in the stability and activity of the
Yersinia effector protein YopE that could be important for Y.
enterocolitica pathogenesis.
Chlamydia trachomatis induced RANTES production
through the activation of the NF-kB host signalling pathway
K. Sommer1, O. Dittrich-Breiholz2, M. Kracht2, A. Klos1
HanoverMedical School (MHH), Medical Microbiology
Hanover, Germany
HanoverMedical School (MHH), Department of
Pharmacology, Hanover, Germany
Chlamydia trachomatis is a highly successful pathogen that
causes a wide variety of disease in humans including pelvic
inflammatory disease, sterility, and blindness. Pathology
associated with this obligate intracellular bacterium is due to
inflammation-associated tissue damage and scaring from
repeated or chronic infection; however, the mechanism by
which the inflammatory response is induced is poorly
inflammation, in part through induced chemokine synthesis.
RANTES, a CC chemokine, is known to be activated and
regulated by NF-kB, a critical modulator of immune function.
We hypothesize that RANTES is induced through activation of
the NF-kB host signalling pathway within chlamydia infected
In chlamydial infected HT1080 cells, NF-kB activation was
characterized by translocation of NF-kB from the cytoplasm to
the nucleus, increased DNA binding activity, and NF-kB
regulated gene expression. NF-kB activation was dose and time
dependent. UV inactivation of Chlamydia significantly reduced
the level of NF-kB activation. Degradation of IkB protein was
detected late in chlamydial infection, and overexpression of the
dominant negative form of IkB-alpha significantly suppressed
NF-kB activation and RANTES expression induced by
chlamydia. RANTES protein and mRNA were detected late in
the infection from cells infected with replicating chlamydia, but
not with UV-inactivated bacteria. The induction of RANTES
was not dependent on a secreted soluble factor of infected cells
and therefore associated with an internal cellular signalling
pathway. Our observations imply that NF-kB activation is a
biological significant aspect of chlamydial pathogenesis. We
conclude that an effector-protein produced by C. trachomatis at
mid-developmental stages induces RANTES within infected
HT1080 cells through activation of host signalling pathways
related to NF-kB. The present study provides a basis for
understanding molecular pathways of pathology and immune
evasion associated with disease caused by C. trachomatis.
Functional analysis of the Coxiella effector AnkG
A. Lührmann1, C. R. Roy1
Yale University School of Medicine, Section of Microbial
Pathogenesis, New Haven, USA
Coxiella burnetii, the causative agent of Q fever, a worldwide
zoonosis, is an obligate intracellular pathogen. After uptake into
its host cell this pathogen resides in a phagolysosomal
compartment. Coxiella modifies this organelle into a very large
vacuole that permits bacterial replication. Although it is
unknown how Coxiella actually creates its replicative niche, its
clearly an acitve process requiring bacterial proteins synthesis,
suggesting bacterial products may be directly involved. One
mechanism by which Coxiella might be able to manipulate its
host cell is by delivering bacterial effector proteins into the host
cell using a chromosomally-encoded type IV secretion system. I
was able to demonstrate, that a subset of Coxiella proteins,
which contains muliple ankyrin repeat domains, are effectors of
the type IV secretion system. One of these effector proteins,
AnkG, shows a vesicular pattern with association to
mitochondria when ectopically expressed. To determine the
function of AnkG, host interacting proteins were isolated by
affinity purification on a column having a purified AnkG protein
attached covalently to Affi-Gel. Host proteins that bound
specifically to the AnkG affinity column were identified by
mass spectrometery. I was able to confirm interactions between
AnkG and the host protein gC1qBP (glycoprotein complement
component 1, q subcomponent binding protein) by coimmunoprecipitation as well as immunofluorescence. The
function of gC1qBP, a protein that resides predominantly in the
mitochondria matrix, is unknown.
Currently, I am investigating how these interaction influences
normal host cell function and more importantly, how Coxiella
benefits from this binding. This analysis should provide unique
insight into how Coxiella modulate the host cell to ensure
survival and multiplication.
Chlamydial Protease-like Activity Factor (CPAF): A Factor
of chlamydial pathogenicity
S. Paschen1, J. Vier1, J. Christian1, S. Ying1, G. Häcker1
Technical University Munich, Institute for Medical
Microbiology, Munich, Germany
Chlamydiae are obligate intracellular bacteria and important
human pathogens. Chlamydia is known to induce massive
changes in the infected host cell, including altered gene
transcription, both inhibition of apoptosis and induction of nonapoptotic cell death and structural alterations culminating in the
release of newly formed bacteria. The molecular basis of how
Chlamydia achieves this is largely unknown. CPAF is a protease
that translocates at later stages of the infection from the
chlamydial inclusion into the cytosol. Although a few CPAF
substrates are known, and although the presence of a free
protease in a human cell’s cytosol can be expected to be of
consequence, it is unclear what the contribution of CPAF to the
cellular effects of chlamydial infection is. During infection,
CPAF is processed by intramolecular cleavage, and this event is
required for its activity, precluding the study of the protease
outside chlamydial infection. We have recently devised an
experimental system where CPAF can be activated through
‘induced proximity’, i. e. the experimental oligomerisation of a
CPAF protein by pharmacological cross-linking of its fusion
partner, gyrase B, in intact cells. We used this system to test for
the physiological consequences of CPAF-activation in
uninfected cells. The activation of CPAF caused the cleavage of
the known CPAF substrates and allowed the identification of
new cellular targets of the protease. CPAF-activation led to the
death of the cell and induced massive morphological changes,
possibly due to the cleavage of cytoskeletal components.
Intriguingly, caspase-activity was also measured upon
prolonged activity of CPAF, indicating the potential of CPAF to
activate the apoptotic pathway. These studies show the capacity
of CPAF to cause massive changes to the host cell and suggest
that this protease is a good candidate for a major pathogenicity
factor of Chlamydia that causes changes to the host cell towards
the end of the bacterial cycle.
Infection with Anaplasma phagocytophilum inhibits IFNsignalling in human neutrophils
U. Bussmeyer1, A. Sakar1, K. Broszat1, G. van Zandbergen1
C. Bogdan2, W. Solbach1, F. von Löwenich2, T. Laskay1
University of Luebeck, Institute of Medical Microbiology and
Hygiene, Luebeck, Germany
University of Freiburg, Institute of Medical Microbiology and
Hygiene, Freiburg, Germany
Anaplasma phagocytophilum (Ap) is a tick-borne obligate
intracellular bacterium that survives and multiplies inside
polymorphic neutrophilic granulocytes (PMN). Previous
findings demonstrated that the bacterium actively subverts
antimicrobial effector mechanisms of PMN including the
oxidative burst after priming with IFN- . The present study
aimed to investigate whether an infection with Ap leads to a
more general impairment of IFN- signaling in PMN that
enables intracellular survival of the bacterium.
The capability of Ap to interfere with IFN- -mediated activation
of PMN was assessed by measuring MIG and IP-10 secretion.
Neutrophils secreted substantial levels of both chemokines when
stimulated with IFN- for 18h. Infection of PMN with Ap
markedly decreased the secretion of MIG and IP-10 by PMN.
To obtain first insights into the molecular events leading to the
diminished secretion of IFN- -induced chemokines, the
phosphorylation of STAT1 was investigated. Western blot
analysis revealed that IFN- -induced STAT1 phosphorylation
was diminished in Ap-infected PMN. In further experiments
flow cytometry analyses showed a markedly decreased
expression of the IFN- receptor alpha chain CD119 on the
surface of Ap-infected PMN. Moreover, using quantitative RTPCR a strong upregulation of the negative regulator SOCS3 was
observed in infected cells.
Taken together our data show that infection with Anaplasma
phagocytophilum results in a decreased CD119 surface
expression, diminished tyrosine phosphorylation of STAT1 and
augmented SOCS3 gene expression in infected cells and,
consequently, results in compromised IFN- -responsiveness of
infected PMN. Impaired IFN- signaling in infected cells is
likely to contribute to intracellular survival of Ap in PMN.
Identification of a new conjugation/ type IVA secretion
system (Trb/Tra) of Legionella pneumophila
K. Heuner1, C. Albert-Weissenberger1, E. Schunder1
M. Steinert2, G. Glöckner3
Institute for Molecular Infection Biology, Wuerzburg
Institute for Microbiology, Brunswich, Germany
Leibniz-Institute for Age Research, Jena, Germany
In the genome sequence of Lp Corby a new conjugation/ T4ASS
(trb/tra) was identified. This system is not present in the yet
sequenced genomes of Lp but similar loci were identified by
DNA hybridization in various non-pneumophila species of
Legionella. It is known, that L. pneumophila is able to
horizontally transfer chromosomal DNA, but no element or
mechanism could be identified so far, responsible for this
observation. L. pneumophila can conjugate RSF1010-related
plasmids in an icm/dot-dependent manner and lvh also
contributes to the ability to mobilize a plasmid.
We will report about two similar versions of trb/tra present in
the genome of L. pneumophila Corby, localized on two different
genomic islands (Trb-1, 42,710 bp and Trb-2, 34,434 bp). Trb-1
and Trb-2 are integrated within the tRNAPro gene (lpc2778) and
the tmRNA gene (lpc0164), respectively. Both islands exhibit an
oriT region and both can be excised from the chromosome
forming episomal circles. The genomic island Trb-1 can be
transferred horizontally to another Lp strain by conjugation and
integrates site-specific into the genome of transconjugants. This
mechanism explains for the first time horizontal DNA-transfer
in Legionella, which is not due to natural competence for DNA
transformation. Thus, here we demonstrate a putative
mechanism of horizontal transfer of large chromosomal DNA
regions in Legionella.
The cell-associated phospholipase PlaB of Legionella
pneumophila is a major virulence factor
E. Schunder1
University, Molekulare Infektionsbiologie, Wuerzburg
Legionella pneumophila is an aquatic environmental bacterium
which replicates inside of protozoa. After inhalation of
contaminated aerosol, Legionella is able to replicate in alveolar
macrophages and can cause an atypical, severe pneumoniaLegionellosis.
Phospholipases are known to contribute to bacterial
pathogenicity, because they hydrolyze cell membranes, generate
second messengers, lysophospholipids and destruct the lung
surfactant. Recently, the PlaB phospholipase has been identified
and characterized as the major cell-associated phospholipase A
with additional lysophospholipase and hemolytic activity.
Experiments in guinea infection model clearly verified that PlaB
is a major virulence factor in vivo, which contributes to bacterial
spreading during infection. Contrary to the yet known
phospholipase activities of Legionella pneumophila, PlaB does
not belong to the known enzyme classes and therefore may
represent a new class of enzymes.
Life within the cytosol - Molecular analysis of the
intracellular life style of Burkholderia pseudomallei
K. Eske1, K. Breitbach1, H. Schalimow1, I. Steinmetz1
University of Greifswald, Friedrich-Loeffler-Institute of Med.
Microbiology, Greifswald, Germany
The gram-negative environmental saprophyte Burkholderia
pseudomallei is the causative agent of melioidosis, an infectious
disease of humans and animals which is now known to be a
major cause of morbidity and mortality in certain areas of the
tropics. Burkholderia pseudomallei is able to invade host cells,
escapes from endocytic vesicles, multiplies intracellularily, and
induces the formation of actin tails and membrane protrusions,
leading to direct cell to cell spreading. Recently, we identified a
number of B.pseudomallei genes which are essential for this
intracellular life cycle (Infect. Immun. 2006. 74:3576-3586). In
this study, we extented the identification of B.pseudomallei
genes responsible for the different steps of the intracellular life
cycle by screening 2344 B.pseudomallei transposon mutants for
reduced ability to form plaques on cell monolayers as a result of
reduced intercellular spreading. 44 mutants showed impaired
plaque formation but no growth defect in culture medium.
Determination of the transposon insertion sites revealed the
disruption of genes encoding for metabolic, structural, and
hypothetical proteins. Mutants were further characterized with
respect to intracellular invasion, survival and replication,
intracellular motility, and in vivo virulence. In a mouse model of
infection, up to now 10 mutants were shown to be highly
attenuated, including a mutant with a defect in an immunophillin
with peptidyl prolyl cis-trans isomerase activity. In summary,
we have identified a number of novel virulence genes which are
important for the intracellular life style of B.pseudomallei.
The Salmochelin siderophore receptor IroN contributes to
invasion of urothelial cells by extraintestinal pathogenic
Escherichia coli in vitro
F. Feldmann1
Max von Pettenkofer-Institute, Munich, Germany
Extraintestinal pathogenic Escherichia coli (ExPEC) strains
possess a diversity of specific virulence factors that enable them
to cause infections outside the gastrointestinal tract. These
infections range from asymptomatic urinary tract infections
(UTIs) to life-threatening diseases, such as pyelonephritis or
sepsis. The acquisition of iron (Fe3+) is a critical step in the
pathogenesis of UTIs, as the concentration of free Fe3+ is
extremely limited at the sites of infection in mammalian hosts.
In order to acquire iron from the host organism, ExPEC strains
have developed a variety of iron uptake mechanisms, such as the
synthesis and transport of small iron chelators, called
Previous studies revealed that several siderophore systems are
more prevalent in ExPEC than in commensal strains and play an
important role in the pathogenesis of UTIs. The presence of
different iron uptake systems in ExPEC strains prompted us to
look for further functions of the siderophore systems in the
pathogenicity of ExPEC strains. Furthermore, lately it has
become evident that siderophore systems of ExPEC strains may
contribute to other virulence traits, such as adherence and
invasion. This is of particular interest, as it has recently been
shown that ExPEC strains are able to form intracellular, biofilmlike structures in epithelial cells of the bladder in mice.
In this study we clearly demonstrate that the salmochelin
siderophore receptor IroN is involved in the invasion of
urothelial cells by ExPEC in vitro. Thus, IroN may play a dual
role in the establishment of urinary tract infections, displaying
an iron uptake receptor as well as an internalization factor.
New Legionella pneumophila genes essential for amoeba
P. Aurass1, B. Pless1, K. Rydzewski1, A. Flieger1
Robert-Koch-Institute, NG5, Berlin, Germany
Legionella pneumophila is an intracellular bacterium and
normally inhabits fresh water systems where amoebae are a site
for replication and transmission. When the bacteria accidentally
enter the human lung, they infect lung cells and cause a severe
pneumonia, Legionnaires’ disease. Colonization of amoebae by
Legionella evolutionary existed a long time before association
with humans occurred and bacterial human-to-human
transmission is unknown; therefore amoeba interactions
basically shaped Legionella intracellular survival strategies.
Understanding of amoeba exploitation by Legionella is for
several reasons interesting: a) amoeba colonization is a common
strategy of many microorganisms, however little is known on
the precise mechanisms; b) danger for increased environmental
Legionella presence arises within the context of global warming
because temperatures > 25°C support Legionella amplification
in amoebae; c) Legionella exploitation of amoebae and lung
macrophages shows similarities; therefore identification of
amoeba colonization strategies allows insights into mechanisms
applied for mammalian cells; and d) Legionella pneumonia
renders more severe when bacteria together with amoebae enter
the lung, mostly because amoeba-released bacteria are more
To discover novel Legionella genes for amoeba colonization, we
generated a L. pneumophila transposon Tn-5 mutated clone
bank of about 26.000 clones, each with a single random
mutation. To handle the big number of different clones, we
developed an Acanthamoeba-based infection assay which
allowed fast screening of L. pneumophila clones for replication
defects. As a result we on the one hand found 21 genes,
including icm/dot virulence genes, already known to promote
Legionella infection of macrophages and/or amoebae. On the
other hand, we identified the high number of 49 novel genes.
Those genes comprised potential regulator, transporter, or
enzyme genes, and also 15 genes with unknown function.
Geno- and phenotypic analysis of asymptomatic bacteriuria
Escherichia coli isolates
J. Zdziarski1, B. Wullt2, C. Svanborg3, J. Hacker1, U. Dobrindt1
University of Wuerzburg, Institute for Molecular Infection
Biology, Wuerzburg, Germany
University Hospital of Lund, Dept. of Urology, Lund, Denmark
Lund University, Dept. of Laboratory Medicine, Div. of
Microbiology, Immunology and Glycobiology, Lund, Denmark
Asymptomatic bacteriuria (ABU) results from the frequently
long term urinary tract colonization by Escherichia coli without
causing typical symptoms of urinary tract infection (UTI). The
role of multiple virulence-associated factors of uropathogenic E.
coli (UPEC) involved in the development of symptomatic and
chronic UTI has been elucidated so far, but only little
information is available on characteristics of ABU isolates. We
compared the geno- and phenotypes of eleven ABU isolates to
characterize this group of organisms in more detail. ABU
isolates represent a heterologous group of organisms.
Accordingly, they differed markedly in genome content, i.e., the
genome size as well as the presence of typical UPEC virulenceassociated genes. Multi locus sequence typing suggested that
certain ABU strains evolved from UPEC variants that are able to
cause symptomatic UTI by genome loss. Reductive evolution by
point mutations, DNA rearrangements and deletions resulted in
inactivation of genes coding for several UPEC virulence factors
thus supporting the idea that a reduced bacterial activation of
host mucosal inflammation supports the ABU lifestyle of these
E. coli isolates without activation of local and systemic
inflammatory response pathways.
Exploitation of vitronectin for pneumococcal adherence and
invasion via v 3-integrins
S. Bergmann1,2, A. Lang1, C. R. Rennemeier1, K. T. Preissner3
S. Hammerschmidt1,2
University of Wuerzburg, Research Center for Infectious
Diseases, Wuerzburg, Germany
University of Munich, Max von Pettenkofer Institute, Munich
Julius-Liebig-University, Institute for Biochemistry, Giessen
The human glycoprotein vitronectin (VN) is a multimeric
extracellular matrix (ECM) protein which exists in two
conformations. Within the blood plasma VN circulates as
globular monomer within blood plasma and the multimeric
isoform is predominantly present in the ECM. Pneumococci
bind multimeric VN and recruit the monomeric isoform of
vitronectin from human plasma as shown by flow cytometric
and immunoblot analysis. Infection studies with human
epithelial cells and human brain-derived microvascular
endothelial cells (HBMEC) demonstrated a significant increase
in pneumococcal adherence and invasion in the presence of
host-cell bound VN. This effect was dose-dependent and
sensitive to protease-treatment of the bacteria, indicating a
proteinaceous bacterial receptor. Flow cytometric analysis and
cell culture infections demonstrated that the interaction of
pneumococci with soluble VN and host-cell bound VN is
inhibited by heparin and heparan sulfate. This suggests that the
heparin binding domains of VN mediate the interaction with
VN. In cell culture blocking experiments, antibodies
v 3-integrins
inhibited internalization of pneumococci in nasopharyngeal
epithelial cells. In addition, vitronectin-mediated internalization
of pneumococci was inhibited by pharmacological inhibitors
cytochalasin D, latrunculin, and jaspaklinolide, respectively.
Finally, vitronectin-mediated uptake of pneumococci in
fibroblast deficient for the integrin?linked kinase (ILK) was
significantly lower compared to ILK-expressing fibroblasts. In
conclusion, our data provide evidence that pneumococci engage
vitronectin as a molecular bridge thereby linking the bacteria
with the v 3-integrin receptors on the host cells. Moreover, the
ILK and actin cytoskeleton are essential molecules during
vitronectin-mediated pneumococcal invasion.
Pertussis toxin enhances the translocation of pathogenic
bacteria in a human blood-brain barrier model
K. Böcker1, J. Schulte1, C. Wewer1, L. Greune1, V. Humberg1
K. - S. Kim2, M.A. Schmidt1
Westfaelische Wilhelms-University Muenster, Institute of
Infectiology - ZMBE, Muenster, Germany
Johns Hopkins University, Department of Pediatrics
Baltimore, MD, USA
The respiratory tract infection whooping cough caused by
Bordetella pertussis can be accompanyed by complications such
as encephalopathies and neurological disorders. A decisive
virulence determinant of Bordetella pertussis, pertussis toxin
(PT), contributes to these sequelae by influencing the integrity
of the blood-brain-barrier (BBB). Human brain microvascular
endothelial cells (HBMEC) were cultured on Transwell filters
and used as an in vitro model for the BBB. We hypothesized
that the PT-mediated increase in BBB permeability might lower
the threshold for pathogenic bacteria to traverse the BBB. To
address whether secondary infections of the CNS might develop
faster or more serious we used E. coli K1 the most common
strain that causes neonatal meningitis in our in vitro BBB model
system. Indeed, following treatment with PT we observed an
increased traversal of these bacteria across the HBMEC
monolayers. E. coli K1 alone did not alter the permeability of
the BBB model system. Enhancement of translocation could not
be detected in studies with the non-pathogenic E. coli strain
C600. Interestingly, in TEM images no invasive E. coli K1 was
found within the endothelial cells- However, E. coli K1 was
found between the HBMEC, indicating the paracellular way for
traversal of the HBMEC monolayer. In addition, no effect of PT
on invasion of E. coli K1 was observed. Furthermore, an
increased migration/translocation of HL60 macrophage-like
cells through PT-treated HBMEC monolayers was examined. As
in these cells we were able to demonsrtate live E. coli K1 the
translocation of E. coli K1-loaded macrophages resembles the
“Trojan horse mechanism”, a possible alternative mechanism
used by these pathogens to infect the CNS in vivo.
Interactions between murein (peptidoglycan) synthases and
the divisome in Escherichia coli
U. Bertsche1, E. Breukink2, W. Vollmer3
University of Tuebingen, Microbial Genetics, Tuebingen
University of Utrecht, Center of Biomembranes and Lipid
Enzymology, Utrecht, Netherlands
University of Newcastle upon Tyne, nstitute for Cell and
Molecular Biosciences, Newcastle upon Tyne, United Kingdom
The murein (peptidoglycan) sacculus represents the stressbearing structure in the cell envelope of most bacteria. Even
though it is a major target for antibacterial treatment, the
knowledge about its synthesis and the involved proteins is still
very limited. During cell division and cell separation, which is a
major requirement for the spreading of diseases, murein
synthesis in Escherichia coli is focused on the septation site to
form two new polar caps.
cross-linking/coimmunoprecipitation and affinity chromatography experiments
to study physical interactions between the proteins involved in
murein synthesis during cell division. This revealed the
existence of direct interactions between murein synthases, cell
division proteins, and murein hydrolases, which cleave the
septum for daughter cell separation. The murein synthase
PBP1B, which catalyzes both the transglycosylase and the
transpeptidase reaction during murein synthesis, was shown to
interact with the essential cell division protein PBP3 (FtsI), a
monofunctional transpeptidase [1]. In addition both proteins
interact with FtsN, a major component of a cell division specific
multiprotein complex called the divisome. Tested by an in vitro
murein synthesis assay the activities of PBP1B [2], were
positively stimulated by the interaction with FtsN. We propose
that septal murein synthesis occurs by multi-enzyme complexes
containing murein synthases and murein hydrolases, which are
controlled by the divisome.
[1] Betsche, U., Kast, T., Wolf, B., Fraipont, C., Arasman, M.
E., Kannenberg, K., von Rechenberg, M., Nguyen-Disteche, M.,
den blauauwen, T., Hoelthe, J.V., and Vollmer, W. (2006) Mol.
Microbiol .61(3), 675-690
[2] Bertsche, U., Breukink, E., Kast, T., and Vollmer, W. (2005)
J. Biol. Chem. 208(45), 38096-38101
Role of agrD for biofilm formation and virulence in L.
C. U. Riedel1,2, I. R. Monk2, P. Casey2, C. G. M. Gahan2
C. Hill3
University of Ulm, Institute of Microbiology and
Biotechnology, Ulm, Germany
University College Cork, Alimentary Pharmabiotic Center and
Microbiology Department, Cork, Ireland
University College Cork, Alimantary Pharmabiotic Center and
Microbiology Department, Cork, Ireland
Autoinducing peptides are used in cell density dependent
quorum sensing of Gram positive organisms. One example is the
agr system, which is involved in virulence gene expression in
staphylococci. The Agr system is composed of four gene operon
with agrB involved in the proteolytic processing/export of the
gene product of agrD, a post transitionally modified peptide,
and agrA/agrC encoding for the histidine kinase/response
Here, we present the phenotypic characterisation of L.
monocytogenes EGDe DargD peptide deletion mutant. The
DargD mutant showed a significant defect in biofilm formation.
Promoter studies in broth cultures using a bioluminescent
reporter system revealed an altered expression profile of hlyA
and plcA, two major virulence factors of L. monocytogenes. In
line with these findings, invasion of DargD into Caco-2 cells
was about 4-fold lower than that of EGDe wild type. Both
biofilm formation and invasion could be complemented by
single copy chromosomal integration of argD under the control
of a constitutive promoter. Additionally, when the DargD
mutant was mixed with wild type EGDe, as little as 1 % of the
wild type in the inoculum was sufficient to completely restore
biofilm formation of EGDe DargD to wt levels. Finally,
bioluminescence in vivo imaging was used to confirm that
EGDe DargD is significantly attenuated in a murine model of
Identifying a pore-forming region within E. coli hemolysin
by means of cysteine substitution mutant analysis
A. Valeva1, I. Valev1, I. Siegel1, M. Wylenzek1
University of Mainz, Institute of Medical Microbiology and
Hygiene, Mainz, Germany
Escherichia coli hemolysin is a pore-forming toxin of the RTX
toxin family. Cysteine scanning mutagenesis was applied to
characterize the proposed pore-forming domain 170 - 400 of
HlyA. A single cysteine residue was introduced at 48 different
positions in this domain and labeled with the polarity-sensitive
dye badan. Spectrofluorimetric analysis of labeled toxin showed
that several amino acids in this domain insert into the lipid
bilayer after pore-formation. An alpha helix domain consisting
of amino acids 272 to 298 was identified that forms the lining of
the aqueous pore. The importance of this alpha helix in the porebuilding domain was confirmed by the introduction of helix
breaker proline at position 284 and 287, which completely
abolished haemolytic activity, despite unchanged binding
Integrated stress response following membrane perforation
M. Husmann1, G. Veerachato1, N. Kloft1, T. Busch1
W. Bobkiewicz1, C. Neukirch1, S. Bhakdi1
Johannes Gutenberg-University, Mainz, Germany
Formation of pores in membranes of cell targets is an archetypal
attack mechanism that is conserved through evolution.
Nucleated cells have evolved poorly understood means to cope
with the potentially lethal threat of membrane perforation. A
pivotal role for MAPK p38 in cellular defense against bacterial
pore-forming toxins has recently been discovered, but the
trigger for p38 activation and for downstream events thereof
have not been elucidated. Here, we show in epithelial cells that
membrane perforation by staphylococcal a-toxin, archetype of
the b-barrel family of pore-forming toxins, elicits the integrated
stress response (ISR), which is characterized by rapid but
transient attenuation of global translation, followed hours later
by restart of protein synthesis as cells recover from the initial
insult. While the ISR has recently been identified as a
primordial response of cells to various forms of stress, no link
has yet been made between ISR and membrane perforation.
Three current findings are of prime interest. First, the ISR in
these cells is under the control of p38 MAPK, which undergoes
rapid and prolonged activation. Within minutes, the stress kinase
induces translational arrest. Subsequently, p38 enhances
expression of GADD34, which is required for translational
restart. Second, activation of p38 is triggered by the loss of
cellular potassium. Third, the ISR delays expression of MKP-1
thus prolonging activation of p38. The data may help to explain
the central role of MAPK p38 in defense against membrane
perforation by pore forming proteins.
CD18 is the T-lymphocyte receptor of the Helicobacter
pylori Vacuolating Cytotoxin
X. Sewald1, S. Prassl1, B. Gebert-Vogl1, E. Weiss1
B. Holzmann2, W. Mothes3, R. Haas1
Ludwig-Maximilian University of Munich, Max von
Pettenkofer-Institute, Munich, Germany
Technical University of Munich, Institut for Medical
Microbiology, Munich, Germany
Yale University School of Medicine, Section of Microbial
Pathogenesis, New Haven, USA
Helicobacter pylori is a bacterial pathogen that colonizes the
human stomach. It persists, if untreated, lifelong in the gastric
mucosa of about 50% of the human population thereby causing
gastritis, peptic ulceration, and gastric carcinoma. To evade and
modulate the immune system H. pylori expresses a set of
virulence factors. The vacuolating cytotoxin (VacA) is one
important factor in modulating the immune system. It represents
a multifunctional toxin with pleiotropic effects on mammalian
cells. Although many data about VacA receptors, intracellular
trafficking routes, and cellular effects of VacA exist for
epithelial cells, little is known about these steps for other cell
types. Because the major immunomodulating effect of VacA is
the inhibition of T-lymphocyte activation, we focus our work on
the interaction between VacA and lymphocytes.
VacA interferes with the T-cell receptor/IL-2 signalling pathway
at the level of the Ca2+-Calmodulin dependent phosphatase
calcineurin. IL-2 transcription and therefore activation of T-cells
is down regulated by blocking the nuclear translocation of
NFAT, a global regulator of immune response. How and where
does the immunomodulating effect of VacA on lymphocytes
take place? To address this issue, we focus our work on
identifying the VacA receptor of lymphocytes and characterize
the route of endocytosis. Functional and microscopy studies
show that the 2-subunit of the LFA-1 integrin is a receptor for
VacA on lymphocyte cell lines and primary CD4+-lymphocytes.
The direct interaction of VacA with CD18 allows internalization
and makes lymphocytes susceptible for the immunomodulating
activity of VacA. With different fluorescence markers for
endocytosis and GFP-fusion marker proteins for endosomal
structures, the transport of VacA in lymphocytes is
characterized in more detail.
Protein subassemblies of the Helicobacter pylori Cag type
IV secretion system revealed by localization and interaction
S. Kutter1, R. Buhrdorf1, W. Schneider-Brachert2, R. Haas1
W. Fischer1
Max von Pettenkofer-Institute, Bacteriology, Munich, Germany
University Ratisbon, Institute for Medical Microbiology und
Hygiene, Ratisbon, Germany
Type IV secretion systems are possibly the most versatile
protein transport systems in Gram-negative bacteria, with
substrates ranging from small proteins to large nucleoprotein
complexes. In many cases, such as the cag pathogenicity island
of Helicobacter pylori, genes encoding components of a type IV
secretion system have been identified due to their sequence
homologies to prototypical systems such as the VirB system of
Agrobacterium tumefaciens. The Cag type IV secretion system
of Helicobacter pylori is responsible for the induction of a
pronounced proinflammatory response and for translocation of
the effector protein CagA into various host cells. It therefore is
considered as an important virulence determinant of H. pylori.
The secretion system contains at least 14 essential apparatus
components and several substrate translocation and auxiliary
factors, but the functions of most components cannot be inferred
from their sequences due to the lack of homologies.
In this study, we have performed a thorough sequence analysis
of all essential or auxiliary Cag components, and we have used a
panel of antisera against several components to determine their
subcellular localization. The results suggest that the Cag system
contains functional analogues to all VirB components except
VirB5. Moreover, we have characterized mutual stabilization
effects and performed a comprehensive yeast two-hybrid
screening for potential protein-protein interactions. To obtain
independent evidence for protein-protein interactions found with
the yeast two-hybrid screen or suggested by the stabilization
data, we performed immunoprecipitation studies using the
antisera against Cag secretion apparatus components. Our data
suggest that distinct subassemblies of secretion apparatus
components and distinct translocation complexes for the effector
protein CagA exist. In conclusion we provide a first structural
model of the Cag type IV secretion apparatus.
Cryo-electron tomography and vitreous sections reveal the
outer membrane of mycobacteria
C. Hoffmann1, A. Leis1, M. Niederweis2, G. Pfeifer1, J. Plitzko1
H. Engelhardt1
Max-Planck-Institute of Biochemistry, Molecular Structural
Biology, Martinsried, Germany
University of Alabama at Birmingham, Department of
Microbiology, Birmingham, AL, USA
Mycobacteria possess a unique cell wall, containing extractable
lipids and long-chain mycolic acids that are covalently linked to
peptidoglycan via an arabinogalactan network. Current models
arrange these lipids in an asymmetrical membrane of a size that
is incompatible with the dimensions and structure of the porin
MspA from Mycobacterium smegmatis. The investigation of
unperturbed M. smegmatis and M. bovis BCG cells embedded in
vitreous ice and rendered visible three-dimensionally by cryoelectron tomography and in projections by vitreous sectioning
now reveals the native organization of the cell envelope. The
mycobacteria are surrounded by a symmetrical outer membrane,
with a bilayer structure apparent in sufficiently thin
cryosections. This outer membrane is only 20% thicker than the
cytoplasmic membrane and agrees with the dimensions but also
physicochemical properties of MspA. The two lipid membranes
define an extensive periplasmic space that occupies about 24%
of the total cell volume in M. bovis. The periplasm harbours
several layers of enhanced density that are attributed to the
peptidoglycan and the arabinogalactan polymers.
To get a better understanding of the location and the role of the
mycolic acids, we analyzed the cell walls of mycolic acid-free
deletion mutants. Since such a knock-out is lethal for
mycobacteria, we used a mutant strain of Corynebacterium
glutamicum, one of the phylogenetic relatives in the distinctive
suprageneric actinomycete taxon, and the respective wild-type.
Electron microscopy of vitreous sections revealed the different
organization of the cell wall in both strains.
Based on these results, a revised model for the mycobacterial
cell wall architecture is proposed.
Use of antisense-technique as an alternative to mutagenesis
for the identification of virulence genes from mycobacteria
A. Lewin1, E. Kamal1, S. Janßen2
Robert-Koch-Institute, Microbiology, Berlin, Germany
Paul-Ehrlich-Institut, Langen, Germany
The genus Mycobacterium comprises the most important
bacterial infectious agent, M. tuberculosis, but also opportunistic
bacteria as M. avium and M. fortuitum. Our knowledge about the
virulence and latency mechanisms of mycobacteria is
insufficient. Therefore, further research aiming at a better
understanding of the interaction between mycobacteria and the
human host has to be performed. The investigation of the
functions of bacterial genes in infection processes usually
involves their mutagenesis by homologous recombination. This
technique proves elusive in mycobacteria due to the high
frequency of illegitimate recombination events and the low
electroporation efficiencies. Our study aimed at testing the
potential of antisense-techniques as an alternative to
mutagenesis for the isolation of mycobacterial virulence genes.
We used a random and a directed cloning strategy to insert gene
fragments from M. fortuitum and M. bovis BCG in antisenseorientation behind the strong promoter from the hsp60 gene in a
shuttle vector. The DNA region behind the hsp60 promoter was
sequenced in all 106 generated recombinant plasmids. We were
able to confirm 33 antisense constructs with M. fortuitum DNA
and 30 containing M. bovis BCG DNA that carried genes
orientated in antisense with respect to the promoter.
Electroporation in either M. fortuitum or M. bovis BCG
succeeded with 33 antisense-plasmids for M. fortuitum and 6
antisense-plasmids for M. bovis BCG. The recombinant
mycobacteria were tested for their colony morphology, the
growth rate in broth culture and their intracellular survival in
mouse macrophages. Impairments in intracellular persistence in
mouse macrophages were found with 6 M. fortuitum
transformants. In this way the following M. fortuitum genes
could be shown to potentially play a role in intracellular
survival: an acetyl-CoA dehydrogenase-like gene, an integrase
gene, a metal-dependent phosphohydrolase gene, an anthranilate
hydratase/isomerise gene and the gene orthologous to Rv1738
from M. tuberculosis. Currently we are confirming the results of
the antisense-experiments by generating mutations in the
corresponding genes.
Swarming of Pseudomonas aeruginosa PAO1 is a complex
adaptation resulting in virulence and antibiotic resistance
J. Overhage1, M. Bains1, R. Hancock1
University of British Columbia, Department of Microbiology
and Immunology, Vancouver BC, Canada
Motility has been strongly implicated in the virulence of P.
aeruginosa. It plays an important role in mobilization to and
colonization of different environments, attachment of bacteria to
surfaces, and biofilm formation. P. aeruginosa is unusual in that
it is capable of three different types of motility: flagellummediated swimming in aqueous environments and at low agar
concentrations (<0.3% [wt/vol]); type IV pilus-mediated
twitching on solid surfaces or interfaces; and most recently
observed, swarming on semisold (viscous) media (0.4 to 0.7%
[wt/vol] agar). Swarming was shown to be dependent on both
flagella and type IV pili as well as the presence of rhamnolipids.
It can be described as a social phenomenon involving the
coordinated and rapid movement of bacteria across a semisold
Recently, we have shown that swarming of P. aeruginosa is a
more complex motility mechanism influenced by a large number
of cooperating genes. To understand more about swarming, we
investigated gene regulation events associated with swarming by
performing microarrays of bacteria at the leading edge of a
swarm zone, compared to bacteria growing in identical medium
under swimming conditions. Analysis of 5 independent
experiments demonstrated that 417 genes were significantly upor downregulated by more than 2-fold. Of these, 309 were
upregulated and 108 downregulated of which about half were
hypothetical or conserved hypothetical genes. We observed
under swarming conditions the up-regulation of numerous
virulence-related genes including exoS, exoT, exoY, etc. and
genes coding for the type III secretion system, pyochelin,
phenazine, and pyoverdine biosynthesis. Furthermore, besides
several genes involved in energy metabolism, nitrogen
assimilation, fatty acid and amino acid biosynthesis and
transport systems, no fewer than 18 were predicted or known
regulators, including e.g. lasR and rhlR, with 2 to 11-fold altered
expression, indicating a complex regulatory network. Overall,
these data indicate that swarming motility of P. aeruginosa is
part of an alternative growth state and a complex adaptation of
this human pathogen to a viscous environment resulting in
changes that reflect altered virulence and antibiotic resistance
and overlap with another complex adaptation, biofilm
formation. Consistent with this notion, swarming cells exhibited
highly elevated resistance to antibiotics including ciprofloxacin,
gentamicin and polymyxin B.
Azithromycin treatment affects siderophore production in
Pseudomonas aeruginosa
Y. Nalca1, F. Bredenbruch1, P. Cornelis2, S. Häußler3,1
Helmholtz-Center for Infection Research, Chronic
Pseudomonas Infections, Brunswich, Germany
Vrije Universiteit Brussel, VIB Department of Molecular and
Cellular Interactions, Brussels, Belgium
Helmholtz-Center for Infection Research, Brunswich
The administration of macrolides such as azithromycin in
chronic pulmonary infection of cystic fibrosis patients has been
reported to be of beneficial value. In a previous study we used a
systematic approach and analyzed the impact of azithromycin
treatment on the global transcriptional pattern and the protein
expression profile of P. aeruginosa PAO1 cultures. The most
remarkable finding was that azithromycin exhibited extensive
quorum-sensing antagonistic activities. In accordance with the
inhibition of the quorum-sensing systems, virulence factor
production was diminished and the oxidative stress response
was impaired, whereas the type III secretion system was
strongly induced.
Since a quorum-sensing antagonistic effect holds great promise
in the management of chronic infections, we became interested
in the molecular mechanisms of action. Here we demonstrate
that azithromycin affects P. aeruginosa siderophore production.
Whereas pyoverdine production of PAO1 and a pyochelin
knockout mutant ( pchE/F) was increased in the early
stationary phase under exposure to azithromycin, the pyochelin
production of PAO1 and pyoverdine knockout mutant ( pvdD)
was diminished. Most interestingly, we observed that the
pchE/F and the PAO1 wild-type strain but not the pvdD and
the pvdD / pchE/F double knockout mutant exhibited an
impaired oxidative stress response upon exposure to
azithromycin as compared to the untreated controls. These
results implicate that an induction of pyoverdine production by
azithromycin accounts for the observed impaired oxidative
stress response. It will be an important task for the future to
identify whether the global proteome and transcriptome profile
in response to azithromycin can be traced back to the induction
of pyoverdine production. We will perform a comparative
analysis of the pvdD mutant and its wild-type upon
azithromycin addition to answer this question.
The Pseudomonas quinolone Siganl (PQS) Balances Life and
Death in Pseudomonas aeruginosa Populations
S. Häußler1, T. Becker2, C. Zaoui1, M. Müsken2
F. Bredenbruch1
Helmholtz-Center for Infection Research, Cell Biology
Brunswich, Germany
Helmholtz-Center for Infection Research, Brunswich
Although bacteria are capable to respond to environmental
challenges with a wide variety of stress responses, under
sufficiently severe conditions it might be favorable to either
undergo a regulated cell death allowing surviving bacteria to
scavenge nutrients from dead siblings or to enter a dormant state
with retained ability to revive when conditions become more
conductive. In this study we provide evidence that the
Pseudomonas quinolone signal (PQS) recently identified as an
interbacterial signal molecule in Pseudomonas aeruginosa
exhibits activities that support both of these assumptions.
The appearance of iridescent plaque-like clearances of aged P.
aeruginosa colonies is a well-known phenomenon and has
previously been implicated to be due to an autolytic process
which involves the production of PQS. In this study we aimed to
identify stressful conditions that trigger a PQS-mediated selfinduced bacterial cell death in P. aeruginosa. We subjected the
bacteria to various antibiotics and monitored bacterial viability
in the P. aeruginosa PAO1 wild-type and its dependency on
PQS signaling. Our results demonstrate that in a PQS nonproducing PAO1 mutant strain the initiation of DNA release and
fragmentation is delayed and the mutant exhibited a drug
tolerant phenotype. In contrast, in wild-type stationary P.
aeruginosa cultures a reversible bacteriostatic effect of PQS
sensitizes the bacteria to bactericidal antibiotic activities.
However, rather than being the effector molecule of autolysis,
the physiological role of PQS seems to be the synchronization of
the entry of P. aeruginosa populations into stationary phase of
growth. We suggest that by balancing life and death in P.
aeruginosa populations PQS shapes the population structure and
significantly contributes to biofilm development. Continued
investigation on the physiological role of PQS will provide
further fascinating insights into bacterial adaptation strategies
that do not only act at the individual level but serve the
multicellular community behavior in P. aeruginosa populations.
Strictly anaerobic bacteria in sputum of patients with cystic
D. Worlitzsch1, C. Rintelen1, K. Boehm1, B. Wollschlaeger2
N. Merkel3, M. Borneff-Lipp1, G. Doering4
Institute of Hygiene, University Hospital Halle-Wittenberg
Halle, Germany
Hospital of Internal Medicine, University Hospital HalleWittenberg, Halle, Germany
Childrens Hospital, University Hospital of Tuebingen, Halle
Institute of Medical Microbiology and Hygiene, University
Hospital of Tuebingen, Tuebingen, Germany
The oxygen partial pressure in sputum plugs in the lung of
patients with cystic fibrosis (CF) is extremely low. Besides
facultatively anaerobic bacteria such as Pseudomonas
aeruginosa, Staphylococcus aureus or Burkholderia cepacia
complex also strict anaerobes can be found in most CF patients.
We analyzed antibiotic susceptibilities and bacterial counts of
strict anaerobes.
From 31 adults and 8 childrenwith CF 92 sputum samples were
obtained.Strictly anaerobic bacteria were identified using an
anaerobic bench and the RapID AnaII®identification system,
and cfu’s were determined by dilution plate counting. Fastidious
anaerobes (141 strains) were submitted to E-test® susceptibility
testing (AB Biodisk, Solna, Sweden) using the anaerobically
active antibiotics ceftazidime, clindamycin, meropenem,
metronidazole, and piperacillin/tazobactam.
In 75.0% of the patients, in addition to facultative anaerobes the
following strict anaerobes were detected with a mean of 6.3x106
cfu/ml (range 2.5x104 to 2.5x107 cfu/ml): Peptostreptococcus
spp., Clostridium spp., Actinomyces spp., Prevotella spp.,
Wolinella spp., Propionibacterium spp., Streptococcus spp.,
Lactobacillus spp., Gemella spp., Bacteroides spp. and
Eubacterium spp.. Identical strict anaerobic species were
detected in 12 out of 19 patients with two or more repeated
sputum samples (63%). E-test® sensitivity testing yielded high
sensitivity for meropenem (5.7% resistant strains),
piperacillin/tazobactam (20.6%), and clindamycin (24.8%), but
not ceftazidime (49.0%) or metronidazole (50.4%).
High numbers of strict anaerobes are present in the majority of
CF patients. The high persistence of identical anaerobic strains
reflects chronic lung infection and may be caused by their
increased resistence against standard antibiotics such as
Predominant CTX-M-15 ESBL type among Escherichia coli
and Klebsiella pneumoniae ESBLs isolates from Giessen
University Hospital.
S. Mshana1,2, C. Imirzalioglu1, H. Hossain1, T. Hain1
E. Domann1, T. Chakraborty1
Institute of Medical Microbiology/ University of Giessen
Giessen, Germany
Bugando University College of Health Sciences, Mwanza
Over an 8-months period, 39 Escherichia coli and 18 Klebsiella
pneumoniae with multiple resistance to cephalosporins were
isolated from clinical samples. All isolates produced ESBLs
when assessed using the disk approximation method. Gene
specific primers were used to amplify and sequence Tem, SHV
and CTX-M ESBL genes. DNA sequencing revealed that 32
(86.5%) Escherichia coli strains and 15 (83.3%) Klebsiella
pneumoniae strains harboured CTX-M genes. Tem genes were
found in 5 (13.5%) and 3 (16.6%) in Escherichia coli and
Klebsiella pneumoniae strains respectively. No isolates
harbouring SHV genes were detected. The CTX-M-15 was the
most common allele detected in both Escherichia coli and
Klebsiella pneumoniae and represented by 21 (65.6%) and 12
(80%) strains, respectively. Isolates with the CTX-M-15 allele
were resistant to cefepime. The majority of strains had a high
level resistance (MIC > 48µg/ml) and were uniformly resistant
to aminoglycosides and quinolones. All these isolates were
sensitive to meropenem and imipenem. Most Klebsiella
pneumoniae strains harbouring the CTX-M-15 allele had a
common PFGE pattern indicating clonal identity. For
Escherichia coli strains the PFGE patterns were heterogenous.
CTX-M-15 allele is common at Giessen University hospital
among ESBL isolates and our data suggest clonal spread of
Klebsiella pneumoniae isolates with CTX-M-15 allele.
Antimicrobial susceptibilities of Ochrobactrum spp.
B. Thoma1
Bundeswehr Institute of Microbiology, Department of
Bacteriology and Toxinology, Munich, Germany
The antibiotic susceptibilities of a representative collection of
one hundred-five Ochrobactrum spp. isolates of both clinical
and environmental origin were examined. Minimal inhibitory
concentrations (MICs) of 20 clinically relevant antimicrobial
agents were determined using Etests™ (AB BIODISK, Solna,
Sweden). Species designation was confirmed using biochemical
differentiation with API® 20NE (bioMérieux, Marcy L’Etoile,
France) and 16S rRNA and recA gene sequencing. All species
except for O. gallinifaecis were highly resistant to all b-lactams.
The tested isolates were all sensitive to ciprofloxacin and to a
lesser extent to trimethoprim/sulfamethoxazole. However, some
of the clinical cases treated with these antibiotics did not
survive. Regarding the susceptibility to colistin we could not
confirm the general susceptibility of all O. anthropi species as
indicated in previous studies. Additionally, we observed
resistance to colistin in O. anthropi isolates. Therefore,
susceptibility to colistin of O. anthropi and resistance of O.
intermedium does not seem to be a reliable phenotypic feature
for differentiating between these two species as proposed
before. We have further assessed the biochemical reaction
profile to determine, whether a differentiation among the species
of the genus Ochrobactrum is feasible using the API 20NE.
Based on our data we suggest that the biochemical identification
of a bacterium as “O. anthropii” should be only interpreted as
“Ochrobactrum spp.” with O. anthropii being the most
important representative. Therefore, reported cases might have
been misclassified. In general, antibiotic susceptibility patterns
as means of phenotypic differentiation in Ochrobactrum species
is questionable due to the great variation of susceptibility within
the species. In conclusion, our in vitro data suggest that
ciprofloxacin and trimethoprim/sulfamethoxazole could be used
for empiric antibiotic therapy of Ochrobactrum spp.
Occurrence and clinical relevance of Mycobacterium
chimaera sp. nov.
B. Schweickert1, O. Goldenberg2, E. Richter3, P. Buchholz4
A. Moter4, U. B. Göbel4
University hospital Charité, Microbiology, Berlin, Germany
Transgenomic LTD, Berlin, Germany
Referenzzentrum für Mykobakterien, Borstel, Germany
Charité Berlin, Institute for Microbiology and Hygiene, Berlin
Recently, a cluster of strains belonging to the heterogeneous
Mycobacterium Avium Complex (MAC) has been described and
named as M. chimaera sp. nov.. As comprehensive
epidemiological data are missing, we performed a retrospective
study to investigate the frequency of its occurrence within the
group of MAC-positive clinical specimens and to determine its
role as a cause of human disease as compared with the closely
related M. intracellulare. Mycobacterial isolates from 166
patients previously identified as M. intracellulare by 16S rDNA based methods have been reassessed by sequencing of the
16-23S internal transcribed spacer region. In addition, we
evaluated Denaturating High Pressure Liquid Chromatography
(DHPLC) and its capacity to distinguish between M. chimaera
sp. nov and M. intracellulare type strain. The mycobacterial
isolates of 97 in house patients of the Charité University
Hospital have been assessed for their clinical significance
according to the criteria of mycobacterial lung disease of the
American Thoracic Society. Genetic analysis revealed that M.
chimaera sp. nov. accounts for the predominant portion 86,1%
of the isolates, whereas M. intracellulare type strain and other
members of the MAC represent 10.2% and 3.6%, respectively.
Only six isolates, each three M. chimaera sp. nov. and three M.
intracellulare type strains, could be classified as clinically
significant. The data indicate that M. chimaera sp. nov. exhibits
a relatively low pathogenic potential in contrast to M.
intracellulare type strains expressing a noticeably higher
virulence. However, further studies with larger sample numbers
are necessary. DHPLC turned out as a reliable low cost method
for the fast processing of a high number of samples.
Increasing prevalence of MRSA isolates with MLST type
ST022 in the Bochum area
F. Szabados1, M. Kaase1, S. Friedrich1, T. Sakinc1, B. Kleine1
K. Henne1, S. Gatermann1
Ruhr-University Bochum, Department of Medical
Microbiology, Bochum, Germany
Understanding the epidemiology of methicillin-resistant S.
aureus (MRSA) is important because therapeutic options are
limited in patients with MRSA infections and measures against
further spread of MRSA are associated with increased costs and
From 1998 to 2006 consecutive non-copy MRSA strains (N =
1516) were collected from patients in four hospitals in the
Bochum area. All strains were typed by pulsed field gel
electrophoresis (PFGE) and grouped together according to the
criteria proposed by Tenover et al.. For each PFGE group
consisting of more than 12 isolates representative isolates were
further characterized by multilocus sequence typing (MLST),
SCCmec-typing and agr-typing.
Average MRSA rates in all four hospitals increased from 18% in
1998 to 36% in 2006. Overall, isolates belonged to PFGE group
35 in 26.7%, to PFGE group 13 in 17.6%, to PFGE group 7 in
11.7%, to PFGE group 11 in 7.7%, to PFGE group 3 in 6.3%
and to PFGE group 16 in 5.4% of all MRSA isolates. Significant
changes over time were observed for most PFGE groups: PFGE
group 35 appeared in 2001 and increased in frequency from
9.7% to 56.5%, PFGE group 13 increased from 1.5% in 1999 to
12.4% with a peak of 27.3% in 2002, PFGE group 7 increased
from 2.2% in 1998 to 20.6% in 2002 and decreased again to
3.1%, PFGE group 11 increased from 0.9% in 2000 to 14% in
2006, PFGE group 3 had its maximum of 23.3% in 2001 and
decreased to 2.6%. PFGE group 35 belongs to ST022, is of agr
type Ia and harbours SCCmec type 4b or 1. PFGE group 13
belongs to ST08, PFGE groups 7 and 3 belong to ST228, PFGE
group 11 belongs to ST225 and PFGE group 16 belongs to
Predominant MRSA clones changed significantly over time.
Since 2001 a MRSA clone characterized by PFGE group 35 and
belonging to MLST type ST022 steadily increased in frequency
in our area with more than half of all MRSA isolates belonging
to this PFGE group.
Genomic mutS-rpoS region polymorphisms in
extraintestinal Escherichia coli (ExPEC) of human and
animal origin correlate with in vivo pathogenicity
C. Ewers1, E. - M. Antáo1, G. Li1, A. Bethe1, S. Kiessling1
I. Diehl1, S. Glodde1, T. Homeier1, L. H. Wieler1
Free University Berlin, Institute for Microbiology and
Epizootics, Berlin, Germany
Currently, three major extraintestinal pathogenic E. coli
(ExPEC) pathovars [uropathogenic (UPEC), newborn
meningitis (NMEC), and avian pathogenic (APEC) E. coli] are
distinguished based on clinical symptoms and virulence
features. Due to Multi locus sequence typing (MLST), ExPEC
are clustered into three major phylogenetic complexes, including
sequence type (ST) complex 95, 23 and 73, as well as ST 62 and
ST 117.
Despite extensive virulence typing of ExPEC strains, no clear
correlation was seen between pathovars and virulence types.
However, we were able to observe associations between unique
mosaic structures of the mutS-rpoS regions among strains from
single STs and pathogenic significance by PCR analyses. The
chromosomal mutS-rpoS region in E. coli is often subjected to
genetic exchange during the evolution of pathogenic lineages,
and high levels of variation in this genomic region have been
suggested as a hallmark for the emergence of E. coli pathogens.
Characterization of this genomic region in NMEC (n = 24),
UPEC (n = 31), APEC (n = 100), and non pathogenic avianderived E. coli strains (n = 78) identified four main structures
based on PCR amplicon sizes of the downstream fhlA-mutS
(1.200 or 1.673bp) and upstream o454-nlpD (1.319, 3.685,
4.546bp or no product) region. Irrespective of the pathovar,
pathogenic strains predominantly harboured identical mutS-rpoS
patterns, whereas non pathogenic strains mostly revealed
structures differing from APEC, UPEC and NMEC strains.
Among 33 virulence associated genes tested, strains with a so
called “pathogenic mutS-rpoS pattern” harboured 19.8, whereas
strains belonging to other patterns possessed less than 10 genes
on average. There was also a clear association of different
genomic structures with certain phylotypes, with the
“pathogenic pattern” being mainly grouped into ST complexes
95 and 73.
Animal infection trials with APEC strains harbouring different
mutS-rpoS regions showed a significant relation between the
“pathogenic mutS-rpoS pattern” and disease outcome in
chickens. Fecal E. coli strains isolated from clinically healthy
chickens bearing this pattern could be re-classified as being
pathogenic, as determined by chicken infection tests providing
strong evidence for a direct link between the mosaic structure of
the mutS-rpoS genomic region and pathogenicity. Further
characterization of the mutS-rpoS region as well as
polymorphisms of their respective genes promise to be the basis
of a future typing tool.
Molecular relationship in Panton-Valentine Leukocidin
(PVL)-positive MRSA and PVL-positive MSSA
F. Szabados1, C. von Eiff2, A. Anders1, M. Kaase1, K. Becker2
S. Gatermann1
Ruhr-University Bochum, Institute for Hygiene and
Microbiology, Bochum, Germany
University Muenster, Institute of Medical Microbiology
Muenster, Germany
The Panton-Valentine Leukocidin (PVL) of Staphylococcus
aureus plays an important role in the pathogenesis of necrotic
pneumonia and recurrent skin and soft tissue infections often
with a fatal outcome in young and otherwise healthy patients.
considerable emergence of community-acquired
methicillin-resistant S. aureus (CA-MRSA) strains commonly
harbouring this toxin has fuelled the discussion on the origin of
this toxin. PVL might have spread from PVL-positive MSSA
into MRSA, on the other hand, PVL-positive MSSA might have
become methicillin-resistant by acquiring SCCmec.
The aim of this study was to investigate the clonal relationship
between PVL-harbouring MSSA and MRSA strains in the
Western part of Germany. We used pulsed-field gel
electrophoresis (PFGE) and multi locus sequence typing
(MLST) to establish possible relationships between CA-MRSA
and MSSA. For MLST, the arcC, aroE, glpF, gmk, pta, tpi and
yqiL genes were amplified by PCR, sequenced, and compared
with reference strains. S. aureus LukF/LukS genes and further
toxin genes, such as enterotoxin genes and tst, were detected
with PCR.
Among PVL-positive MRSA, ST80 was the predominating type
followed by isolates of types ST022, ST36, and ST152.
Irrespective of the possession of PVL, MRSA were mostly
represented by the following types: ST228, ST022, ST008,
ST225 and ST045. ST228 and ST008 strains were not found to
carry PVL. Among PVL-positive MSSA strains, we found 18
strains of ST030 (8 PFGE groups), 11 strains of ST120 (7 PFGE
groups), 2 strains of ST217, and 3 strains (ST profile
1,13,1,1,5,3) with relationship to ST14 (or ST259 or ST795 ) as
well as 5 singletons.
Two PVL-positive MRSA and MSSA strains were clonally
related according to PFGE, but were different applying MLST
(MSSA ST030 and MRSA ST036). Other PVL-positive MSSA
strains were different according to MLST and PFGE, except for
two singletons. PVL-positive MRSA strains were often related
to PVL-negative MRSA strains according to PFGE, but not
corresponding to MLST types.
These data indicate that acquisition of methicillin resistance by
PVL-positive MSSA strains seems to be less likely and that
PVL toxin genes might have spread from PVL-positive MSSA
strains into MRSA.
A condensed LightCycler based spa typing method for S.
B. Schwenz1, U. Vogel2, W. Bautsch1, H. Hannig1
Staedtisches Klinikum Brunswich GmbH, Institute for
Microbiology, Immunology und Hygiene, Brunswich
University of Wuerzburg, Institute for Hygiene and
Microbiology, Wuerzburg, Germany
Molecular typing methods for methicillin-resistant S. aureus
(MRSA) become more important in view of the unchecked
increase of the MRSA problem in German hospitals. However,
most typing methods require special equipment, such as pulsedfield gel electrophoresis equipment (PFGE) or sequencing
facilities (MLST, spa typing) which are not readily available in
most microbiological laboratories. In contrast, real-time PCR
methods gain more and more access to the routine laboratory.
We therefore developed a novel typing method for S. aureus
based on LightCycler technology. The scheme exploits the wellknown polymorphisms in the X-region of the protein A gene,
which is also the basis of the spa typing method. This region
consists of a polymorphic 24 bp sequence repeated head-to-tail a
variable number of times. Instead of sequencing the whole X
region (as in spa typing), we only identify the first and last
repeat sequences by melting point analysis of a suitable
reference detection probe after amplification of the X-region by
LightCycler. In addition, the total length of the PCR product is
determined to obtain the total number of repeat sequences.
Analysis of several reference strains revealed the method to be
reproducible and of sufficiently high discriminatory power to
allow for differentiation of the most common MRSA clones.
Currently, a collection of 40 different S. aureus isolates obtained
from the routine work-up in our laboratory is analyzed by this
method to check its suitability for routine use. Results will be
presented at the meeting.
Quinolone susceptible MRSA in the Hamburg area:
Phenotypic and genotypic characterization
J. K. Knobloch1, J. C. von Freyberg2, S. Scherpe2
University of Luebeck, Institute for Medical Microbiology and
Hygiene, Luebeck, Germany
University Medical-Center Hamburg-Eppendorf, Institute for
Medical Microbiology, Hamburg, Germany
Background: Most MRSA in Germany display resistance
against quinolones. However, we observed a lower prevalence
of quinolone resistance in MRSA isolates associated with skin
and soft tissue infections. We investigated the clonal relation of
quinolone susceptible MRSA and the presence of the lukS/F
genes encoding the Panton-Valentine leukocidin.
Methods: 50 Ciprofloxacin (CIP) susceptible MRSA isolates
were collected from February 2001 to April 2005. Clonal
relationship between these isolates was determined by spatyping. Representative isolates were further characterized by
multi locus sequence typing (MLST). MIC values of Oxacillin
(OXA), CIP, Moxifloxacin (MOX), Daptomycin (DAP), and
Linezolid (LIN) were determined for all isolates.
Results: 45 isolates were divided into 7 clonal complexes using
the BURP algorithm for the determined spa-types, whereas 5
isolates were identified as singletons. The lukS/F genes were
identified in 19 isolates associated with three different spaclonal complexes. Representative isolates of the three spa-clonal
complexes revealed to belong to the MLST sequence types
ST80 (13 isolates), ST30 (3 isolates) and ST8 (3 isolates). 37
isolates, including all lukS/F-positive MRSA, displayed a MIC
for OXA of 64 mg/L or lower, whereas only 5 isolates displayed
MIC values for OXA greater than 256 mg/L. In contrast to the
primary disc diffusion testing two isolates displayed a MIC for
CIP of 2 mg/L, whereas all isolates were MOX susceptible, with
a four to eightfold higher activity of MOX compared to CIP. In
the study period no quinolone resistant lukS/F positive MRSA
isolates were detected in the Hamburg area. All investigated
MRSA displayed MIC values in the susceptible range for DAP
and LIN.
Conclusions: In the Hamburg area three different clones of
lukS/F positive MRSA were identified representing CA-MRSA
clones of different regions of the world. Thereby, the MLST
ST80 isolates had the highest prevalence. The lack of MOX
resistant lukS/F positive MRSA during the study period
indicates that modern chinolones could be used for the therapy
of severe skin and soft tissue infections even if CA-MRSA rates
will increase in the Hamburg area.
the hospital level in both cases reveals substantial differences in
resistance rates as well as in trends.
In gramnegative bacteria, proportions of resistance for several
species-antibiotic-combinations varied significantly over time
but only for cefotaxim in E. coli from blood cultures and
meropenem in P. aeruginosa the trends represented linear
increases. For E. coli there is a remarkable difference in
ciprofloxacin resistance with regard to origin of isolates: in
blood cultures the level of resistance was approx. 30% whereas
in non-blood isolates an increase from 10.1% to 19.1% was
Data from the GENARS project give important clues on
changes in antimicrobial resistance of bacteria isolated from
patients of participating university hospitals within the past five
years. To obtain representative evidence, to distinguish local
from overall trends and to carry out more sophisticated analysis
an expansion of the surveillance is urgently needed.
Figure 1:
Five Years of german network for antimicrobial resistance
surveillance: Trends in resistance 2002-2006.
I. Noll1, J. Beer2, W. Pfister3, S. Schubert4, N. Wellinghausen5
H. Wisplinghoff6, S. Ziesing7, T. Eckmanns1
Robert-Koch-Institute, Infectious Disease Epidemiology, Berlin
University of Leipzig , Institute for Medical Microbiology and
Epidemiology of Infectious Diseases, Leipzig, Germany
University of Jena, Institute of Medical Microbiology, Jena
University Medical Center Schleswig-Holstein Campus Kiel
Institute for Infection Medicine, Kiel, Germany
University Hospital of Ulm, Medical Microbiology and
Hygiene, Ulm, Germany
University Hospital of Cologne, Medical Microbiology
Immunology and Hygiene, Cologne, Germany
Medical School Hanover, Medical Microbiology and Hospital
Epidemiology, Hanover, Germany
Six laboratories of university hospitals representing the German
Network for Antimicrobial Resistance Surveillance (GENARS)
have been collecting data continuously for all clinical relevant
pathogens in a widely standardized and quality controlled way
for the past five years. The objective is to analyze trends in
antimicrobial resistance for the most frequent species to selected
antimicrobials within this period.
Antimicrobial susceptibility is determined as minimal inhibitory
concentrations by broth microdilution method performed by
automated test systems for antibiotics of various classes.
Proportions of resistance are calculated employing breakpoints
of DIN 58940-4 (2005). Analysis has been made yearly for nonduplicate isolates of E. coli from blood cultures (N=2115), E.
coli from non-blood cultures (N=29234), K. pneumonia
(N=7388), P. aeruginosa (N=12003), E. faecium (N=5047) and
S. aureus from blood cultures (N=2018).
The most important findings are related to grampositive
bacteria: The overall resistance rate of S. aureus from blood
cultures to oxacillin increased significantly from 12.8% in 2002
to 20.1% in 2006; an even higher increase from 1.2% to 16.6%
was found for vancomycin in E. faecium. However, analysis on
Genetic characterization of Methicillin-Resistant
Staphylococcus aureus (MRSA) isolated from clinical
specimens of various animal species investigated by
molecular typing procedures
B. Walther1, C. Ruscher1, A. Lübke-Becker1, L. H. Wieler1
University Berlin, Veterinary Microbiology, Berlin, Germany
S.aureus is one of the major causes of infectious diseases in
veterinary medicine, especially in bone and soft-tissue
infections. Similar to the threatening development of
Methicillin-resistant Staphylococcus aureus (MRSA) in human
medicine, these pathogens reached increasing importance in the
field of veterinary medicine. MRSA by definition are resistant to
the antibacterial activity of the entire class of ß-lactam
antibiotics. Frequently, other resistance genes are associated
with MRSA, which decreases the options for a successful
chemotherapeutic intervention in many cases.
In this study, a total of 43 MRSA isolates from infected sites of
various animal species (horses, small animals) from different
geographical regions in Germany were investigated. MRSA was
confirmed by a multiplex PCR, including species verification
and the presence of the resistance gene mecA. All MRSA
isolates were typed by pulsed field gel electrophoresis (PFGE)
after genomic macrorestriction. Further genetic characterization
of representative PFGE types (PFT) was performed by
multilocus sequence typing technique (MLST) and PCR
procedures for analysing the mecA-harbouring genomic cassette
SCCmec. As a result, 13 different PFT occurred among the 43
MRSA isolates. Furthermore, five sequence types (ST), which
were already known from human isolates, were assigned to the
13 representative PFT: ST225, ST22; ST8, ST254 and ST239.
Analysis of SCCmec typing procedures showed genetic
elements in different combinations and composition.
Characteristic elements (ccrAB genes, mecI, mecRA-mecRB) of
SCCmecIII and SCCmecIV were detected, but also additional
ccr-Genes in some strains. These findings supported the
suggestion of recombinant SCCmec types in MRSA isolates of
animal origin, like it has been reported for human
staphylococcal strains before. For factual epidemiological
considerations, further investigation of the mobile genetic
elements SCCmec derived from MRSA strains of animal origin
in detail is needed.
Bands vs. bases: A critical view on clonal analyses in
Escherichia coli
H. Wilking1,2, C. Ewers1, M. Achtman1, L. H. Wieler1
Free University Berlin, Institute for Microbiology and
Epizootics, Berlin, Germany
Friedrich-Loeffler-Institute, Institute for Epidemiology
Wusterhausen, Germany
Avian pathogenic E. coli (APEC) are the causative agent of coli
septicaemia, associated with high morbidity and mortality in
poultry. In addition, evidence is increasing that these extraintestinal pathogenic E. coli (ExPEC) are zoonotic, as they share
various virulence features with ExPEC strains causing disease in
human, namely E. coli causing urinary tract infections in
humans (UPEC) as well as strains causing meningitis (NMEC)
in children (Ewers et al. 2007 Int. J. Med. Microbiol., 297, 163176). In this study, we initially typed 67 APEC-isolates sampled
from different outbreaks mainly in Germany by the use of multi
locus sequence typing (MLST) to reveal their phylogenetic
relationship with sequence types (STs) of human ExPEC. We
revealed a total of 23 different STs. Interestingly, these APEC
strains were particularly prominent in two sequence type
complexes (ST95 and ST23), which due to the MLST database
( also contain human
In addition, we analyzed the 67 strains by pulsed field gel
electrophoresis (PFGE) as a highly discriminatory fingerprinting
method to compare MLST with PFGE results. Comparing the
results of both analytical methods, utilizing TreeMap as a tool to
compare two dendrograms of different origin, the clustering
trees of both methods revealed significant inconsistencies. This
study ascertains the need for appropriate tools in molecular
epidemiology of E. coli. These analyses highlight the fact that
capabilities and limits of different methods have to be chosen
with care when comparing unrelated strains of the species E.
coli, otherwise clonal analysis is prone to lead to problematic
recommendations regarding risk assessment.
Typing of German F. tularensis isolates using Multi Locus
VNTR Analysis (MLVA) and different mass spectrometric
E. Seibold1, K. Mätz-Rensing2, W. D. Splettstoesser1,3
Bundeswehr Institute of Microbiology, Immunology, Munich
German Primate Center, Pathology, Goettingen, Germany
Institute of Medical Microbiology, Virology & Hygiene
Medical Microbiology, Rostock, Germany
Background: Differentiation of the highly virulent F. tularensis
subspecies tularensis from the less virulent subspecies
holarctica is of substantial clinical interest in areas were both
subspecies occur naturally (North America) but maybe even
more important regarding the potential use of F. tularensis as a
biological warfare agent. For epidemiological studies
investigating modes of transmission within enzootic disease
circles and routes of the geographical spread of F. tularensis,
molecular tools which are able to discriminate between different
strains are mandatory. We characterized and typed 16 F.
tularensis strains recently isolated in Germany, where tularemia
re-emerged in several federal states.
Methods: We analyzed 16 different F. tularensis strains isolated
between 2004 and 2007 in threes different states from nonhuman primates, hare and water voles by VNTR employing 25
different markers. Results were compared to our BionumericsTM
data base comprising more than 200 strains from a worldwide
collection of F. tularensis strains. Additionally, protein
preparations of representative strains of this collection were
analyzed by SELDI-TOF and MALDI-TOF mass spectrometry.
Results: Whereas phenotypic spectrometric methods were able
to discriminate strains on the subspecies level, differentiation of
single strain was not possible. MLVA revealed at last five
different genotypes within the group of isolates from Germany.
Three genotypes grouped within the Eurasia I cluster, while the
remaining strains could not attributed to recently published
genotype clusters but showed similarities to single isolates from
Conclusion: Mass spectrometry may be a valuable tool to
identify and discriminate F. tularensis to the subspecies level,
which might be sufficient in the clinical setting. For
epidemiological or forensic purposes, even the currently applied
molecularbiological methods based on MLVA are not sufficient
to resolve F. tularensis holarctica to the strain level. However,
it was possible to show that distinct genotypes were responsible
for two different enzootics in geographically adjacent regions
within one county.
Toxin equipment of Methicillin-rResistant Staphylococcus
aureus (MRSA) strains circulating in Germany
C. von Eiff1, F. Hasenberg1, A. W. Friedrich2, G. Peters1
S. Gatermann3, K. Becker4
University Hospital Muenster, Institute of Medical
Microbiology, Muenster, Germany
University Hospital Muenster, Institute for Hygiene, Muenster
University of Bochum, Department of Medical Microbiology
Bochum, Germany
University Hospital Muenster, Institute for Medical
Microbiology, Muenster, Germany
Objectives: Methicillin-resistant Staphylococcus aureus
(MRSA) strains are one of the leading causes of nosocomial
infections. In addition, community-onset infections due to this
pathogen continue to be a major public health issue. While
MRSA strains are increasingly reported in many countries
worldwide, representative nation-wide data in Germany on
prevalent clones and their characteristics are not available.
Methods: Altogether, 36 Centers throughout Germany
(laboratories associated with university and general hospitals,
outpatient clinics) were enrolled to collect 50 consecutive
MRSA isolates and to respond to a questionnaire. Only one
isolate per patient was included. All isolates were spagenotyped and characterized concerning their equipment with
virulence factors.
Results: In this study, MRSA strains as well as demographic and
clinical data from 1753 patients were collected. While only 19
isolates from 13 Centers were found positive for the PantonValentine leukocidin (PVL)-encoding genes, mainly from
patients with skin and soft tissue infections (n = 10), nearly all
strains (98.5%) were positive for at least one of the pyrogenic
toxin superantigen (PTSAg) genes. In contrast to frequently
detected enterotoxin genes, only a few strains (n=58; 3%)
harbored the TSST-1 encoding gene. Of particular interest,
associations between defined PTSAgs and single spa types were
revealed, e.g. strains belonging to spa type t008 (n=131) were
mainly positive for sea (n=94; 72%), strains belonging to spa
type t003 (n=664) were mainly positive for sed (n=600; 90%).
Also of note, all t002 strains (n=116) were seg/sei-positive
whereas only one fifth (21%) of the t008 strains harbored this
gene combination.
Conclusion: These data collected during the course of a German
multicenter study offer a reliable overview on currently
circulating MRSA strains and their toxin possession. While
strains harboring genes encoding PVL or TSST-1 are rare,
nearly all MRSA strains are positive for at least one of the
classical and/or newly described enterotoxin genes.
New methods to estimate incidence of HIV infections: a pilot
study in Berlin 2005 to 2007 – Results and implications for
HIV surveillance
J. Bätzing-Feigenbaum1, S. Loschen2, S. Gohlke-Micknis1
C. Kücherer2, O. Hamouda1
Robert-Koch-Institute, Department for Infectious Disease
Epidemiology, Berlin, Germany
Robert-Koch-Institute, HIV Variability and Molecular
Epidemiology, Berlin, Germany
Background: After a decrease of newly diagnosed HIV
infections in Germany the number of reported cases increased
by >70% from 1.444 in 2001 to 2.486 in 2005. However, these
cases do not reflect the true HIV-incidence defined as newly
acquired infections. Only part of incident infections is detected
by routine clinical carebecause symptoms frequently lack during
acute infection and the asymptomatic period can vary
considerably. Furthermore attitudes and access to HIV
counselling and testing affect detection of infections. Since there
are new serological methods (e.g. BED-CEIA) available we
aimed to measure recency of HIV infections in newly diagnosed
patients. Patients found infected recently were considered as
proxies for true incident HIV cases. The data allow real-time
analysis ofincident HIV cases in terms of clinical features,
attitudes towards HIV/AIDS and risk behaviour.
Methods: Venous blood and clinical data were sampled from
adults ( 18 years) with newly diagnosed HIV-infections by
practitioners and clinics in Berlin. To determine recency of
infection samples were tested at the RKI using BED-CEIA
(recent: 20 weeks). Data on knowledge, attitudes and
behaviour towards HIV/AIDS were collected through patients’
questionnaires (KAB survey).
Results: 132 individuals have been enrolled and 118 were MSM
(90%) indicating over-sampling of this transmission group. The
proportion of recent infections in MSM is 47% (CI [95%]: 38;
56) and 20% (CI [95%]: 0; 41) in patients stating other risks of
HIV infection. 30% of recently infected patients had a VL
>500.000 copies/ml, 57% had CD4 cells >500/ l compared with
7% and 31%, respectively, in prevalent cases. Symptoms of
primary acute infection were reported by 60% of recently
infected and 26% of prevalent patients. 66% of recently HIV
infected MSM had anonymous sex in the past 6 months. 59%
specified not having used condoms because they thought not to
be at risk or believed their sexual partner not to be HIV infected,
28% stated problems with condom use.
Conclusions: Results suggest a possible delay to test for HIV in
women and groups at risk for HIV other than MSM.
Asymptomatic seroconversion might contribute to delay in
counselling and testing. There are indicators for high risk
behaviour with regards to condom use in recently HIV infected
MSM. These findings can help adapting prevention strategies to
present-day circumstances. In general they underline the
importance of extending second generation HIV surveillance in
Germany. (Funded by BMG).
Penicillin resistance of Neisseria lactamica and its impact on
H. Claus1, A. Karch1, U. Vogel1
Institute for Hygiene and Microbiology, Wuerzburg, Germany
Neisseria spp. are commensals of the human upper nasopharynx.
It has been shown that the species share a common gene pool
horizontally transferred by transformation. N. lactamica strains
isolated in Spain exhibited an intermediate susceptibility to
penicillin (Arreaza et al., 2002). Horizontal exchange of the
penA gene encoding the penicillin binding protein 2 between
commensal and pathogenic Neisseria has been proposed;
however, most meningococcal isolates are susceptible to
penicillin. It is unclear whether the differing susceptibilities
towards penicillin are due to infrequent co-colonization of the
host with N. meningitidis and N. lactamica, or the result of
major differences at the level of genes involved in penicillin
In this study, we estimated the minimal inhibitory concentration
(MIC) for penicillin of more than 100 N. lactamica strains
isolated from Bavarian children and teenagers and furthermore
sequenced the transpeptidase region of penA. For German
isolates, the Spanish observation of elevated MICs in
comparison to meningococci was confirmed. PenA sequences
obtained in this study were compared to 154 penA alleles
identified in meningococcal strains deposited in an international
database ( We did not identify mutations in
the transpeptidase region of the N. lactamica penA explaining
elevated MICs, suggesting a role of other genes in penicillin
resistance. Comparative sequence analyses and in vitro selection
of penicillin resistant meningococci after transformation with N.
lactamica DNA will unravel their identity.
Infection with serotype 23F is an independent risk factor for
pneumococcal meningitis in adults
I. Seegmüller1, F. Burckhardt2, M. van der Linden3
R. R. Reinert3
University Heidelberg, Hygiene, Heidelberg, Germany
Robert-Koch-Institute, Berlin, Germany
University Aachen, NRZ für Streptokokken, Aachen, Germany
Pneumococcal meningitis is a subgroup of invasive
pneumococcal disease (IPD) with a case-fatality rate of up to 50
percent and long-term sequelae in up to 60 percent of cases in
adults. We wanted to determine risk factors for this form of
pneumococcal disease.
We conducted a prospective population-based study of invasive
pneumococcal disease in North-Rhine-Westphalia, Germany
from February 2001 until August 2006. All isolates underwent
serotyping and resistance testing in the National Reference
Center for Streptococci in Aachen, Germany. Data were
analysed using multiple logistic-regression.
1043 isolates from bacteraemia and 131 isolates from meningitis
could be included into the study. Age, serotype and gender were
independent risk factors. Old age (more than 60 years) was
associated with a reduced odds ratio for pneumococcal
meningitis whereas being female was associated with and
increased odds ratio. Although serotype 14 was the most
common serotype it was not associated with an increased odds
ratio for central nervous system (CNS) involvement. Serotype
23F, however, increased the odds ratios for CNS involvement
twofold whereas serotype 1 decreased the odds ratio by four.
Season, penicillin and macrolide resistance were not statistically
associated with CNS involvement.
Infection with serotype 23F is an independent risk factor for
pneumococcal meningitis. IPD with serotype 1 and old age
protect against pneumococcal meningitis, whereas being female
increases the risk for central nervous system involvement in
invasive pneumococcal disease. Neither season of infection nor
antibiotic resistance have influence on CNS invasion.
Seroprevalence and reservoirs of leptospirosis in Conakry
S. Zimmermann1, A. ter Meulen2, E. Fichet-Calvet3
L. Koivogui4, O. Sylla4, M. Goris5, R. Haartskerl5
J. ter Meulen6
Institute of Hygiene, Heidelberg, Germany
GTZ, Frankfurt am Main, Germany
Museum National d’Histoire Naturelle, Paris, France
University of Conakry, Department of Micorbiology, Conakry
Rep. Guinea
Koninklijk Instituut voor de Tropen, Amsterdam, Netherlands
Leiden University Medical Center, Dept. of Microbiology
Leiden, Netherlands
From September to December 2001, an urban outbreak of
febrile jaundice revealed 107 cases in Conakry. Among them,
16 were diagnosed with acute leptospirosis. Because this disease
is probably underdiagnosed in this country, a pilot study was
undertaken in 2004 in Conakry, with an investigation of both
humans and small mammals. Sera from 1200 human subjects
were screened for leptospirosis antibodies to estimate the
incidenceand identify risk factors for transmission. In parallel,
rodents were trapped in households to identify the reservoir
animals of the disease.
A cross-sectional serologic survey was carried out in 5 resource
poor urban neighbourhoods in Conakry, Guinea. A detailed
questionnaire was completed to document demographic and
environmental risk factors. Leptospirosis specific IgM and IgG
levels were detected by ELISA and confirmed with MAT
testing. The trapped rodents were taxonomically identified and
one kidney was collected for detection of leptospires by culture
and PCR testing. A nested PCR with a high sensitivity was
performed. PCR positive samples were confirmed using
different typing PCRs.
7 percent of study subjects were positive for leptospira
antibodies. Preliminary epidemiological analysis revealed as
risk factors for leptospira IgM antibodies: (i) living in a
neighbourhood from which leptospirosis cases were reported in
2001 (ii) use of tap water for washing and bathing (iii) living
close to a waste pipe (iv) history of hospitalisation during the
past rainy season. 330 rodents were trapped within the 5
neighbourhoods. Rattus rattus and Mus musculus were the most
frequent species, but in addition some Crocidura and Mastomys
spp. were identified. In five of the kidney samples leptospiral
DNA was detected by nested PCR.
This survey shows that a significant percentage of the
population of Conakry is exposed to leptospirosis. Transmission
probably occurs through leptospira infested water on the
domestic compounds, with rodents as one possible reservoir. As
the outbreak in 2001 shows, there is an urgent need for further
identification of the main reservoir(s) of the disease and
environmental control measures.
Emergence of MRSA spa type t011 in the Dutch-German
border region EUREGIO Twente/Muensterland
R. Köck1, A. Mellmann1, M. G. R. Hendrix2, I. Daniels-Haardt3
H. Karch1, R. Lärberg1, C. von Eiff4, G. Peters4, K. Becker4
A. W. Friedrich1
University Hospital Muenster, Institute for Hygiene, Muenster
Laboratory Microbiology Twente-Achterhoek, Enschede
Landesinstitut für den oeffentlichen Gesundheitsdienst
(LOEGD) NRW, Muenster, Germany
University Hospital Muenster, Institute for Medical
Microbiology, Muenster, Germany
The project EUREGIO MRSA-net comprises the Dutch-German
border area Twente/Muensterland in which the prevalence of
methicillin-resistant Staphylococcus aureus (MRSA) is
unequally distributed. On the German side of the border, MRSA
strains are widespread, whereas the prevalence in the Dutch
neighbouring region remains 20-fold lower since many years.
Nevertheless, more recently, in Netherlands an increase of
MRSA isolated from patients in the community not belonging to
typical risk groups for MRSA carriage has been observed.
Since regional surveillance of the molecular MRSA
epidemiology provides the basis for controlling MRSA
dissemination and the prevention of cross-border transmission,
MRSA recovered from screening samples and clinical
specimens of hospital inpatients in the EUREGIO
Twente/Muensterland ( were collected
from September 1st, 2005 to February 28th, 2007 using a
standardized protocol. To assess the molecular epidemiology, all
MRSA isolates were spa sequence-typed.
During the 18-month period, we observed an increase of MRSA
isolates exhibiting spa-type t011 on both sides of the border.
During three half-year terms, the t011 clone represented 3.0%,
9.4%, and 9.3% of all isolates in the German and 0%, 4.8%, and
35% in the Dutch part of the region, respectively. Of 45 MRSA
isolates representing t011, 36 were recovered from
nasopharyngeal screening samples while only 9 derived from
various clinical specimens (wound swabs, n=4; tracheal fluids,
n=2; others n=3).
So far, MRSA t011 has not been reported as a frequent clone in
German hospitals. In contrast, several recent reports described
an emergence of MRSA t011 colonisations and infections in
Dutch pigs. As transmissions between pigs and humans have
already been shown in Netherlands, the impact of animal
sources as potential reservoirs of MRSA t011 in the community
and the prevalence of t011 in human carriers within the DutchGerman EUREGIO Twente/Muensterland warrants further
Phylogenetic analysis of extraintestinal E. coli (ExPEC)
reveals a close relationship between isolates of human and
animal origin, urging a zoonotic potential of distinct
C. Ewers1, H. Wilking1,2, M. Achtman3, L. H. Wieler1
Free University Berlin, Institute for Microbiology and
Epizootics, Berlin, Germany
Friedrich-Loeffler-Institut, Institute for Epidemiology
Wusterhausen, Germany
Max-Planck-Institut for Infection Biology, Department of
Molecular Biology, Berlin, Germany
Although extraintestinal E. coli (ExPEC) are a common cause of
morbidity and mortality in man as well as in animals, reliable
identification, pathogenicity, as well as risk assessment of their
zoonotic potential is far from being defined. So far, single
ExPEC pathovars (uropathogenic E. coli; UPEC, newborn
meningitis E. coli; NMEC, avian pathogenic E. coli; APEC)
have been defined based on the host species and infection site as
well as by their virulence properties. Virulence typing has led to
the identification of several pathogenicity islands and virulence
associated factors shared among strains of each single pathovar,
however this typing was unable to define unique virulence
features, explaining the host specificity of each pathovar. Multi
locus sequence typing (MLST) of 150 ExPEC strains revealed a
remarkable dichotomy with some strains spreading all over the
E.coli population and the majority accumulating in distinct
phylogenetic clusters [clonal complex ST95 (CC-ST95), CCST23, CC-ST73, ST117 and ST62]. Highly pathogenic avian
E.coli strains cluster at three different points on the evolutionary
tree (ST95-Cplx; ST29-Cplx, and ST117), strongly suggesting
the possibility of multiple origins of APEC. Likewise, the
MLST-database has revealed the co-existence of APEC, NMEC
and UPEC strains in common sequence types, while identical
phylotypes have mostly been grouped under ST95, a sequence
type that is highly linked to capsular polysaccharide K1 and
several ExPEC-associated virulence genes.
The appearance of multiple clusters within the three ExPEC
pathovars, which are nowadays universally distributed, indicates
an independent and parallel evolution over millions of years,
putatively leading to different pathogenic mechanisms and
special epidemiological behaviour of single phylotypes. MLSTanalysis ascertains the zoonotic potential of APEC as a direct
infectious agent, a reservoir for recurrently emerging clones, and
as a distributor for virulence factors. Preliminary data derived
from animal infection studies also indicate the pathogenic
significance of NMEC in chickens, corroborating the indistinct
determination of ExPEC pathovars.
Mycobacteria MIRU-VNTRplus: Online database for
identification of M. tuberculosis complex isolates based on
MIRU, SPOLIGO, and regions of difference data
S. Niemann1, T. Weniger2, D. Harmsen2, P. Supply3
Research Center Borstel, Natl. Reference Ctr. Mycobacteria,
Borstel, Germany
Univ. Hosp. Muenster, Dept. Periodontology, Muenster
Inst. Pasteur de Lille, Lille, France
PFGE types of isolates of Listeria monocytogenes originating
from human cases have been compared with those of isolates
coming from various food items. The human isolates do not
belong to a single cluster but are rather individual in each
patient. Furthermore, there are no genetic similarities between
human and environmental isolates. Obviously, no outbreak due
to a particular food item has been documented. But definitely
more human isolates are required to describe the actual
epidemiology of listeriosisin Germany.
Background: Molecular typing of bacteria from the
Mycobacterium tuberculosis complex (MTBC) is essential for
epidemiological purposes such as investigating the spreading of
specific genotypes. Recently, mycobacterial interspersed
repetitive units (MIRU) typing has become an important
method, as it allows high-throughput, discriminatory and
reproducible analysis of clinical isolates. Because of its portable
data format, MIRU typing has the potential to be a versatile tool
for individual strain identification based on large reference
databases. However, so far no public MIRU database with well
characterized reference strains is available.
Methods: A collection of 177 strains representing the major
MTBC lineages was used to build up an internet based database.
For each strain epidemiologic and genotype information was
stored together with copy numbers of 24 MIRU loci,
spoligotyping patterns, regions of difference (RD) profiles, and
IS6110 RFLP fingerprint images.
Results: Via the freely accessible new service users can compare
their strain(s) with the reference strains or analyze their strains
without using the database content. Comparisons can be based
on single MIRU-, spoligo-, RD-typing data, or by a combination
of different datasets. If a combined analysis is performed,
weights can be assigned to the different methods. For each
comparison, a list of the reference strains most similar to the
submitted strain can be displayed, thereby allowing MTBC
species and lineage classification. Several distance coefficients
are available, including Nei’s DA, and categorical. Based upon
the respective distance matrix, a dendogram can be calculated
using UPGMA or neighbor-joining clustering algorithms. The
resulting trees may be exported in various data formats.
Conclusion: The new MIRU-VNTRplus database offers an easy
way to compare user strains against a collection of well
characterized reference strains. As one additional novel feature,
combinations of typing methods can be used for comparison.
The open database can be accessed via the internet at
The effect of hospital volume on surgical site infection rates
following orthopeadic procedures: What seems to be the
most appropriate threshold?
D. Weitzel-Kage1, S. Dorit1, M. Behnke1, P. Gastmeier2
Charité-Berlin-University Medicine, Institute for Hygiene and
Environment Medicine, Berlin, Germany
Medical University Hanover, Institute for medical
Microbiology and Hygiene, Hanover, Germany
Epidemiology of listeriosis - a changing pattern
H. Hof1, B. Becker2
University Hospital Mannheim, Inst. Med. Microbiology
Mannheim, Germany
Bundesforschungsanstalt für Ernaehrung, Inst. Hygiene
Karlsruhe, Germany
In the last few years the numbers of registered cases of
listeriosis in Germany has dramatically increased. This is
particularly due to the incidence in aged patients. Hence, the
A lot of individual patient risk factors for surgical site infections
(SSI) have been analysed in the past, but the number of studies
investigating the influence of structural factors like annual
procedure volume on SSI rates is limited.
To investigate the effect of hospital volume on the SSI rate for
hip and knee replacement and arthroscopic operations.
Data of the German national surveillance system for nosocomial
infections (KISS) have been used for this analysis. A total of
34,579 hip replacement procedures from 77 hospitals, 29,237
knee replacement procedures from 56 hospitals and 16,642
arthroscopic operations from 24 hospitals (between January
2001 and June 2006) have been included in this analysis.
Thresholds of 50 and 100 procedures per year have been used to
distinguish between high and low volume institutions. SSI rates
were calculated according to these thresholds and logistic
regression analyses have been performed controlling for age,
gender, duration of operation, wound class, ASA score and
hospital volume.
Using an annual number of 50 procedures per year as a
threshold, the SSI rate following knee replacement was 1.9% in
lower volume hospitals and 1.0% in hospitals with more than 50
procedures per year (p=0.02). The logistic regression analysis
confirmed the significant association between higher provider
volume and lower SSI rates for knee replacement (OR= 1.67),
but not for hip replacement and arthroscopic operations. When
using 100 procedures as a threshold, a sigificant association
between higher volume and low SSI rates was found for knee
replacement (OR=1.42), hip replacement (OR=2.04) and
arthroscopic procedures (OR= 1.98).
From the viewpoint of SSI prevention an uncontrolled
expansion of orthopeadic procedures to small hospitals should
be avoided, the annual hospital volume should be at least 50
procedures, preferably about 100.
Emergence of monophasic Salmonella enterica serovar
4,5,12:i:- phage type DT193 in Germany with a
characteristic 19kb island in thrW locus
W. Rabsch1, J. Laverde-Gomez1
Robert-Koch-Institute, Department Wernigerode, Wernigerode
Salmonella enterica are one of the most common causes of
foodborne gastroenteritis with 52000 cases in 2005 and 2006 in
Germany. Recently in Europe, most human cases have been due
to serovars S. Enteritidis, typically associated with hen-eggs and
chicken and S. Typhimurium, typically associated with pork or
beef. Monophasic multidrug-resistant Salmonella enterica
serovar 4,5,12:i:- phage type DT193 has become one of the
dominant serovar during 2006 in Germany causing some large
diffuse outbreaks with increased hospitalization. Locally
produced pork meat is suspected to have been the vehicle for
transmission causing substantial morbidity in children and
elderly people. We look for virulence differences to the
sequenced S. Typhimurium (4,[5],12:i:1,2) LT2 to which it is
closely related to but lacks the second-phase flagellar antigen. In
the t-RNA thrW locus a 19kb island with lower G+C content is
present. We produced a PCR product by long range PCR and
sequenced that fragment. The sequence analysis shows some
similarities with enterobacterial phage sequences and some
possibly pathogenicity related genes. A multilex PCR for
occupancy of the thrW locus by other phages or this island is
Report of the national reference Center for Streptococci,
M. van der Linden1, R. R. Reinert1, C. Heeg1, R. Lütticken1
I. Seegmüller1, A. Al-Lahham1
University Hospital RWTH Aachen, National Reference Center
for Streptococci, Aachen, Germany
In the years 2005 and 2006 the NRCS received 5.249
streptococcal strains for identification and as part of
epidemiological studies. The NRCS was actively involved in
national and international surveillance studies on invasive and
non-invasive streptococcal infections. In 2006 the surveillance
study on invasive Streptococcus pneumoniae in North RhineWestphalia was extended to Saxony and Bavaria. Several
quality management and assurance measurements were
established: SOPs are now available for most of the
standardized test procedures in the field of streptococci.
Since 1997 the level of penicillin G resistance in streptococci in
Germany has been rising. However, in 2005 we observed a
small decrease in the level of resistance, and this decrease
continued in 2006. Macrolide resistance is increasingly seen in
isolates from invasive pneumococcal disease. Especially in very
young children, macrolide resistance is a growing problem, with
more than 30% of isolates now being resistant. The macrolide
resistance levels also show a slightly decreasing tendency in
2005 and 2006.
Furthermore, in 2006 a new surveillance system of vaccine
failures after a 7-valent pneumococcal conjugate vaccination
was initiated and determination of pneumococcal antibodies
against single antigens according to the WHO standard is now
provided by the NRCS.
Nine outbreaks and cases of suspected hospital transmission of
streptoccocci and enterococci were carefully analysed by the
NRCS. The strain collection of the NRCS now comprises
29.796 isolates, including 25.016 Streptococcus isolates.
Reference strains have been distributed to several working
groups in Germany and abroad.
Of note, the surveillance systems of the NRCS were adapted to
the recent STIKO recommendation to vaccinate all children
against pneumococcal disease with the 7-valent pneumoccal
conjugate vaccine. The surveillance in children and adults was
extended to detect herd immunity effects, replacement, cases of
vaccine failures and reduction in the overall incidence of
pneumococcal disease.
Several projects are planned for the future: Surveillance on S.
agalactiae disease in Germany, international cooperation in the
7th EU framework (CAREPNEUMO), two streptococcal
genome projects, full MLST data on invasive isolates in
Lessons from ResiNet and treatment recommendations
provided by the national reference Center for Helicobacter
N. Wüppenhorst1, H. - P. Stüger2, B. Hobmaier1, M. Kist1
Institute for Medical Microbiology und Hygiene, Nationales
Referenzzentrum für Helicobacter pylori, Freiburg
AGES, Österreichische Agentur für Gesundheit und
Ernährungssicherheit, Graz, Austria
In 2001 the German National Reference Center for H. pylori
(Hp) launched a nationwide sentinel study on the surveillance of
development and risk factors of antimicrobial resistance in Hp
(ResiNet). A database was established containing information
about antibiotic resistance of the individual isolate, prior
eradication regimes, and socioeconomic characteristics of study
patients. In addition gastric biopsies from routine patients were
investigated. From all isolates, which were cultured from those
patients, resistance pattern was determined using the Etest®
method. To investigate the outcome of the patients involved,
and to get more information concerning the efficacy of the
individually applied treatment regimes, we decided to develop a
follow-up study. The aim of this study is to determine which
eradication therapy is most successful in a given setting of
antimicrobial resistance, and finally to establish evidence-based
recommendations for Hp eradication therapy. For this purpose
gastroenterologists participating in ResiNet as well as such
sending routine samples are asked to complete respective
questionnaires for each investigated patient three months after
gastroduodenoscopy. In the questionnaire they are asked to
provide information on the treatment scheme applied, as well as
on the outcome of the therapy. Furthermore they are asked
whether confirmation of Hp eradication was performed. At the
time point of abstract submission 158/190 and 167/256
questionnaires were completed and resent by gastroenterologists
participating in ResiNet and those sending routine samples
Clinical management of Helicobacter pylori–infection:
Impact of first multivariate analysis data from the
nationwide sentinel study ResiNet
M. Kist1, E. Glocker1, B. Hobmaier1, N. Wüppenhorst1
H. - P. Stüger2
Institute for Medical Microbiology and Hygiene, Nationale
Reference Center for Helicobacter pylori, Freiburg
AGES, Österreichische Agentur für Gesundheit und
Ernaehrungssicherheit, Graz, Austria
The recently published International Maastricht Consensus III
recommends proton pump inhibitors combined with
clarithromycin (CLA) and amoxicillin or metronidazole (MTZ)
as first line regimes in H. pylori (Hp)-infection. Furthermore,
culture and sensitivity testing of Hp is not recommended before
second treatment failure. On the other hand, use of CLA is
discouraged in settings with resistance rates of more than 1520% without prior sensitivity testing. The same is true if
resistance to MTZ exceeds 40% in the respective population.
The aim of the nationwide study ResiNet, which was launched
by the National Reference Center (NRZ) for Hp in 2001, is to
monitor and to prospectively investigate risk factors for the
development of antimicrobial resistance in Hp. For this purpose
the NRZ recruited 16 microbiological Centers (MC), each
collaborating with 3 to 7 gastroenterologists (GE) in clinical
practice. At the end of April 2007 a total of 850 patients were
completely investigated. Overall 39.4% and 21.8 % of isolates
were resistant against MTZ or CLA respectively, and 15.3%
showed double-resistances against both drugs. The proportions
of primary resistance (N=570) were 28.1% (MTZ), 6.3%
(CLA), 3.3% (MTZ and CLA) and 14.7% (CIP). However, in
patients pre-treated once (N=94) resistance rates had increased
significantly to 52.1% (MTZ), 52.1% (CLA), and 29.8% (MTZ
and CLA). Repeated pre-treatment (N=91) was associated with
further increase of double-resistances up to 69.2% to MTZ and
CLA, clearly indicating that a significant increase in resistance
to MTZ and CLA already occurs after the first treatment failure.
Multivariate analysis in addition revealed pre-treatment as
highly significant independent risk factors for developing
resistance to CLA (OR 89.8) as well as to MTZ (OR 29.6). In
conclusion, these results underline the hypothesis that
development of resistance in Hp in contrast to other pathogens is
nearly exclusively dependent on treatment strategies in the
individual patient. Further, the data clearly demonstrate that
culture and sensitivity testing of Hp from gastric biopsies – in
contrast to Maastricht 2005 - should be mandatory already after
the first treatment failure, because already at that stage
resistance rates exceed 50% for both drugs, CLA and MTZ.
Field investigations for the detection of animal reservoirs of
Francisella tularensis in three different endemic areas of
P. Kaysser1, E. Seibold1, K. Maetz-Rensing2, S. Essbauer3
W.D. Splettstoesser1,4
Bundeswehr Institute of Microbiology, Immunology, Munich
German Primate Center, Pathology, Goettingen, Germany
Bundeswehr Institute of Microbiology, Virology, Munich
Institute of Medical Microbiology, Virology & Hygiene
Medical Microbiology, Rostock, Germany
Background: Within the last three years, tularemia re-emerged
in different regions of Germany. Two outbreaks affected nonhuman primate research facilities in the vicinity of Goettingen,
Lower Saxony. An outbreak in humans occurred in a group of
hare hunters in the county of Darmstadt-Dieburg, Hessen. The
reason for the re-emergence of this highly virulent pathogen in
Germany as well as the potential reservoir in wildlife is
Methods: Field investigations including the description of the
ecological and geographical features of the outbreak regions,
live trapping of different rodent species and on spot sample
preparation (blood samples) were conducted. Tissue and organ
preparation were performed in a standard laboratory
environment. All specimens were screened by a highly sensitive
real-time PCR and different antigen detection assays (hand-held
test kit, ELISA). Positive samples were additionally cultured
according to standard microbiological protocols supporting the
growth of F. tularensis. Detection of F. tularensis LPS specific
antibodies was performed employing an ELISA.
Results: All outbreak areas showed several geographic and
ecological characteristics known to favor long-term presence of
F. tularensis (Alluvial forests, low precipitation, mean annual
temperature above 10°C). A total of more than 400 rodents of
several different species from all three areas were analysed. In at
least 14 different rodents, F. tularensis could be directly
detected by PCR or other diagnostic methods. In two cases F.
tularensis could be grown from the spleen or liver from infected
mice. Water voles represented the predominantly affected
Conclusions: We could prove that tularemia is still endemic in
wildlife in Germany. In at least one affected region, the
infection rate in live trapped rodents was as high as 7%. From
the lack of detection of specific antibodies, we presume that
tularemia is an acute, mainly fatal infection in rodents. The role
of water, free living amoebae and ticks as a reservoir for F.
tularensis between consecutive enzootics as well as the
geographical distribution of this pathogen in different German
states is currently investigated.
OPS 8-987: useful tools in documentation and surveillance
T. Pietzcker1, B. Gaertner2, E. Kniehl3, L. Leitritz4, H. Mauch5
E. Straube6
University Hospital, Ulm, Germany
University Hospital Saarland, Institute for Virology
Homburg, Germany
Clinic Karlsruhe, ZLMT – Department of Microbiology and
Hygiene, Karlsruhe, Germany
bioscientia Labor Ingelheim, Ingelheim, Germany
HELIOS Clinic Emil-von-Behring, Institute for Microbiology,
Immunology and Laboratory Medicine, Berlin
Friedrich-Schiller-University Jena, Institute for Medical
Microbiology, Jena, Germany
OPS 8-987 (complex treatment of colonisation or infection by
multiresistant bacteria) was introduced in 2006 and is
reimbursed since 2007 in G-DRG. Adherence to infection
control regulations and documentation of at least 2 hours daily
additional workload per patient are required for coding OPS 8987. We present several edp-supported tools to document
adherence and to document the workload as well as the new
recommendation of "Deutsche Krankenhausgesellschaft" and
"Spitzenverbaende der GKV" which facilitates workload
National reference Center for tropical infections
S. Kramme1, M. Panning1, S. Günther1, E. Tannich1
C. Drosten1, B. Fleischer1
Bernhard-Nocht-Institute, Hamburg, Germany
The National Reference Center for Tropical Infections deals
with tropical viruses including the haemorrhagic fever viruses,
Protozoa, helminths, tropical bacteria such as M. leprae and M.
ulcerans, rickettsia and tropical fungi. For many of these agents
few established and standardised assay systems do exist. To
meet the demands of modern diagnostic assays the NRC works
on the establishment and improvement of systems and
cooperates with industry. Among others new PCR assays with
improved specificity and sensitivity were validated. These
include assays for the detection of intestinal parasites,
Filoviruses, Lassavirus and rickettsia. A real-time PCR test to
measure the viral load of tropical HIV-1 subtypes was
established and evaluated in several developing countries.
Several of these assays were brought to the market as test kits in
cooperation with industry, e.g. real-time PCRs for Filoviruses or
The NRC is a WHO Collaborating Center for Arbovirus and
Hemorrhagic Fever Virus Reference and Research. Furthermore
it is a member of European networks for the diagnosis of
imported viral diseases and the WHO SARS Laboratory
Network. A close collaboration with the United Nations exists
for the analysis of suspected cases of haemorrhagic fever in the
context with the UNAMSIL-Mission in Sierra Leone.
The NRC has set up a reporting office for infections with
Entamoeba histolytica to obtain information about the
occurrence of imported and autochthonous infections. Since this
infection is not notifiable no data about its prevalence in
Germany exist. In the reporting period 67 reports were received.
The NRC was a leading participant in the clarification of
transmission of leishmaniasis and rabies after organ
transplantation and of several cases of rare parasite infections in
Germany. A case of imported Lassa fever in 2006 and imported
Rabies in 2007 were diagnosed and investigated at the NRC.
The NRC is strongly engaged in various quality assurance
activities. It is the organizer of the quality assessment exercises
for parasite detection in Germany and participated in large
international studies in parasitology.
Several external quality assurance studies were performed in
collaboration with Robert Koch Institute, e.g. on CrimeanCongo Hemorrhagic Fever virus, Chikungunyavirus and
poxvirus infections. The first international quality assessment
study for the detection of West Nile virus was performed and
evaluated in collaboration with RKI.
The national reference center for retroviruses
H. Walter1, K. Korn1, K. Metzner1, R. Grassmann1
B. Fleckenstein1
University Hospital Erlangen, Virologisches Institut
Erlangen, Germany
The National Reference Center for Retroviruses has pursued its
broad spectrum of activities in the fields of HIV/AIDS and
human T cell leukemia viruses (HTLV) by the development of
additional methods for the analysis of the relatively uncommon
retroviruses HIV-2, HTLV-1, and HTLV-2. and specific
methods for the monitoring of infected patients are usually not
available as commercially available test kits.
Another major epidemiologic focus is the investigation of
transmitted drug-resistant HIV-1. A very fruitful cooperation
with the Robert-Koch-Institute has been established in national
and international seroconverter studies. The development of
methods for the detection and quantification of minority species
of HIV-1 allowed more sensitive analyses of transmission
events and the dynamics of viral infection during antiretroviral
therapy. We could show that the transmission of drug-resistant
viruses may be substantially underestimated by the use of
conventional methods alone. Current investigations aim to
evaluate the clinical relevance of drug resistant minorities. Most
recently we could show that the occurrence of drug resistance in
very early phases of treatment did not necessarily lead to
therapy failure.
Additonally, the interpretation of complex HIV resistance data
became more and more important. Thus, the freely available
geno2pheno system for the prediction of resistance was fortified
by a clinical validation. However, such database-driven
bioinformatic system have some disadvantages, e.g. with the
interpretation of rare mutational patterns. Therefore, a new
rules-based interpretation system for genotypic HIV-1 drug
resistance data (HIV-GRADE) has been develpoed and
validated by a group of German clinical virologists under the
guidance of the NRC. HIV-GRADE is online freely available
and may become a standard for laboratories performing HIV-1
drug resistance testing in Germany.
With regard to advisory activities and visibility, the quarterly
“Retrovirus-Bulletin” provides scientific and clinical
information focusing on HIV and AIDS and other retroviral
infections. Its readership comprises physicians specialized in
HIV medicine, public health and laboratory professionals and
members of the HIV community.
Consulting laboratory for Hemolytic-Uremic-Syndrome
A. W. Friedrich1, M. Bielaszewska1, W. Zhang1, O. Böhler1
A. Lagemann1, R. Fischer1, A. Mellmann1, H. Karch1
University of Muenster, Institute of Hygiene, Muenster
Enterohemorrhagic Escherichia coli (EHEC) are the major
cause of hemolytic-uremic syndrome (HUS) in childhood. E.
coli O157:H7 is the most important pathogenic serotype
worldwide, and accounts for more than 50% of all HUSassociated EHEC infections in Germany. Furthermore,
additional 15% of HUS cases are caused by sorbitol-fermenting
(SF) EHEC O157:H-. This serotype differs from classical EHEC
O157:H7 in its pheno- and genotype, its epidemiology and
clinical importance, and was responsible for several large
outbreaks in Europe. In contrast to EHEC O157:H7, SF
O157:H- do ferment sorbitol and are therefore not detected on
sorbitol MacConkey agar frequently used for the isolation of
EHEC O157:H7.
Major tasks of the consulting laboratory are bacteriological,
serological and molecular biological analyses of specimens from
patients with EHEC infections. In addition, we perform detailed
phenotypical (e.g. toxin expression) and molecular
characterisation of the clinical isolates. We use a molecular
assay based on the fimbriae-encoding sfpA gene to detect SF
EHEC O157:H- directly from the patient’s stools.
Subtyping of Shiga toxin genes (stx) is of a clinical importance,
since specific stx-subtypes (stx1, stx2, stx2c and stx2dact) are
significantly associated with the development of HUS. stxsubtyping can therefore be used for risk assessment and as a
prognostic marker for the clinical outcome of the infection.
Furthermore, we investigate the presence of IgM antibodies
against the lipopolysaccharides of the major EHEC serotypes
associated with HUS, which is especially useful in HUS patients
with EHEC-negative stool cultures. Finally, we provide
consulting services for patients, physicians, and public health
Because of the clinical impact and the lack of specific therapy
for EHEC-associated diseases, the development of new
diagnostic tools, the identification of reservoirs and transmission
routes, and the characterisation of pathogenicity of different
EHEC are of major importance. The consulting laboratory for
HUS in Muenster works on these tasks in close collaboration
with the National Reference Center for Salmonella and other
enteric pathogens at the Robert Koch Institute in Wernigerode.
Sequence based typing of Legionella pneumophila strains
isolated from patients in Germany: Common feature and
differences to other countries and regions
P. C. Lück1, F. Görlach1,2, J. Borchardt2, J. Helbig1
Technical University of Dresden, Medical Microbiology and
Hygiene, Dresden
Legionella pneumophila is commonly detected in environmental
specimens. If transmitted to susceptible persons it can cause
pneumonia (Legionaires’ disease). In order to detect the source
of the infection reliably and rapidly genetic typing of clinical
and environmental strains must be performed. Currently a seven
locus sequence-based typing scheme (flaA, pilE, asd, mip,
ompS, proA, neuA) for the epidemiological typing of clinical
and environmental isolates of Legionella pneumophila has been
developed by the European Working Group on Legionella
Infections and made available on the web (
This typing scheme is applicable to all L. pneumophila
serogroups and can be performed using cultivated Legionella
strains but also directly from DNA preparations from clinical
We typed 185 L. pneumophila strains isolated from patients
with travel-associated, community- acquired, or nosocomial
legionellosis. 136 of these strains belonged to serogroup 1, and
49 to other serotypes. In 36 cases sets of related strains proved
the environmental source to be the source of the infection. The a
significant number of clinical isolates (33%) strains belonged to
five sequence types which has been found in clinical isolates
different countries. However there are differences in the
distribution in different regions in the world. Thus several
clones found in Germany seem to be unique. Our study supports
the hypothesis that a significant part of clinical cases in
Germany is caused by defined L. pneumophila clones that are
distributed throughout the world.
Clostridium difficile - molecular and functional analysis of
naturally occurring variations in the putative negative toxin
regulator Gene tcdC
J. Fritzsche1, B. Waidner1, T. Giesemann2, B. Scherer1, M. Kist1
University Hospital Freiburg, Institute for Med. Microbiology
and Hygiene, Department for Microbiology and Hygiene
Nationale Reference Center for Helicobacter pylori, Freiburg
University Hospital Freiburg, Institute for Experimental and
Clinical Pharmacology and Toxicology, Freiburg, Germany
Since 2001 ongoing spreading of a highly virulent Clostridium
difficile (Cd) strain (Ribotyp 027, Toxinotyp III, 18 bp-deletion
in the putative negative regulator gene tcdC) is observed. The
epidemic strain was first detected in the United States, from
there spreading over Canada and England, and reaching Western
Europe in 2005. In the meantime it has been detected in
Netherlands, Belgium, France, Switzerland, and recently in
Austria and Poland.
During the same period a case-control-study on epidemiological
risk factors and molecular virulence of nosocomial Cd153
associated diarrhea was carried out at the Freiburg University
Medical Center (granted by the BMBF), resulting in Cd isolates
from 600 study patients, and 400 patients investigated routinely
during the same time period. From all isolates (N=1000),
including three epidemic strains from Belgium and Switzerland
as positive controls, PCR-based ribotyping, and in addition,
molecular analysis of the putative negative regulator gene tcdC
was carried out using PCR and molecular sequencing. Isolates
with respective variations were further analysed phenotypically
applying a Vero cytotoxicity assay, and a quantitative ELISA
toxin assay, which was adapted for this purpose. In a total of 35
isolates the same 18 bp deletion as found in the epidemic strains
was detected. However in no case those isolates belonged to the
epidemic ribotype 027. A total of 70 isolates presented with an
already known 39 bp deletion, but in 19 isolates a 54 bp deletion
was detected, which had not been described before. All “novel”
deletions were further investigated by molecular sequencing.
Growth curves performed from truncated and wild type strains
were not significantly different. The already known 18 bp
deletion was associated with an up to 2.2 fold increase in
toxicity measured by the ELISA, however the toxic effect was
more pronounced (about 10fold) if the verocell cytotoxicity test
was applied. Surprisingly, cytotoxin production of isolates with
deletions of 39 and 54 bp was markedly reduced, reaching zero
in some cases.
In conclusion, the functional impact of the putative regulator
gene tcdC needs further investigation.
Biofilm-grown Klebsiella pneumoniae capsular type 2
induces higher stimulation of polymorphonuclear leucocytes
than planktonic bacteria
R. Podschun1, S. Hany1
Christian-Albrechts-University, Institute for Infection
Medicine, Kiel, Germany
The biofilm-growth mode of bacteria is considered to mediate
enhanced resistance to antibiotics and to modify virulence. In
Klebsiella pneumoniae, the major virulence factor is the
production of a polysaccharide capsule, capsular types K1 and
K2 being the most virulent serotypes. K2 inhibits phagocytosis
and killing by polymorphonuclear leucocytes (PMNL). The
present study investigates whether biofilm grown and planktonic
K2 bacteria differ with respect to interaction with PMNL.
Two clinical K. pneumoniae K2 isolates and their noncapsulated variants were examined for their ability to induce
respiratory burst induction in human PMNL. The
chemiluminescence response (CL) was taken as a gauge of
leucocyte stimulation. Surprisingly, stimulation by biofilmgrown bacteria was significantly increased compared to
planktonic cells (median CL values 38.3 vs. 22.3; P<0.001). The
effect observed was independent of composition of the culture
media used. Biofilm enhanced PMNL stimulation was not due
to reduced capsular production as the amounts of glucuronic
acid in capsular preparations from planktonic and biofilm-grown
bacteria were equivalent (3.8 vs. 4.1 µg/109 bacteria; P>0.1). In
contrast to K2 capsulated strains, in biofilm-grown noncapsulated variants a significantly decreased stimulation of
PMNL compared to planktonic cells was observed (median CL
values 53.6 vs. 78.5; P<0.01). The expression of K2 capsules
furthermore diminished the early phase of biofilm formation as
determined by measuring bacterial adhesion to microtitres plates
(OD578nm 0.03 vs. 0.17; P<0.0001).
Our findings suggest that expression of Klebsiella capsules in
the biofilm-growth mode might render the bacteria more
susceptible to interaction with PMNL, but the nature of this
finding remains unclear at this stage and needs to be further
Analysis of a cross-border rise in Incidence of serogroup B
invasive meningococcal disease affecting Netherlands and
J. Elias1, L. Schouls2, I. van de Pol2, M. Schroeter3, M. Frosch1
U. Vogel1, A. van der Ende4
Institute for Hygiene and Microbiology, Wuerzburg, Germany
National Institute of Public Health and the Environment
Bilthoven, Netherlands
Landesinstitut für den Oeffentlichen Gesundheitsdienst NRW
(loegd), Muenster, Germany
Netherlands Reference Laboratory for Bacterial Meningitis
(AMC/RIVM), Amsterdam, Netherlands
Automated finetype-specific spatiotemporal analyis repeatedly
identified an unusually high concentration of meningococci of
the finetype B:P1.7-2,4:F1-5:ST-42:cc41/44 in the region of
Greater Aachen (counties of Aachen, Dueren, and Heinsberg,
population: 1.1m). Further analyses confirmed the
predominance of this finetype within this region (63% compared
to 13% in the rest of Germany, 2005) and an approximately
threefold (2005) incidence of invasive meningococcal disease
compared to the rest of Germany. The question was raised
whether this community outbreak represented a delayed
spillover of the hyperendemic situation starting in Netherlands
in the 1980ies.
Serogroup B strains isolated in Netherlands, in North-Rhine
Westphalia and in Lower Saxony in 2000-2006 were screened
for ST-41/44 using a lineage specific PCR (Bart et al FEMS
Microbiol Lett 2000). The PCR positive strains were subjected
to MLST and to MLVA analysis (Schouls et al. J Clin Microbiol
2006). Results were visualized on geographic maps.
MLST revealed an accumulation of ST-42 in four counties
around Aachen, and in the province of Limburg. Moreover,
MLVA suggested an increased case number due to MT-19 in the
Aachen area. Only the combined use of MLST and MLVA
clearly segregated a clone with ST-42 and MT-19, which
accounted for the majority of cases in Greater Aachen, while
being almost absent in the rest of the two German Federal states.
The clone was more prevalent in the province of Limburg than
in the rest of Netherlands, but geographic mapping suggests that
it emerged as an independent descendant of the ST-41/44
complex in the Aachen area.
Antigen fine typing was of pivotal importance for the
identification of the outbreak of meningococcal disease. In this
particular scenario, combined application of MLST and MLVA
was crucial for the elucidation of the emerging strains origin.
The visualization of bi-lateral geographic data in this
investigation enhanced data interpretation, arguing for
international geographic mapping of laboratory surveillance
data. It furthermore paves the grounds for newly designed
investigation of epidemic waves and international spread of
meningococcal strains.
Second Helicobacter pylori multiresistant clinical isolate
including tetracycline resistance in Germany
N. Wüppenhorst1, E. Glocker1, M. Kist1
University Freiburg, Institute for Medical Microbiology and
Hygiene, National Reference Center for Helicobacter pylori
Freiburg, Germany
In 2006 the National Reference Center for H. pylori (Hp)
reported the first Hp strain with reduced susceptibility to
tetracycline, which was isolated from a multimorbid 70-year-old
woman in Germany. In February 2007 we isolated the second
tetracycline-resistant Hp strain from a 50 year-old woman in our
routine-laboratory. Antimicrobial susceptibility testing (Etest®
method) showed sensitivity to amoxicillin (0.016 mg/L) and
rifampicin (1 mg/L) but resistance to metronidazole (16 mg/L),
clarithromycin (16 mg/L), ciprofloxacin (32 mg/L) and
tetracycline (8 mg/L). Sequencing of the 16S rRNA encoding
genes revealed a double nucleotide exchange at positions 926928 (AGA926-928ATC) which confirms the tetracyclineresistant phenotype in Hp. The patient reported preceding
antimicrobial treatments due to unrelated bacterial infections
which included minocycline and clarithromycin. Further genetic
analysis of the isolate and more detailed information concerning
the patient’s medical history and antibiotic pre-treatment
regimes due to Hp infection and further unrelated bacterial
infections are in progress.
In conclusion, this case clearly demonstrates that rare resistant
phenotypes, like resistance to tetracycline, which up to now
have been observed predominantly in Asia and South America,
may also occur in Germany. These findings further underline the
importance of antimicrobial susceptibility testing in H. pyloriassociated infections and the need for surveillance studies on the
development of antimicrobial resistance.
How can the national reference Center be helpful in
combating the resistant tuberculosis in Germany and the
S. Rüsch-Gerdes1
Research Center Borstel, National Reference Center for
Mykobakteria, Borstel, Germany
Large scale sequence based typing of Staphylococcus
aureus: a reference Center’s experience
B. Strommenger1, C. Braulke1, D. Heuck1, W. Witte1
Robert-Koch-Institute, Wernigerode Branch, Wernigerode
Objectives: In May 2006 we introduced spa-typing in our
laboratory. This method replaced phage typing in combination
with SmaI-macrorestriction analysis for routine S. aureus strain
typing. This study reviews typing results for six month with
respect to typeability, reproducibility, discriminatory power and
concordance with alternative typing methods. Methods: A total
of 1459 S. aureus isolates were characterized. A spa-type was
assigned using the Ridom StaphType software. The algorithm
BURP (based upon repeat patterns) was used to cluster resulting
spa-types into different groups. To verify the results a subset of
isolates was subjected to SmaI-macrorestriction analysis and
MLST. Results: Among 1459 S. aureus isolates 221 different
spa-types were defined. The twenty spa-types most frequently
found comprise more than 60% of all isolates and include most
types linked to predominant clonal lineages of MRSA in Central
Europe, previously defined by phage typing and SmaImacrorestriction analysis. Among those clonal lineages distinct
differences in spa-type variability were seen; while some
lineages (e. g. the Barnim epidemic MRSA, t032, ST22)
encompassed a variety of closely related spa-types, others (e. g.
a subclone of Rhine-Hesse epidemic MRSA, t003, ST225) are
represented only by a single type. For assessment of widely
disseminated lineages exhibiting only a single spa-type,
additional markers must be applied. One hundred and twentyone spa-types were determined only once. For 90% of these
isolates BURP proved to be a valuable tool to assign them to
particular clonal groups. Thereby, results of BURP grouping
were overall concordant with those of SmaI-macrorestriction
analysis and MLST. Conclusion: spa-typing in combination
with BURP based grouping of isolates proved to be a valuable
tool for strain typing with regard to the demands of a national
reference Center. The BURP algorithm revealed extremely
useful for grouping new and uncommon spa-types. However, to
overcome limitations in terms of typing accuracy and
discriminatory power, additional markers, like SCCmec, lineage
specific genes or alternative DNA polymorphisms are
Prion diseases in Germany: epidemiological data and risk of
U. Heinemann1, B. Meissner1, I. Zerr1
Georg-August-University, Neurology, Goettingen, Germany
Creutzfeldt-Jakob disease is the most frequent form of human
transmissible spongiform encephalopathies. It is caused by
accumulation of pathological prion protein PrPsc. Beside
sporadic, there are also genetic (genetic CJD, FFI, GSS),
iatrogenic (e.g. by dura mater grafts, growth hormone) and
variant CJD (probably due to uptake of BSE contaminated food)
known. Susceptibility and clinical manifestation are modified by
the disease subtype composed of genotype at codon 129
(methionine or valine) and prion protein type (1 or 2). In
Germany, since 1993 a systematic prospective surveillance of
prion disease is performed by the National Reference Center in
Incidence of sporadic CJD is slightly increasing up to 1.7 per
million per year in 2006. Especially the frequency of atypical
cases (young age at onset, longer disease duration, atypical
technical findings) is increasing. By several mutation of the
prionprotein gene on chromosome 20 gentic prion disease can
be caused. The most frequent mutation in Germany is D178N
coupled with methione at codon 129 (FFI). Variant CJD is not
recognized in Germany up to now.
Additionally, ten patients with iatrogenic CJD were identified (9
by dura mater grafts, 1 by corneal transplant). Instead, no
transmission by cadaveric growth hormone treatment werde
found, what is the most frequent cause of iatrogenic CJD
worldwide. Whether neurosurgical intervention can transmit
CJD is not yet resolved. In the litertaure only two cases are
reported. Due to the long incubation time up to decades this rare
phenomenon might be caused by an anamnestic problem. In a
german case-control study seven patients with CJD were
identified who had anamnestic neurosurgical internvention.
There was no significant difference to the frequency in control
population and therefore brain surgery is no statistical risk
factor. Nevertheless, it is remarkable that three patients with
iatrogenic CJD by dura mater grafts were treated within 17
months within the same hospital.
In summary, the results of prospective surveillance for prion
diseases in Germany will be presented with special emphasis on
diagnostic and
differential diagnostic characteristics.
Additionally, potentially transmission by iatrogenic procedures
will be discussed.
Molecular epidemiology of respiratory syncytial virus
infections in Underfranconia between 2002 and 2006
D. Schneiderbanger1, F. Neske1, K. Blessing2, H.W. Kreth2
B. Weissbrich1
University of Wuerzburg, Institute of Virology and
Immunobiology, Wuerzburg, Germany
University of Wuerzburg, Childrens Hospital, Wuerzburg
Background: The respiratory syncytial virus (RSV) is the most
common viral cause of respiratory infections in infants and
children. Reinfections occur lifelong. Two RSV groups (A and
B) have been described, each with several genotypes. The
available data on the molecular epidemiology of RSV in
Germany are only limited.
Material and Methods: Between January 2002 and July 2006,
221 respiratory samples of infants and children treated in the
Children?s Hospital of the University Wuerzburg were found to
be positive for RSV antigen by routine testing with
immunofluorescence assays. Phylogenetic analysis was
performed on 211 of these samples by amplification and
sequencing of the second variable region of the RSV G gene.
Results: The group distribution of the 211 RSV positive samples
was 69.5 % group A and 30.5 % group B. RSV group A was the
predominating virus in all seasons except for the winter season
2002/03. Over the whole observation period, three different Agenotypes (GA2, GA5, GA7) and four different B-genotypes
(GB3, SAB3, BA, and a novel genotype) were detected. The
RSV genotypes GA2, GA5, SAB3, and BA were most
frequently found and were prevalent in almost all seasons.
Among the B-genotypes, the proportion of the genotype BA
increased from 25 % in 2002 to 91 % in 2005/06. Three group B
sequences were assigned to a novel genotype, which was
tentatively named BWUE. One reinfection with the same
genotype (GA5) was observed in a child who was hospitalised
with RSV infection at the age of 12 and 28 months.
Conclusion: The results of our study are in agreement with the
molecular epidemiology of RSV in other geographical regions.
We found both genotype persistence and genotype shifting
during the observation period. In addition, we detected a novel
B genotype.
Role of Game in the natural cycle of Borrelia burgdorferi
sensu lato
V. Fingerle1, U. C. Schulte-Spechtel1, C. Hizo-Teufel1, A.
König2, B. Wilske1
Max v. Pettenkofer , National Reference Center for Borrelia,
Munich, Germany
Wissenschaftszentrum Weihenstephan, Wildbiologie und
Wildtiermanagemnt, Munich, Germany
B. burgdorferi s.l. is maintained in nature in complex cycles
involving hard bodied ticks as vectors and warm blooded
animals as hosts. Several authors have suggested that certain B.
burgdorferi s.l. species are preferentially or exclusively
maintained in specific vertebrate host – tick cycles. B. afzelii
and some B. garinii strains appear to use rodents as the main
reservoir, while B. valaisiana and most B. garinii strains were
found in enzootic cycles with birds. Furthermore, it was shown
that these associations are mirrored in the complement
sensitivity of these species or strains. However, data from
Germany of mice or of game on the influence on the host-vector
cycle of B. burgdorferi s.l. are poor so far.
To gain more information we investigate in a still ongoing study
skin biopsies (ear) and ticks from hunted animals. Deers, wild
boars, and foxes were investigated for infection with B.
burgdorferi s.l. species and OspA types by restriction fragment
length polymorphism (RFLP) analysis of the ospA gene. The
method enables differentiation of both single and multiple
infections with B. burgdorferi s.s. (OspA type 1), B. afzelii
(OspA type 2), B. garinii (OspA types 3 - 8), B. valaisiana
(subgroupI and II), B. lusitaniae, B. bissettii, and B. spielmanii.
In the frame of this study skin biopsies from 209 roe deer
(Capreolus capreolus), 55 red deer (Cervus elaphus), 127 fallow
deer (Dama dama), 49 wild boars (Sus scrofa), and 92 red foxes
(Vulpes vulpes), as well as 550 ticks were investigated. In
general, feeding on all deer species clearly reduced borrelia
prevalence in ticks. However, young roe deer may serve as host
for B. valaisiana and B. garinii OspA types 4 and 6 and red deer
possibly for B. garinii OspA type 4. In skin biopsies from Wild
boars B. burgdorferi s.s. and B. garinii OspA types 4, 5, and 6
were present and from red foxes B. garinii OspA types 5 and 6
and B. spielmanii. Detection of B. spielmanii from red foxes is
of special interest, since this only recently described new species
was only detected from dormice so far.
The results of the present study argue for a relevant role of game
in distinct natural cycles of different B. burgdorferi s.l. species
and subtypes. However further studies are necessary to
substantiate these findings.
Accreditation of microbiological laboratories according ISO
A. Steinhorst1
DACH Deutsche Akkreditierungsstelle Chemie, Frankfurt am
Main, Germany
In the last years the importance of quality has increased in all
parts of the society. In medical laboratories quality has a special
importance. It is not only the implementation of a quality
management system, but moreover to a high degree that the
medical laboratories are competent to provide valid results,
including their interpretation. These additional quality feature is
the essential part of the International Standard ISO 15189:2003
“Medical laboratories - Particular requirements for quality and
competence” and the main difference to ISO 9001:2000.
The ISO 15189 covers all elements, which are essential to
patient care. Such elements include arrangements for
requisition, patient preparation, patient identification, collection
of samples, transportation, storage, processing and examination
of clinical samples, together with subsequent validation,
interpretation, reporting and advice.
The International Standard has been elaborated for all different
types of medical laboratories. Therefore many German societies
in the field of medical laboratory diagnostics – the DGHM as
well – have decided some years ago to complement the standard
for the different laboratory diagnostic disciplines. The
complements are available in form of special checklists. Special
checklists for microbiological laboratories are:
1. Microbiology and Hygiene - General requirements
2. Bacteriology
3. Mycobacteriology
4. Mycology
5. Parasitology
6. Virology
7. Infection serology
8. Molecular biology of infection diagnostics
9. Hygiene
That the requirements of ISO 15189 are fulfilled can be
demonstrated through the accreditation from the German
Accreditation body of Chemistry (DACH). Important part of the
accreditation is the on-site assessment, carried out by experts.
The experts are recommended from the various medical
societies, correspondingly the microbiological experts are
recommended from the DGHM.
With the accreditation and therefore with the conformation of
competence the confidence in the medical laboratories should be
strengthened. Many laboratories are already accredited against
ISO 15189 and many others are on the track.
Antibiotic prescribing indicators in German ICUs
E. Meyer1, P. Gastmeier2,3, B. Schrören-Börsch1
H. Rüden4,3, U. Frank1, F. Schwab4,3
Freiburg University Hospital, Institute of Environmental
Medicine and Hospital Epidemiology, Freiburg, Germany
Hanover School of Medicine, Institute of Medical Microbiology
and Hospital Epidemiology, Hanover, Germany
National Reference Center for Surveillance of Nosocomial
Infections, Berlin, Germany
Charité-University Medicine Berlin, Institute of Hygiene and
Environmental Medicine, Berlin, Germany
Objective: To determine and to analyse antibiotic prescribing
parameters of intensive care units (ICUs) which are not only
measures for quantity but also for quality in the sense of
picturing diversity of antibiotic use.
Methods: From January through December 2005, 43 ICUs
participating in SARI (Surveillance of Antibiotic Use and
Bacterial Resistance in Intensive Care Units) provided data and
were included in the analysis. For antibiotic prescribing
indicators we choose the following parameters: 1. The number
of different antibiotics used 2. The most frequently antibiotic
used of each ICU (top1) and the three and ten most frequently
antibiotics used (top3, top 10) and 3. The percentage of total
antibiotic use of penicillins, of quinolones, of cephalosporins, of
carbapenems and of glycopeptides. Antibiotic use was expressed
as defined daily doses (DDD) and normalised per 1000 patientdays (AD=antimicrobial usage density), one DDD being the
standard adult daily dose of an antimicrobial agent for one day’s
treatment defined by the WHO (index 2006).
Results: The data covered 182,565 patient days. The median of
total antibiotic use was 1156 DDD/1000 pd and ranged from
450 to 1799 DDD/1000 pd. The median number of different
antibiotics used was in 27 (range 19 - 37). The most frequently
single antibiotic used per ICU (top1) accounted for almost 20%
(median) of the total antibiotic use. The top 3 antibiotics used
made up 40% of total use and the top 10 of 80% (range 62.5 –
93.8). Maximum the top 3 single antibiotics used accounted for
over 60% of total antibiotic use in an ICU. Total antibiotic use
and the percentage of different antibiotic groups used were
extremely heterogeneous in single ICUs. At single ICU level
quinolones could make up more than 35%, broad spectrum
cephalosporins almost 40% and carbapenems 30% of their total
antibiotic use. However, at median penicillins and
cephalosporins accounted each for a forth of total use.
Conclusion: Prescribing parameters of antibiotic use differ
considerably from ICU to ICU. The three most frequently used
antibiotics account for 40% of the total antibiotic use.
Prescribing parameters provide additional and easy to
understand information for the analysis of antibiotic
consumption and of the selective pressure applied by those
Immunodiagnosis of cystic and alveolar echinococcosis:
antibody screening and species differentiation
I. Reiter-Owona1, M. Frosch2, P. Kern3, B. Grüner3
A. Hörauf1, D. Tappe2
University Clinic Bonn, Institute of Medical Microbiology
Immunology and Parasitology (IMMIP), Bonn, Germany
University of Wuerzburg, Institute of Hygiene and
Microbiology, Wuerzburg, Germany
University Hospital and Medical Center Ulm, Division of
Infectious Diseases, Ulm, Germany
Differential diagnosis between cystic echinococcosis (CE) due
to Echinococcus granulosus (Eg), and alveolar echinococcosis
(AE) due to E. multilocularis (Em), has significant implications
for the treatment and follow-up of infected individuals. Whereas
imaging methods mostly provide sufficient criteria for
differentiation, selected serological assays back-up the diagnosis
and are considered as highly specific. Recent results of external
quality control measurements (INSTAND), however, did not
confirm the expected specificity when applied in routine
diagnostic laboratories. Therefore, the diagnostic performance
of the Em2plus-ELISA (Bordier Affinity Products, Switzerland)
was evaluated using 56 sera from 30 CE patients and 39 sera
from 35 AE patients from two different Centers (Bonn and
Ulm). The following standardized in-house techniques were run
in parallel: IHAT (Eg human hydatid cyst fluid), IFT (Eg
protoscolex antigen), and ELISA (Em crude larval extract
antigen) for screening, and the Em10-ELISA for species
differentiation (recombinant Em antigen).
Results: 3 sera out of 26 (11.5 %) from 20 CE patients (Bonn)
and 4 sera out of 30 (13.3 %) from 10 CE patients (Ulm)
showed a cross-reaction in the Em2plus-ELISA, whereas no
cross-reactivity was observed with the Em10-ELISA. From 9
sera of 5 AE patients (Bonn), 3 sera (one single patient with
long-term chemotherapy) were negative in both the Em2plus
and Em10-ELISA, whereas from 30 sera of 30 AE patients
(Ulm), 10 sera were negative in the Em2plus-ELISA (33 %) and
11 sera were negative in the Em10-ELISA (37 %). Of these
negative sera, 4 were from patients who had undergone curative
surgery. The remaining 7 negative sera were from patients who
had received long-term chemotherapy.
Conclusion: Cross-reactivity of the Em2plus-ELISA on sera of
CE patients tested (11.5 % and 13.3 %, respectively) was lower
than anticipated by the manufacturer (20-25%). The Em10ELISA proved to be very specific. As the individual antibody
profiles of CE and AE patients may change during antiparasitic
chemotherapy and after parasite resection, a definitive speciesspecific diagnosis is not always possible.
Acknowledgment: The Em2-ELISA kits were kindly provided
by Bordier Affinity Products
Proficiency testing program for bacterial genome Detection
(PCR/NAT by INSTAND e.V.): Why to participate?
U. Reischl1
University Hospital Ratisbon, Institute of Medical
Microbiology and Hygiene, Ratisbon, Germany
Numerous methodological and technical advances combined
with manifold efforts to automate and to standardize the most
critical steps within sample preparation, amplification and
sequence-specific detection of amplicons, have turned nucleic
acid-based assays from a highly demanding and sophisticated
technology into almost routine analytical procedures.
With the increasing acceptance of this novel technology in the
field of diagnostic microbiology and the broad availability of
individual open platforms to perform a permanently growing
spectrum of in-house or commercially prefabricated PCR / NAT
assays, there is also a growing demand for appropriate quality
control (QC) activities. A program for external validation was
timely activated by the DGHM and is now organized by
INSTAND e.V., Duesseldorf, Germany (
This quality control initiative termed Bacterial Genome
Detection (PCR / NAT) started in early 2003 with a panel of 5
pathogens. At present we are conducting bi-annual ring trials for
12 bacterial pathogens or pathogenicity factors including
Neisseria gonorrhoeae, Chlamydia trachomatis, Bordetella
pertussis, Legionella pneumophila, Chlamydia pneumoniae,
Helicobacter pylori, EHEC, Borrelia burgdorferi, Salmonella
enterica, Listeria spp., and MRSA / cMRSA. In order to keep up
with the current scope of routine PCR testing in medical
microbiology, our spectrum of PCR/NAT ring trials will be
extended by Mycoplasma pneumoniae soon. Then we can offer
the first CAP-panel worldwide covering the most frequently
PCR-tested pathogens involved in community acquired
pneumonia (L. pneumophila, C. pneumoniae, and M.
Here we present the concept of our unique sample design and
discuss some practical aspects as well as noticable problems
experienced during the past rounds of PCR/NAT proficiency
testing. Relevant benchmarks are summarized and the direct as
well as the indirect benefits for participating laboratories are
Diagnostic accuracy of chlamydia serology as assessed from
14 sentinel surveys performed by the German Infection
serology proficiency testing program between 2000 and 2006
I. Müller1, K. - P. Hunfeld1, M. Frosch2, H. - J. Hagedorn2
H. Hlobil2, C. Schörner2, G. Stanek2, E. Straube2
C. - H. Wirsing von König2, V. Brade1
University Hospital of Frankfurt am Main, Central Laboratory
of the Bacteriologic Infection Serology Study Group of Germany
(BISSGG), Institute of Medical Microbiology and Infection
Control, Frankfurt am Main, Germany
Bacteriologic Infection Serology Study Group of Germany
(BISSGG), Frankfurt am Main, Germany
Serological methods such as enzyme immuno assay (EIA) and
microimmunofluorescence test (MIFT) are frequently used in
the routine medical laboratory to confirm or to rule out possible
Chlamydia infection. The poor quality of Chlamydia serology in
the non-specialist laboratory is a question of growing concern,
although, actual data dealing with this issue are rare. Here, the
results of 14 proficiency testing sentinel surveys for diagnostic
laboratories in Germany were analyzed. From 2000 to 2006, the
average number of laboratories participating in each survey was
n=207 for C. trachomatis serology and n=176 for C.
pneumoniae serology. A total of 56 clinically and/or
serologically characterized serum samples were provided by
voluntary donors (two per survey) and distributed to the
participating laboratories in order to determine the accuracy of
diagnostic methods such as complement fixation test (CFT),
EIA, and MIFT. Assessment of reference test results for each
trial was performed according to the provisional guidelines for
proficiency testing in infection serology as proposed to the
German general council of physicians. The use of EIA for
specific antibody detection was four times more frequent than
that of CFT and MIFT. For specific IgG- and IgA-testing, mean
accuracy ranged from 77 to 90% for EIA and from 70 to 83%
for qualitative MIFT. Quality of quantitative MIFT was poor
with mean accuracy rates between 55 and 70%. The accuracy of
diagnostic comments was even lower: On average, only 60%
(range:15 - 92%) of participants provided correct statements
when asked to classify their findings as active, past or no
infection. These findings suggest that serological test results
alone are not sufficient for a clear cut diagnosis when
Chlamydia infections are suspected. Standardization of testing
procedures and the implementation of standard sera are urgently
needed to improve the diagnostic value of Chlamydia serology
in the routine microbiological laboratory.
Safety of laboratory tests for infection diagnostics –
Experience of the BfArM until end 2005
R. Siekmeier1, J. Lütz1, D. Wetzel1
Bundesinstitut für Arzneimittel und Medizinprodukte, Abteilung
Medízinprodukte, Bonn, Germany
University Hospital, Dept. of Med. Microbiology, Virology and
Hygiene, Rostock, Germany
Helios Hospital, Institute of Microbiology, Immunology and
Laboratory Medicine, Berlin, Germany
In the years 2006 and 2007, the MiQ series of Standards has
been amended with novel editions addressing “Respiratory
Infections in Cystic Fibrosis” (Corresponding Author, M.
Hogardt)(MiQ 24), “Diagnosis of Liver Infections”
(Corresponding Author, S. Schaefer)(MiQ 25), and an newly
written edition of “Blood Culture Diagnosis: Sepsis,
Endocarditis, and Catheter Infections” (Corresponding Author:
H. Seifert)(MiQ 3a and 3b). With these Standards, an expert
consensus has been reached on various analytic procedures
including preanalytic issues, basic and extended microbiologic
analysis in the diagnostic laboratory, reporting and interpretative
aspects as well as quality assurance. The consensus statements
are supported by an extensive literature review, and these new
Standards are edited with the approval and support of other
Medical Societies of the respective fields. New MiQ projects
currently planned or in preparation include “Bioterrorism”,
“Ocular Infections”, “Myocarditis”, “Validation in the
Microbiology Laboratory”, and “Multiresistant Organisms”.
Furthermore, new editions on the following MiQs are planned or
in progress: “Wound Infections”, “Gastrointestinal Infections”,
“Peritonitis”, “Mycobacteria”, “Lower Respiratory Infections”,
“Genital Infection”, and “Parasitology”.
In this symposium, select aspects of the recently published
MiQs with interest to a larger audience will be presented as well
as a perspective into the MiQ editions currently under
preparation. The MiQ Procedures Quality Standards are an
integral part of the Quality Assurance Initiatives of the Deutsche
Gesellschaft für Hygiene und Microbiology and provide solid,
expert- and literature-based practical recommendations
applicable in all diagnostic microbiology laboratories.
The European Directive 98/79/EC for in-vitro diagnostic
medical devices (IVD) regulates the marketing and post
marketing surveillance of IVD in the European Economic Area.
Manufacturers have to inform the responsible Competent
Authorities about incidents and field corrective actions related to
IVD. In Germany, the Federal Institute for Drugs and Medical
Devices (BfArM) is the responsible Competent Authority for
most IVD, while a small subset of IVD, specified in Annex II of
the Directive 98/79/EC, for immune hematological and
infectiological testing as well as tissue typing, is under the
responsibility of the Paul-Ehrlich-Institute (PEI). Until the end
of 2005 the BfArM received a total of 653 notifications
regarding IVD. From these 115 related to IVD for analysis of
the infection status (tests, control materials, calibrators, culture
media and analyzing equipment). Most of the reports originated
from manufacturers (57.4 %), while other sources of reports
played only minor roles. Product failures of tests, control
materials, calibrators as well as culture media were frequently
caused by manufacturing errors and biological contaminations.
Analyzing equipment was typically affected by software
malfunction. Through the investigations of the manufacturers
product failures were confirmed in most cases and consequently
corrective actions were performed in the large majority of
incidents. The corrective actions frequently included customer
information, product recalls, changes in the production process
and/or the quality management or software upgrades for the
analyzing equipment. Our data suggest that the existing system
for post marketing surveillance is a sufficient tool to ensure
product safety of IVD.
Antifungal use in intensive care units
E. Meyer1, F. Schwab2,3, P. Gastmeier3,4, H. Rüden2,3
A. Heininger5
Freiburg University Hospital, Institute of Environmental
Medicine and Hospital Epidemiology, Freiburg, Germany
Charité-University Medicine Berlin, Institute of Hygiene and
Environmental Medicine, Berlin, Germany
National Reference Center for Surveillance of Nosocomial
Infections, Berlin, Germany
HanoverSchool of Medicine, Institute of Medical Microbiology
and Hospital Epidemiology, Hanover, Germany
Tuebingen University Hospital, Department of Anaesthesiology
and Intensive Care Medicine, Tuebingen, Germany
The Microbiology Procedures Quality Standards (MiQ) –
New topics
M. Herrmann1, E. Kniehl2, A. Podbielski3, H. Mauch4
University Hospital, Institute of Medical Microbiology and
Hygiene, Homburg, Germany
City Hospital, Dept. of Med. Microbiology, Virology and
Hygiene, Karlsruhe, Germany
Objectives: To provide benchmarking data on antifungal use in
ICUs, to analyse risk factors and to look for correlations with
antibiotic use data and structure parameters.
Methods: Antimicrobial use data for 13 ICUs were obtained
from computerised databases from 1/2004 through 6/2005.
Antimicrobial usage density (AD) is expressed as daily defined
doses/1000 patient-days. Correlations were calculated by the
Spearman correlation or for binomic variables by the two-sided
Wilcoxon test. A multivariate regression analysis was performed
to identify independent risk factors for the outcome ‘antifungal
Results: Mean systemic antifungal drug use was 93.0, the range
being between ADs of 18.9 and 232.2. ICUs treating transplant
patients had a significantly higher mean antifungal usage at
152.9 compared with ICUs not treating transplant patients where
the AD was 46.0. Fluconazole was the most frequently
prescribed antifungal (mean AD 69.6) followed by amphotericin
B (11.4) and voriconazole (6.2). Antifungal use correlated
significantly with the consumption of quinolones, carbapenems
and penicillins with extended spectrum, but not with total
antibiotic use and not with the type of ICU or university status.
In the multivariate linear regression analysis, two parameters i.e.
high quinolone use (p 0.002) and ICUs which treat transplant
patients (p 0.027) were independent risk factors for a high level
of antifungal use.
Conclusion: Antifungal use was heterogeneous in German ICUs
with the mean lying at 93. Benchmarking data might provide a
useful method for assessing strategies that aim to reduce
antifungal use in ICUs. However, data should be stratified for
ICUs with and without transplant patients. Keywords:
Surveillance, defined daily doses, benchmarking data,
fluconazole, amphotericin B, voriconazole
In our opinion, ATP bioluminescence may be a simple to use
surrogate parameter to control the effect of cleaning surgical
ATP bioluminescence – An alternative method for cleaning
control of surgical instruments
D. Hansen1, M. Hilgenhöner1, W. Popp1
University Hospital, Hospital Hygiene, Essen, Germany
Success of cleaning is critical in reprocessing of medical devices
and has to be verified. Visual assessment has limitations;
therefore quality of reprocessing actually is checked by
performing microbial cultures or by determination of residual
blood or protein of real instruments after reprocessing.
Unfortunately these methods have considerable disadvantages:
Results of microbiological cultures are not available in time for
corrective action to be taken at once. Available methods for
detection of residues of protein or blood are too cumbersome to
be performed by non technical staff, dosage of reagents risks to
be imprecise because of improper equipment or interpretation of
results is subjective. Methods are needed which can be routinely
performed on site at reprocessing of medical devices by non
technical staff and which deliver results at once. ATP
bioluminescence, a method which is widely applied to control
surface cleanliness in food industry and kitchen hygiene, may be
a suitable method to control the quality of reprocessing of
medical equipment too. The method of ATP bioluminescence
and our experiences of applying a commercial available ATP
bioluminescence assay to verify the cleaning of real
contaminated surgical instruments in the context of validation of
washer-disinfectors are presented. A total of 235 samples were
collected. In 6.4 % measured ATP bioluminescence was above
the chosen threshold and thus induced immediate troubleshooting. For single instruments ATP bioluminescence indicated
problems of reprocessing when no residual protein was detected.
Detection of antibodies by flow cytometry and ELISA
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Author Index
Baljer, G.
Bandi, C.
Bethe, A.
MSP05, MPP38
Bandt, D.
Betzel, C.
Bär, W.
Bhakdi, S.
Barbuddhe, S.
Biedermann, T.
Barekzai, J.
Biehler, K.
Bartel, M.
Bielaszewska, M.
Aas, F. E.
Abu-Qatouseh, L. F.
Achtman, M.
MSV06, MSP11
Adam, S.
Barthel, M.
Adam, T.
Bastian, M.
Adler, B.
Batra, A.
DVP11, KMV04, MPP64,
Äpfelbacher, M.
Aichinger, C.
Aktan, I.
AlDahouk, S.
AlbertWeissenberger, C.
Alderborn, A.
MPV24, MPP70
Aldick, T.
Aleík, E.
Al-Lahham, A.
FTP02, FTP04, FTP05, FTP07,
FTP12, FTP13
Almeida, R.
Alpers, K.
Alterio, V.
Anders, A.
KMP12, MSP06, MPV06
Aniwatangkoora, Y.
Batzilla, C.F.
BaetzingFeigenbaum, J.
Bauer, B.
Baum, C.
Biundo, T.
Bautsch, W.
Blahout, B.
Bauwens, A.
Blass, D.
Becher, D.
Blaut, M.
Becher, K.
Bleich, A.
IIP02, MPV13
Becken, U.
Bleich, E.
Bleiß, W.
Becker, B.
DVP09, MPP18, MPP22,
MPP23, MSV05, HYV10,
MSP06, MSP13
MPP56, MPV43, MPP41
Becker, K.
Becker, T.
Beer, J.
Behrens, S.
Antonenka, U.
Behrens, W.
Benedek, O.
Beninati, T.
Benner, D.
IIP14, IIV12, KMP06, HYP03,
MPP10, IIP09, FTP18, GIP14
Autenrieth, S. E.
Bensel, T.
Benz, A.
Aurass, P.
Autenrieth, I. B.
Berchtold, S.
Bereswill, S.
GIV07, MPV10, IIV01, IIP13
Berger-Bächi, B.
Babikir, R.
Bachmann, T. T.
Backert, S.
GIV05, MPV11, GIP10, GIV02
MSP05, MPP38
Arvand, M.
Bischoff, M.
Bittner, T.
Antáo, E. - M.
Appel, B.
Bin jasass, F. M.
GIP16, MPP08, GIP17,
RKP11, GIP19, GIP18
FTP22, HYP10, MPP49
HYV06, RKP02, HYP09
Anonsen, J. H.
Biertz, F.
MPP46, MPP50
Bierbaum, G.
IIP16, IIV13
Biswas, R.
BehnckKnoblau, S.
Behnke, M.
Anjum, M. F.
Bessler, M.
Berghoff, J.
Bergmann, S.
MPP02, MPP15
MPV31, MPV02
Bergmans, A. M. C.
Bergstrom, K.
Blessing, K.
Blöcker, H.
Blokhin, B. M.
Böckenholt, A.
Böcker, K.
Bodmer, T.
Böhnke, U.
Bogdan, C.
IIV08, MPV22, IIP22
Bögli-Stuber, K.
Bogner, K. - H.
Böhler, O.
Böhm, K.
Bohn, E.
Bohne, W.
Bönemann, G.
Bonn, M. L.
Borchardt, J.
Borges, F.
MPP04, MPV44
Bader, O.
Bernauer, J.
Bahr, U.
Bertsche, U.
Zepelin, M.
Borkenhagen, G.
Borneff-Lipp, M.
Bertz, H.
Bains, M.
Besier, S.
MPV05, MPP58
HYP08, HYV01
MPP10, IIP07
Bolbot, Y. K.
Bader, L.
Baier, M.
Bobkiewicz, W.
Borrmann, E.
Boye, K.
Busch, U.
KMP02, GIP03
Daniels-Haardt, I.
Brade, H.
FTP34, FTP35
Buschmann, J.
MPP15, MPP50
Danschke, A.
Brade, L.
FTP34, FTP35
Buss, A.
Dasti, J.I.
Brade, V.
HYP11, IIV11, IIV11a
MPV05, QSV03, MPP58
Buss, C.
de Bör, A.
IIV09, EKV11
Bussmeyer, U.
de Groot, P. W.
EKV11, IIV09
de Hoog, G. S.
Decker, E. - M.
Brakhage, A.
Brands, B.
Büssow, K.
Brandt, C.
HYP11, HYV04
Brandt, S.
MPV11, GIP10, GIV02
Brattig, N.
C., P.
Brauers, J.
Cantero, P.
Deckert, M.
Braulke, C.
Carniel, E.
Brecx, M.
Carsauw, H.
Carstens-Slim, A.
Bredenbruch, F.
Breitbach, K.
Breitenstein, A.
MPV42, MPV43, MPP32,
MPV26, KMP05, IIP03, IIV03,
DVV02, DVP28
Brenneke, B.
GIP15, MPV12
Breukink, E.
Brocker, T.
Brockmann, D.
Brockmeyer, J.
Brodegger, T.
Carter, B.
Casey, P.
Cassone, M.
Chakraborty, T.
HYV02, HYV11, HYV09,
IIV05, DVV09, MSP01
Changtrakun, Y.
Chaberny, I. F.
MPP08, GIP17
Charpian, S.
Chatterjee, I.
MPP51, MPV09
Chen, L.
Broich, M.
Bröker, B.
IIV10, MPP65
Chhatwal, G. S.
MPP09, MPP60
IIP04, IIP06
Chikkaballi, D.
Bröker, B. M.
Brosius, J.
Chinni, S. V.
Broszat, K.
Christian, J.
Bruns, H.
Claus, H.
Buchal, A.
Cleland, S.
FTP15, FTP17
Cloud, J.
Buchholz, P.
Cohrs, A.
Buchrieser, C.
Colgan, S. P.
Buchwald, A.
Conrads, G.
Buchbauer, G.
DVP18, DVP19
Buckendahl, A.
Coops, S.
Buder, I.
Cordes, T.
Cornelis, G. R.
Buer, J.
Buhrdorf, R.
Cornelis, P.
Bünger, J.
Cosmina, M.
Bunk, S.
Cramer, N.
Burckhardt, F.
Creuzburg, K.
Burggraf, S.
Cuny, C.
Burghardt, I.
Czerny, C. - P.
Burian, M.
MPP55, MPP20
Burkert, B.
Busch, D.
IIV05, MPP48
Busch, T.
FTP21, GIP12
KMV05, DVV10
Dehghani, F.
MSV05, HYV10
Dehio, C.
Deiwick, S.
DellEra, S.
Denk, S.
Dettenkofer, M.
Deyneko, I.
Diehl, I.
Dierich, M. P.
MSP05, MPP38
EKV06, IIP11
Diestel, A.
Dieterich, G.
Dietz, A.
Dilling, S.
Disqué, C.
Distler, M.
Dittmann, S.
Dittrich-Breiholz, O.
Ditzen, A.
MPP01, MPP52
IIP02, MPV19
Dobrindt, U.
MPP33, MPV29, MPP47
Domann, E.
DVV09, MSP01
Donat, S.
Döring, G.
MPP04, MPV44
Dorit, S.
Dötsch, A.
Dreier, J.
Dreisewerd, K.
Drexler, H. G.
Drögemüller, K.
Drosten, C.
Duchmann, R.
DVP01, DVP13, KMV09
RKP09, DVP24
Dumke, R.
Dunn, J.
Durmus, N.
Dykstra, T.
Dalpke, A.
Daniel, W. G.
Ebel, F.
EKV04, EKP03
Fedtke, I.
Gärtner, B.
Feldmann, F.
Gastmeier, P.
Feldmeier, E.
Eberhard, S.
FerreiradaSilva, M.
Eberhardt, C.
Feucht, H.
Ebert, S.
Fichet-Calvet, E.
Ebner, S.
Eckart, M.
Eckmanns, T.
FTP27, MSP09, HYP05
Egge-Jacobsen, W.
Ehricht, R.
Eichberg, J.
Eichler, P.
Eick, S.
Fingerle, V.
Fink, K.
Finlay, B.
IIV11, IIV11a, RKV07
IIV12, GIP14
IIV10, IIP06
Fischer, R.
GIV07, IIV01, DVP09,
MPP22, MPP23
LMP02, LMP03, LMP04,
LMP05, LMP06
KMP19, KMP20
Fischer, W.
MPV38, GIV08
Eickhoff, M.
Eikmann, T.
HYP13, HYP14, HYV05
Fischer, A.
Fischer, M.
Fleck, R.
Fleckenstein, B.
Fleischer, B.
RKP09, DVP24
Eikmanns, B. J.
FTP19, MPP28
Eisenblätter, M.
Flenner, E.
IIP16, IIV13
Elias, J.
Flieger, A.
Eltzschig, H. E.
Flunker, G.
Elwin, S.J.
Forestier, C.
Engelhard, K.
Forsbach-Birk, V.
Engelhardt, H.
Frangoulidis, D.
Engelhardt, W.
Frank, C.
Engelmann, S.
FTP24, IIV10, MPV08,
IIP04, MPP65, MPV09,
Frank, R.
Engstler, M.
MPP07, KMP05
IIP17, DVV03, FEMS-P08
Frank, U.
Franke, G.
Frankenberg, T.
Frenzel, E.
Ergin, A.
Freytag, C.
Erttmann, K.
Frick, I. - M.
Epis, S.
Erck, C.
Eske, K.
Essbauer, S.
Essig, A.
Ettischer, N.
IIV10, MPV26, IIV03
FTP26, FTP25
Evans, S.
Ewers, C.
DVP10, MSP05, MSV06,
MPP38, MSP11
Fabbri, M.
Fabri, M.
Fahl, E.
Faigle, M.
Fälker, S.
Färber, J.
DVP13, KMV09
Frick, J.-S.
IIP14, IIV12, IIP07, GIP14
Friedrich, A. W.
MSV05, DVV11, HYV10,
MSP13, GIP17, GIP18,
KMP09, MSP04, KMP12,
MPP68, MPP69
DVV08, DVV13
Friedrich, S.
Fritzsche, W.
Gatermann, S.
Fröhlich, J.
Frosch, M.
Fuchs, S.
HYP02, RKV01, MPP17,
QSV03, QSV01
Fulde, M.
Ge, Z.
Gebert, S.
Gebert-Vogl, B.
Gebhardt, F.
MPV37, MPP48
Gehde, N.
IIV06, HYP06, EKP04,
Geiger, T.
MPV04, MPP21
Geipel, U.
Geginat, G.
Geisel, J.
IIP14, IIV12, IIP07
Geißdörfer, W.
Georgi, C.
Gerstenbruch, S.
Gerten, B.
Gessner, E.
Gfrörer, S.
Ghebremedhin, B.
Giedrys-Kalemba, S.
Giese, A.
MPP27, MPP31
Gieseler, S.
Glocker, E.
RKP06, RKV02
Glöckner, G.
Glodde, S.
Gnad, T.
Göbel, U. B.
Görke, C.
Göthe, R.
MSP05, MPP38
GIV07, IIV01, MSP03
MPP55, MPP02, MPP20,
MPV04, MPP21, MPP39,
Götzfried, M.
Gohlke-Micknis, S.
Goldberg, J.
Goldenberg, O.
MSP03, IIP01, LMP01
Gore, M.
Goris, M.
Görlach, F.
Görtz, H. - D.
FTP11, RKP08, FTP32
HYV02, HYV11, HYV06,
HYV03, QSV06, RKP02,
QSP02, HYV08, HYV09,
HYP09, HYP09
KMP09, MSP04, KMP12,
MPP34, MPP35, MSP06,
MPV06, MPP43, MSP13,
MPP68, MPP68, MPP71
Götz, F.
MPP15, MPP46, MPP50,
Gabdoulkhakov, A.
Gowda, A.
MPV14, MPP25, MPP26
Gahan, C. G. M.
Gräme M, W.
DVP06, DVP07
Garcia Diez, M.
Graham, R.
Grassl, G.
Hancock, R.
Hermann, C.
Grassmann, R.
Handrich, C.
Hermann, G.
Hannig, H.
Herr, C.
Greiner, A.
Hansen, D.
FTP08, QSV07
Greune, L.
Hansen, S.
Hanski, E.
Hany, S.
Hardegen, C.
Grauling-Halama, S.
Grif, K.
Grimm, V.
Gröbner, S.
FTP02, FTP04, FTP05,
FTP07, FTP12, FTP13,
Gronow, S.
Harpel, S.
HYP13, HYV05
EKP07, FEMS-V04, EKV02,
GIP11, EKP06, EKV11
Hartig, R.
GIP10, MPP27
Grobbel, M.
Gross, U.
Grosse, R.
Hardt, W. – D.
Harmsen, D.
MPV15, MPV16
DVV04, DVV11, MSV07
Hartmann, M.
Herrmann, I.
Herrmann, M.
Herzberger, P.
HYP13, HYP14, HYV05
MPP51, MPP55, HYV12,
MPP11, QSV05, MPV09
IIV11, IIV11a
Heuck, D.
Heudorf, U.
Heuner, K.
Heusipp, G.
Heussler, V.
Hildebrandt, A.
Hilgenfeld, R.
MPV24, MPP70
MPV14, MPP12, MPP25,
MPP26, MPP44
Grosskinsky, U.
Hasenberg, F.
Gruber, I.
Haslinger-Löffler, B.
Hilgenhöner, M.
Hauser, N.
Hill, C.
MPP56, MPV42, MPV43,
MPP41, FTP23, MPP32
Hill, J.
Hiller, M.
Hilty, A.
Hinsching, A.
Grumann, D.
IIP04, IIP06
Grüner, B.
Häußler, S.
Günther, S.
Heckenbach, K.
Gürra Roman, B.
Hecker, H.
Güngör, N.
Hecker, M.
Günther, S.
KMP08, DVP10, RKP09
FTP24, MPP07, IIV10,
MPV08, IIP04, MPP65,
Güntner, S.
Güntsch, A.
KMP19, KMP20
Heeg, C.
RKP04, HYP04
Gunzer, F.
Heeg, P.
Gutjahr, R.
Heesemann, J.
Gutmann, D.
Heczko, P.
Haas, A.
Haas, R.
Haase, G.
GIV03, MPV37, MPV38,
MPP48, IIP20
DVP23, DVP26
Habold, C.
Häcker, G.
EKV02, MPV21, IIV04
Häcker, H.
Hacker, J.
Hafke, H.
Hagedorn, H. - J.
Hain, T.
Hall, G.
Hallier, E.
Hammerschmidt, S.
MPP33, MPV29, MPP47,
DVV09, MSP01
MPV31, MPV02
Hamouda, O.
Han, S. - R.
Hlobil, H.
Hobmaier, B.
Hochrein, H.
RKP06, RKP05
GIV07, IIV01
Hoffmann, C.
Heinbockel, L.
Hoffmann, R.
Heinemann, U.
Hoffmann, T.
Heimesaat, M. M.
Hoffmann, A.
Hehl, J.
Haartskerl, R.
Hizo-Teufel, C.
RKP01, HYP06, EKP04,
MPP53, MPP52, HYP08,
MPP42, MPP59, MPV18,
Hippe, D.
Heininger, A.
Heiser, V.
KMP06, QSV06
Heisig, P.
Helbig, J.
MPP40, MPP72, RKP12
Helmuth, U.
Hempel, K.
Hendrix, M. G. R.
MSV05, HYV10
Henke, C.
Henker, J.
Henn, A.
Henne, K.
Henrich, B.
Hense, B. A.
Hensel, M.
Henseler, K.
Hentschke, M.
Hof, H.
Hogardt, M.
MPP42, MPP59, ESV09
Hogg, T.
Holland, G.
Holtfreter, S.
IIV10, IIP04, MPP65
Holzmann, B.
Homburg, S.
Homeier, T.
Hörauf, A.
Hörmansdorfer, S.
Hornbach, A.
Hornef, M.
Horré, R.
Horstkotte, M.
Horz, H. - P.
Hossain, H.
MSP05, MPP38
KMP02, GIP03
IIP07, GIP08
DVP18, DVP19
KMV04, MPV18
Hott, U.
Hube, B.
Huber, I.
KMP02, GIP03, HYP08
Huber, S.
Hünger, F.
Hultgren, S. J.
Humberg, V.
Hunfeld, K. - P.
KMV03, QSV03, ESV17
Husmann, M.
Hussain, M.
Hutzler, M.
Ignatius, R.
Kaase, M.
Kabisch, H.
Imirzalioglu, C.
Ingersoll, M.
Irma, O.
Irtenkauf, C.
Iwen, P.
KMP09, MSP04, KMP12,
MPP34, MSP06, MPV06
Käding, J.
Kahl, B.
Kahl, B. C.
Kahl, F.
IIP14, IIV12, GIP14
Kaiser, P.
Kalbacher, H.
IIP16, IIP18, IIV13
Kalteis, T.
Kamal, E.
Kaminski, E. I.
Kanageswaran, N.
Karapetyan, A.
Karch, A.
Karch, H.
GIP16, MPP08, MSV05,
GIP17, GIP18, RKP11,
Karhausen, J.
Klade, M.
Kadlec, K.
Kalka-Moll, W.
Igel, L.
Kist, M.
Kaspar, H.
Jäckel, K.
Kasper, H.
Jacobi, S.
Kästner, N.
Jacobs, E.
Jansen, A.
MPP37, MPP40, MPP72,
HYP10, MPP49
Janßen, S.
Jaros, M.
Jasny, E.
Jellbauer, S.
Jensch, I.
Jensen, V.
Jentsch, H.
Jiang, H.
Johansson, H. M.
Johswich, K.
Jonas, D.
Joost, I.
MPP55, MPP11
Jores, J.
KMP08, GIP06
Josenhans, C.
GIP15, MPV12
Jung, S.
IIV05, IIV02
Juretzek, T.
Just-Nübling, G.
Juuti, K. M.
Katzowitsch, E.
GIP15, GIV10
Klar, S.
Klare, I.
Klebba, P. E.
DVP01, DVP13, KMV09
Kleine, B.
KMP09, MSP04, KMP12,
MPP34, MPP35, MPV06,
MPP43, MPP68, MPP69
Klein-Günther, A.
Klemm, A.
Klemmer, A.
Kleta, S.
Klis, F.
Kloft, N.
Klos, A.
IIP02, MPV19
Klotz, M.
Knabbe, C.
Knappe, D.
Knauer, M.
Knaust, A.
HYP13, HYP14, HYV05
Kniehl, E.
FTP11, QSV05, RKP08,
Knobloch, J. K.
Köberle, M.
Kaysser, P.
IIP17, DVP22, RKP07
Ködel, U.
Kempf, V.
Kepp, O.
Kerksiek, K.
Kohlenberg, A.
FTP24, IIP04
DVP02, MPP03
Köhler, H.
Köhler, J.
IIV03, MPV08
Kohler, T.
Kern, Y.
DVP15, DVP17
Köhn, B.
Koivogui, L.
Kokryakov, V. N.
Kola, A.
Khan, A. - B.
Khang, D. D.
Kolata, J.
Kiehntopf, M.
Kolditz, M.
Kim, K.S.
MSV05, HYV10
Kohler, C.
Kiessling, S.
Kern, P.
Kesztyues, B.
DVV05, DVP08
Knabner, D.
Köck, R.
Kemper, B.
Kline, K.
Kleesiek, K.
Kaur, S. J.
Keckevöt, U.
RKP06, RKP05, HYV02,
RKV02, MPV10
MSP05, MPP38
HYV02, HYV08
KolobovJr., A.
Kondruweit, M.
Kim, K.-S.
Kongdoung, P.
Kipp, F.
Konietzny, A.
Kirsche, H.
König, A.
König, B.
IIP14, IIV12
König, W.
DVP06, DVP07, MPP27,
DVP06, DVP07, GIV05,
MPV11, GIP10, GIV02,
Kirschnek, S.
Kirschning, C. J.
Kulauzovic, E.
Liese, J.
Konkel, M. E.
MPP27, MPP31
Kunert, A.
Liesenfeld, O.
Konstabel, C.
Kunz, S.
Kooistra-Smid, A. M. D.
Kupfahl, C.
Koomey, M.
Kurt, K.
Kopf, S.
Kurtz, D.
Lindner, B.
Kurz, C. - L.
Lingelbach, K.
Kopron, K.
Korableva, E.
Kurzai, O.
Körber-Irrgang, B.
Kurzmann, C.
HYP06, EKP04, EKP05
EKV03, MPP17
Liliana, T.
FTP21, GIP12
Linde, H. - J.
Lindenstrauß, A.
Linke, D.
DVP02, MPP03, FTP18
Lo, N.
Loddenkemper, C.
Körfer, R.
Kusch, H.
Korn, K.
Kutter, S.
Lössner, M. J.
Kuusela, P. I.
Löffler, J.
Kortsik, C.
Kösel, S.
Kosma, P.
Kostrzewa, M.
DVV09, DVV04
Kozjak-Pavlovic, V.
Kracht, M.
IIP02, MPV19
Kraiczy, P.
IIV11, IIV11a
Kramme, S.
KMV10, RKP09, DVP24
Krammer, S.
Kraus, D.
Krause, M.
Krause-Gruszczynska, M.
Kraushaar, B.
Krausse, R.
Kreft, J.-U.
Kreidenweiss, A.
Kreikemeyer, B.
MPP16, MPV01
Kremer, M.
Kremsner, P.
Kresken, M.
Kreth, H. W.
Kretzschmar, H.
Krickhahn, C.
Krickhahn, D.
Krist, S.
FTP15, FTP17
Krüger, S.
Krüger, W. A.
Krumbholz, A.
Krumbholz, G.
Kuchenbecker, U.
Kücherer, C.
Kuczius, T.
Kuhlicke, J.
Kuhlisch, E.
Kühlmann, G.
La Ragione, R. M.
Lärberg, R.
Lagemann, A.
Laib Sampaio, K.
Lakner, A.
FTP26, FTP25
Landré, J.
Landt, O.
Lang, A.
Lohoff, M.
Loitsch, S.
Löns, D.
MPP32, MPP41
Loof, T.
Lorenz, B.
Lorenz, M.
Lorenz, U.
Lorenzen, T.
Loschen, S.
Lübke-Becker, A.
Langehanenberg, P.
Lardon, L.
Larsen, U.
Lüder, C.
La Sala, P. R.
Ludwig, A.
Laskay, T.
Lugert, R.
Laugks, R.
Laverde-Gomez, J.
Layer, F.
Le, T. T. H.
Lupas, A.
DVP06, DVP07, MPP27,
Lüthje, P.
KMP11, MPP30
Leiendecker, J.
Leinberger, D. M.
Leis, A.
Lembke, C.
Lendeckel, U.
Lewin, A.
Li Stephen, T.
MPP19, RKP03
Lehmann, M.
Leitritz, L.
Leithäuser, F.
Lück, P. C.
MSP10, FTP02, FTP04,
FTP05, FTP07, FTP12,
HYP07, KMP21, MPP57,
EKV02, EKP07
Lührmann, A.
Léchenne, B.
Lehn, N.
Lütticken, R.
Lütz, J.
RKP04, DVP26
Luu Duc, H.
Lyytikäinen, O.
Ma, L.
Maass, M.
FTP11, RKP08, FTP32
MacKenzie, C.
MacKenzie, R.
MPP13, GIV01
Li, G.
MSP05, MPP38
Li, H.
Macpherson, A. J.
Mader, D.
Madlener, K.
Mätz-Rensing, K.
Mai, M.
MSP12, RKP07
Maier, G.
Möller, R.
Nilsson, M.
Maier, T.
DVV09, DVV04
Monecke, S. .
Nimptsch, A.
Mainiero, M.
MPV04, MPP21
Monk, I. .
Nitsche-Schmitz, D. P.
Mordmüller, B.
Noll, I.
Moter, A.
Nordhoff, M.
Marsch, S.
Martin, M.
Martinez-Quiles, N.
Mothes, W.
Nübel, U.
Massow, I.
Mshana, S.
Nürnerger, T.
Matern, Y.
Mücke, I.
Matten, J.
Müller, A.
HYV02, HYV09
Müller, D.
Müller, I.
Mattner, F.
Matysiak-Klose, D.
Mauch, H.
Maucher, H.
Maydannik, V. .
McNamara, P.
Medina, E.
FTP11, QSV05, RKP08,
IIV12, LMV01, GIP14
Müller, P.
Müller, W.
MüllerLoennies, S.
Mülverstedt, A.
FTP34, FTP35
KMV05, DVV10
Oberdorfer, K.
Obermaier, B.
Ölschläger, T.
MPP33, GIP07
Özkaya, I.
Ohlsen, K.
MPV05, MPP73, MPV07
Olbermann, P.
Olgemöller, B.
Münch, R.
Orland, A.
Münter, W.
Orth, D.
Meissner, A.
Muselmann, C.
Ostin, M.
Meissner, B.
Müsken, A.
Oswald, S.
Müsken, M.
MPP56, MPV43
OtvosJr., L.
Menge, C.
DVV04, MSV05, DVV11,
HYV10, RKP11
Müthing, J.
GIP16, GIP18, GIP19
Overhage, J.
Merkel, N.
Mertens, T.
Messler, S.
Nagarajan, K.
Metzner, K.
Nagel, M.
Meukow, N.
MPP07, KMP05
Naim, A.
LMP08, LMV02
Nalca, Y.
Paschen, S.
Nau, R.
Pasierb, P.
Meemboor, S.
Meier, S.
Mellmann, A.
Meussdörffer, F.
Müller, M.
IIP16, IIP18
Meyer, E.
HYP01, QSV06, QSP02
Meyer, F.
Meyer, F. J.
FTP21, GIP12
Nauerth, M.
Naumann, M.
Panning, M.
Panthel, K.
Park, S.F.
KMV10, RKP09, DVP24
IIV05, IIP05
Patten, A.
MPP13, GIV01
Pattis, I.
Meyer, H.
Nega, M.
Paul, R.
Meyer, O.
LMP08, LMV02
Neske, F.
Pelkmans, L.
Michalski, N.
MPP34, MPP68
Neuenhahn, M.
PeltrocheLlacsahuanga, H.
Perbandt, M.
Michel, D.
Michel, R.
Neukirch, C.
Neumayer, W.
Michelmann, M.
Newton, S. M. C.
Middendorf, B.
Nguyen, T. T. H.
Middendorf, D.
Miller, T.
Mircea Gabriel, S.
Nicholson, G.
Nickel, D.
Peschel, A.
Peter-Katalinic, J.
Peters, G.
Petzl, W.
MPV03, MPP15, MPP63
GIP19, GIP16, GIP18
DVP09, MPP18, MPP22,
MPP23, MSV05, MPP36,
RKV06, MPP62
FTP21, GIP12
Nicolaisen, S.
Petzold, A.
Misselwitz, B.
Niederführ, A.
Pfaffinger, G.
Moder, K. - A.
DVP06, DVP07
Niederhausen, M.
Pfeffer, K.
Mogk, A.
Niederweis, M.
Pfeffer, M.
Mohsenipour, I.
Niemann, S.
Pfeifer, G.
Pfeifer, Y.
Reichelt, R.
Pfepper, K. - I.
Reichholf, U.
Runge, C.
Reidel, A.
Rupp, J.
Rupp, S.
Ruppert, T.
Pfister, W.
MSP09, KMP19, KMP20
Phuong, D. M.
Reimann, J.
Piening, B.
Reinert, R. R.
RKP04, MSV03, DVP16,
Piper, C.
FTP11, KMV11,
RKP08, FTP32
Pless, B.
Reischl, U.
Plitzko, J.
Reißmann, S.
Reiter-Owona, I.
Pietzcker, T.
Plum, G.
Podbielski, A.
Polidori, M.