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POLSKIE TOWARZYSTWO MIKROBIOLOGÓW
POLISH SOCIETY OF MICROBIOLOGISTS
Polish Journal of Microbiology
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(JCR)/Science Edition.
Stanis³awa Tylewska-Wierzbanowska
Editor in Chief
2010
Polish Journal of Microbiology
formerly Acta Microbiologica Polonica
2010, Vol. 59, No 3
CONTENTS
MINIREVIEW
Bacteriophage receptors, mechanisms of phage adsorption and penetration into host cell
RAKHUBA D.V., KOLOMIETS E.I., SZWAJCER DEY E., NOVIK G.I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
ORIGINAL PAPERS
Interactions between Borrelia burgdorferi and mouse fibroblasts
CHMIELEWSKI T., TYLEWSKA-WIERZBANOWSKA S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Clonal analysis of Staphylococcus aureus strains isolated in obstetric-gynaecological hospital
SZCZUKA E., SZUMA£A-K¥KOL A., SIUDA A., KAZNOWSKI A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Simultaneous detection and differentiation of pathogenic and nonpathogenic Leptospira spp. by multiplex real-time PCR
(TaqMan) assay
BEDIR O., KILIC A., ATABEK E., KUSKUCU A.M., TURHAN V., BASUSTAOGLU A.C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Methanogenic diversity studies within the rumen of Surti buffaloes based on methyl coenzyme M reductase A (mcrA)
genes point to Methanobacteriales
SINGH K.M., PANDYA P.R., PARNERKAR S., TRIPATHI A.K., RAMANI U., KORINGA P.G., RANK D.N.,
JOSHI C.G., KOTHARI R.K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Optimisation of synthetic medium composition for levorin biosynthesis by Streptomyces levoris 99/23 and investigation
of its accumulation dynamics using mathematical modelling methods
STANCHEV V.S., KOZHUHAROVA L.Y., ZHEKOVA B.Y., GOCHEV V.K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Chromate reduction by cell-free extract of Bacillus firmus KUCr1
SAU G.B., CHATTERJEE S., MUKHERJEE S.K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
Occurence and chracterization of Colletotrichum dematium (Fr.) grove
MACHOWICZ-STEFANIAK Z. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Cytotoxic activity of Serratia marcescens clinical isolates
KRZYMIÑSKA S., RACZKOWSKA M., KAZNOWSKI A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Antibiotic susceptibility and genotype patterns of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa
isolated from urinary tract infected patients
ABOU-DOBARA M.I., DEYAB M.A., ELSAWY E.M., MOHAMED H.H. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
SHORT COMMUNICATIONS
rDNA- based genotyping of clinical isolates of Candida albicans
NAWROT U., PAJ¥CZKOWSKA M., W£ODARCZYK K., MECLER I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
INSTRUCTIONS TO AUTHORS AND FULL TEXT ARTICLES (IN PDF FORM) AVAILABLE AT:
www.microbiology.pl/pjm
Polish Journal of Microbiology
2010, Vol. 59, No 3, 145–155
MINIREVIEW
Bacteriophage Receptors, Mechanisms of Phage Adsorption
and Penetration into Host Cell
D.V. RAKHUBA1, E.I. KOLOMIETS1, E. SZWAJCER DEY2 and G.I. NOVIK1*
1 Institute
of Microbiology, National Academy of Sciences of Belarus, Minsk, Belarus
University, Pure and Applied Biochemistry, Lund, Sweden
2 Lund
Received 4 April 2010, accepted 2 July 2010
Abstract
Bacteriophages are an attractive tool for application in the therapy of bacterial infections, for biological control of bacterial contamination
of foodstuffs in the alimentary industry, in plant protection, for control of water-borne pathogens, and control of environmental
microflora. This review is mainly focused on structures governing phage recognition of host cell and mechanisms of phage adsorption and
penetration into microbial cell.
K e y w o r d s: bacteriophage receptors, phage penetration mechanism
Introduction
Currently, the bacteriophage phenomenon may be
regarded from different viewpoints. On one hand,
bacterial viruses pose a grave challenge to industries
based on bacterial agents applied in dairy processing,
production of enzymes, antibiotics, solvents, insecticides, lactic and acetic acid, and various bacterial
preparations when massive concentrations of biomass at active growth phase create favorable conditions for propagation of phages often responsible for
lysis of industrial cultures. On the other hand, bacteriophages are objects that are attractive for application
in medicine and veterinary practice for therapy of
bacterial infections in humans and domestic animals.
Bacterial viruses may also be used for biological control of bacterial contamination of foodstuffs in alimentary industry, agriculture; for control of water-borne
pathogens, clinical pathogens causing aerogenic infections; control of environmental microflora, etc.
Solution of industrial bacteriophage problems and
search for practical virus application require fundamental studies to analyze interactions between bacteriophage and host cell and to elucidate correlations of
viral infection process in bacterial cell. Such interactions are rather complicated and do not always result
in cell lysis. Now phage-cell relations are considered
as process consisting of several sequential stages:
phage adsorption on host cell surface and penetration
of phage nucleic acid into cell, intracellular synthesis
of virus components and assembly of virions, lysis of
bacterial cell and phage release.
Adsorption is a key stage in virus recognition of
sensitive host cell, i.e. specificity of phage infection
is defined at this moment. Since bacteriophages, like
any other viruses are obligate intracellular parasites,
successful penetration into bacterial cell is an essential
condition for continuation of their life cycle. This review is mainly focused on structures governing phage
recognition of host cell and mechanisms of phage adsorption and penetration into microbial cell.
Bacteriophage receptors on cell surface
A specific bacteriophage strain is known to be able
to infect a narrow host range or a concrete microbial
species or strain. Such specificity in interaction of
phage with bacterial cell is determined by specificity
of adsorption, which in turn is dependent on the nature
and structural peculiarities of receptors on bacterial cell
surface (Braun and Hantke, 1997). In addition, a vital
role is attributed to receptor localization on cell surface, their amount and density at various cell wall sites.
* Corresponding author: G. Novik, Institute of Microbiology, National Academy of Sciences of Belarus, Kuprevich 2,
220 141 Minsk, Belarus; phone (+375-17) 2678620; e-mail: [email protected]
146
Rakhuba D.V. et al.
The nature of receptors contacting bacteriophages
is different for representatives of diverse taxonomic
groups and is largely defined by composition of host
cell wall and surface structures.
Receptors localized in cell wall of gram-negative
bacteria. The outer membrane of gram-negative bacteria differs in structure from the inner membrane and
from the plasma membrane of gram-positive microorganisms. One of the distinctive features is its high
permeability caused by increased levels of integral
proteins forming transport channels – up to 20 000 per
cell (Nikaido, 2003). Another distinction is the presence in external lipid layer of a unique glycolipid
– lipopolysaccharide (LPS) – typically exclusive for
gram-negative bacteria. Proteins localized in membrane and various LPS sites may serve as bacteriophage receptors. In many cases phages require molecules of both types for adsorption (Lindberg, 1973).
Protein receptors. Proteins of outer membrane
may be subdivided into 5 classes: 1) structural proteins interacting with peptidoglycan layer; 2) specific
and non-specific porins forming membrane channels;
3) enzymes; 4) substrate receptors with high affinity;
5) transport proteins responsible for secretion.
Among structural proteins serving as receptors for
virus adsorption, transmembrane protein OmpA was
characterized. This protein comprises 8 antiparallel
$-structures fixed inside membrane by non-covalent
link to peptidoglycan layer with the free C-terminal
vertex (Koebnik, 1999a; 1999b; Vogel and Jahnig,
1986). Mutants lacking the protein are distinguished
by spherical shape and labile outer membrane. It was
also shown that OmpA is involved in process of bacterial conjugation (Schweizer and Henning, 1977).
OmpA protein-LPS complex is capable to inhibit
phage Tulb (coliphage isolated from effluents). Binding of bacteriophage with protein-LPS complex occurs
reversibly, and precipitation of this complex with Mg+2
leads to irreversible phage attachment. Apart from protein, the LPS molecule is not able to inhibit phage particles (Datta et al., 1977). OmpA protein inhibits bacteriophage K3 in the solution, while mutants defective in
this protein are resistant to phage infection (Van Alpen
et al., 1977; Scurray et al., 1974). Such findings testify
to the receptor role of the protein with respect to Tulb
and K3, yet phage-recognising sites are located at different areas of the molecule. This assumption is supported by the existence of mutants producing sufficient
amount of OmpA protein and sensitive to phage K3 but
showing resistance to phage Tulb (Henning et al., 1978).
Porins were one of the first outer membrane proteins of gram-negative microorganisms to be characterized in detail (Nakae, 1976). These protein complexes are composed of 3 subunits forming the channel
in bacterial membrane. Major proteins of this type in
E. coli cells are OmpC and OmpF.
3
OmpC serves as a receptor for phages Hy2, ss4,
Tulb and T4 (Scurray et al., 1974; Yu and Mizushima,
1982). Phage T4 utilized the protein as a receptor
in combination with cell wall LPS. It was shown
in experiments with LPS and OmpC mutants that
absence of at least one receptor resulted in reduced
efficiency of infection, whereas loss of both receptors
induced bacteriophage resistance. Protein gp37 shaping tail fibers governs receptor recognition in phage T4
(Montag et al., 1990; Heller, 1990). The region is made
up by approximately 14 amino acid residues and contains a large amount of histidine residues, responsible
for OmpC recognition.
OmpF is a receptor for phage T2 (Riede et al.,
1985; Hantke, 1978). In contrast to T4 phage, the
receptor recognizing site is located within the hypervariable region of protein gp38 attached to terminal
part of protein gp37. Instead of hystidine bases, gp38
includes glycine sequences (up to 9 residues) at terminal and internal loci. Protein receptors in the cell
wall of Shigella and E. coli were revealed for bacteriophage T6 (Jesaitis and Goebel, 1952; Michael,
1968). Manning and Reeves demonstrated that E. coli
with tsx gene mutation displayed resistance to bacteriophage T6 infection and in subsequent papers they
isolated and purified protein Tsx (product of tsx gene)
controlling the transport of nucleotides and proved its
receptor function (Manning and Reeves, 1976; 1978).
Selective transport protein LamB is the receptor
for phage 8 (Randall-Hazelbauer and Schwartz, 1973).
Unlike non-selective porins OmpC and OmpF, this
protein forms a narrow channel specific for transport
of maltose and derived polymers, using aromatic positively charged aminoacid residues (Charbit et al.,
1998). Bacteriophage 8 recognizes LamB via protein
gpJ – the factor defining host range of this phage.
Among enzymes localized in the outer membrane
are proteases OmpT and OmpX which may serve as
receptors for T-like phages with host range mutations
M1 and Ox2, respectively (Hashemolhosseini et al.,
1994a; 1994 b). Proteins TonA (later renamed FhuA)
and TonB serve as receptors for phages T7, T5 and n80.
Receptors with high substrate affinity are components of active transport system. Their function is to
carry out solid binding of substances below demand
by passive transport system, namely vitamin B12 and
iron ions as chelating agents (Frost and Rosenberg,
1975; Hancock et al., 1976; Bassford et al., 1976).
Secretory transport proteins accomplish the function similar to substrate receptors, but in opposite
direction, i.e. they transport diverse compounds out of
the cell. So far phages using this protein type as receptors have not been detected.
Lipopolysaccharide receptors. In addition to proteins, LPS is another constituent of the outer membrane
in gram-negative bacteria serving as a receptor for
3
Receptors, adsorption and penetration of bacteriophage
bacteriophage adsorption. LPS is a complex polymer
made up of monosaccharides and fatty acids. Structurally, it incorporates 3 parts – lipid A, core and
O-chain (side chain, O-antigen). Lipid A usually is
a disaccharide composed of two D-glycosamine moieties linked by $-1,6-bond with attached fatty acids (up
to 8 residues) mediated by ester or amide group. Lipid
A performs the role of hydrophobic anchor fixing in
plasmatic membrane the whole construction bound
via short oligosaccharide core to O-chain consisting
of polymeric carbohydrate links (Wilkinson, 1996).
There are two types of LPS: Smooth (S) type is
characterized by typical LPS structure, i.e. comprising lipid A, core and side chain.
Rough (R) type lacks O-chain but contains lipid A
and the core.
Some bacteriophages might adsorb to both LPS
types. Phages specific to S-type LPS display an extremely narrow host range specificity determined by
large variability of O-antigen structure in bacteria of
different taxonomic groups. Bacteriophages recognizing R-type and vice versa show a broader host range
since the structure of LPS core is rather conservative in
various species and genera of gram-negative bacteria.
A common feature of bacteriophages fixing to LPS
O-chain is that their adsorption results in specific enzymatic cleavage of polysaccharide chain. g15 and P22
may be referred to such phages possesing endorhamnosidase activity and ability to lyse the bond
Rha-1 → 3-Gal in O-antigen of Salmonella anatum
and Salmonella typhimirium, respectively (Takeda and
Uetake, 1973; Kanegasaki and Wright, 1973; Iwashita
and Kanegasaki, 1973; 1976; Eriksson and Lindberg,
1977; Eriksson et al., 1979). Bacteriophage n1 (Reske
et al., 1973) infecting Salmonella johannesbury is
characterized by endo-1,3-N-galactoseaminidase activity (Chaby and Girard, 1980; Girard and Chaby, 1981).
Bacteriophage S8 adsorbed on the surface of E. coli
O8 shows endomannosidase activity, breaking down
Man-1 → 3-Man link between repeating oligosaccharides and releases prevailing levels of hexa- and
nonasaccharides (Reske et al., 1973; Prehm and Jann,
1976; Wallenfels and Jann, 1974). It was demonstrated for bacteriophage Sf6 isolated from strain
Salmonella flexneri serotype 3a that its adsorption is
associated with hydrolysis of Rha-1 → 3-Rha bond in
O-chain of LPS (Lindberg et al., 1978). Virus H-F6S
is able to bind to S. flexneri strains containing O-chain
in LPS. Mutant strains lacking O-antigen are resistant
to phage H-F6S but they are sensitive to other phages,
like T3, T4, T7, with the respective receptors lying in
the area of LPS core. It seems in wild-type strains
these areas are hidden by O-chain complicating access
for bacteriophages.
In common, bacterial viruses adsorbing to O-antigen chain of LPS in gram negative bacteria recognize it
147
via enzyme localized at the tail end, which upon recognition and attachment hydrolyzes one of the bonds in
polysaccharide chain of O-antigen. Besides, described
bacteriophages have a similar morphology – hexagonal head, short tail with base plate where spikes are
localized. According to Bradley classification they are
referred to group C (Bradley, 1967), and to podoviridae
family according to modern classification.
Position of a receptor in O-chain of S-type of LPS
is described for phage 2. This bacteriophage infecting
wild type strain of Pseudomonas aeruginosa B1 is
affiliated to group B according to Bradley classification and to siphoviridae family according to modern
classification because it has a long expanded tail
(Bartell et al., 1971). This phage displays depolymerase activity owing to the constituent enzyme. After
treatment of bacterial cells with purified enzyme isolated from virion, bacteria P. aerurinosa lose phage
sensitivity (Castillo and Bartell, 1976).
As mentioned above, structure of R-type LPS is
limited by lipid A and the core region. In some mutant strains the structure of LPS core could be incomplete which according to a series of reports may result
from disruption in core biosynthesis process occurring
at different stages. Such structural aberrations could
severely affect bacteriophage adsorption. For instance,
phage F0 lysing wild-type Salmonella strains containing LPS with complete core. N-acetylglucosamine
residue linked to the rest of the chain with "-1,2-bond
is located at its terminal position. Mutants lacking this
terminal glucosamine moiety are resistant to viral infection, and LPS isolated from such strains would not
inactivate bacteriophage F0 (Lindberg, 1967; Lindberg
and Hellerovist, 1971).
Phages MX174, S13 and 6SR also require full LPS
core for Salmonella and Shigella adsorption, with certain distinctions. Phage MX174 infects S. typhimirium
strains showing on outer membrane surface LPS with
complete core not protected by O-antigen. Mutants
lacking terminal N-acetylglucosamine are still able to
bind virus, although at lesser degree. On the other hand,
absence of terminal glucosamine residue does not influence adsorption of phage S13 (Jazwinski et al.,
1975). Whole core terminating in glucosamine is
essential for optimal adsorption of phage 6SR to cells
of S. typhimirium and S. flexneri. Yet, mutants of
S. typhimirium defective in core biosynthesis or mutants of S. flexneri containing LPS with disaccharide
composed of glucose and heptose moieties at its terminal position are also sensitive to phage infection,
but at a lower rate (Lindberg and Hellerovist, 1971).
The afore-mentioned phages behave in different
way with respect to E. coli. Bacteriophage MX174
lysing E. coli C loses ability to adsorb to LPS lacking
in its core terminal galactose residue (Feige and Stirm,
1976). Similarly, strain E. coli K12 sensitive to phage
148
Rakhuba D.V. et al.
6SR contains glucose moiety in the terminal position
of LPS core (Picken and Beacham, 1977).
It is well known that receptors for T-phages, specifically T3, T4 and T7 are components of R-type LPS
of Shigella and Esherichia (Jesaitis and Goebel, 1952;
Michael, 1968; Weidel, 1958). Phage T3 is adsorbed
on the surface of S. flexneri mutants harboring core
terminated with glucose linked to heptose by glycoside bond. The LPS isolated from these strains possesses the highest inactivating capacity towards this
virus. Phage T7 adsorbs best on S. flexneri mutants
with core terminated with galactose residue bound to
glucose. Mutant strains E. coli K12 with core ending
up in heptose and glucose are able to adsorb phages
T3 and T7 (Picken and Beacham, 1977). The highest
inactivating potential for phage T4 was displayed by
LPS isolated from S. flexneri possesing in the core
terminal disaccharide glucose-heptose. Mutants with
complete core are also sensitive to phage T4 infection but inactivating ability of their LPS is lower. In
E. coli B cells the optimal receptor proved to be LPS
containing Glu-1→3-Glu-1→3-Hep in core terminal
position (Prehm et al., 1976).
Summing up, structure of LPS core responsible
for recognition of the same bacteriophage may differ
in bacteria of various microbial species and genera
as demonstrated above by phages MX174, 6SR, T3,
T4 and T7. It appears that major role in the receptor formation is played by spatial configuration
around terminal glycosidic bond rather than terminal
residue in polysaccharide chain of the core (Feige and
Stirm, 1976).
Receptors localized in cell wall of gram-positive
bacteria. Cell wall of gram-positive bacteria significantly differs from the gram-negative species both in
structure and chemical composition. The main component is peptidoglycan making up from 40 to 90% of the
cell dry weight. Peptidoglycan is a heteropolymer composed of disaccharide monomer formed by N-acetylglucosamine and N-acetylmuramic acid. A tetrapeptide
most often having the following structure: L-alanine
– D-glutamic acid – L-diaminopimelic acid – D-alanine
is attached to a hydroxy group of N-acetylmuramic
acid. This tetrapeptide mediates covalent links between
peptidoglycan fibers so that cell wall represents a solid
cover adjacent to the cell plasma membrane.
Teichoic acids are the other vital constituents of
gram-positive microorganisms. They are water-soluble
polymers comprising glycerol or ribitol moieties linked
together by phosphodiester bond and traversing peptidoglycan layer in direction perpendicular to the surface of plasmatic membrane. Most teichoic acids contain large ratio of D-alanine bound to free hydroxy
groups, but other substitutes, like N-acetyl-D-glucosamine or D-glucose are found more often. Teichoic
acids constitute the bulk of bacterial surface antigens.
3
Examined bacteriophages specific to Staphylococcus aureus, namely phages 3C, 52A, 71, 77, 79 and
80 are irreversibly inactivated by a complex of peptidoglycan and teichoic acids supplemented in addition
by tetrapeptide attached to muramic acid. Reversible
adsorption may be achieved during phage binding
with teichoic acids connected with glycan fibers but
irreversible procedure requires presence of tetrapeptide in the complex. Presence of N-acetylglucosamine
in teichoic acid formula and O-acetyl groups in muramic acid residue is also essential for phage adsorption (Lindberg, 1973; Coyettl and Gheysen, 1968;
Chatterjee, 1969; Gheysen et al., 1968; Murayama
et al., 1968; Shaw and Chatterjee, 1971).
Microorganisms of the genus Bacillus have the
structure of peptidoglycan and teichoic acids similar
to that of S. aureus. The only distinction is that
N-acetylglucosamine as component of teichoic acids
is substituted for D-glucose (Jazwinski et al., 1975).
Due to this structural resemblance phages specific for
S. aureus may adsorb on the surface of B. subtilis
(Rakieten and Rakieten, 1937). D-glucose moiety
plays a key role for adsorption of bacteriophages specific for B. subtilis. Phages M1, M25, M29, SP3, SP10,
SP02 and µ were not able to adsorb on the surface
of B. subtilis mutants lacking D-glucose in teichoic
acid composition (Glacer et al., 1966; Hemphill and
Whiteley, 1975; Young, 1968; Lindberg, 1973). Yet,
some phages could infect bacterial cell without
glycosylated teichoic acids in case growth occurred
on the surface of solid nutrient media rather than in
submerged culture (Yasbin et al., 1976).
Protein GamR involved in adsorption of phage (
was identified in cell wall of B. anthracis. This protein is probably the component of cobalt transport system. B. cereus and B. thuringiensis also display on the
surface GamR-like proteins. Only B. cereus is sensitive to phage ( infection although electron microscope studies have shown adsorption of phage particles
to cells of both microbial species. It appears, missing
additional surface structures in B. thuringiensis cells
are indispensable for cell penetration and further
phage propagation (Davison et al., 2005).
Bacteriophages specific for Lactobacillus delbrueskii are inactivated by lipoteichoic acids isolated
from cell wall of this microbial species. Inactivation
degree depends on available D-alanine and L-glucose
residues bound to fee hydroxyl groups of teichoic
acids. An increase in D-alanine level results in reduced
inactivating ability of lipoteichoic acids and their preliminary incubation with glucose-specific lectin ConA
leads to complete inhibition of phage adsorption
(Raisanen et al., 2007).
Bacteriophages infecting Lactococcus lactis initially adsorb to polysaccharide cell wall. For some
phages this step is irreversible (Monteville et al., 1994;
3
Receptors, adsorption and penetration of bacteriophage
Schafer et al., 1991; Valyasevi et al., 1990; Valyasevi
et al., 1994). Rhamnose, glucose and galactose moieties, as a part of extracellular polysaccharides are
responsible for primary recognition and attachment of
phage virions. Phage eb7 is characterized by adsorption to glucosamine or galactosamine residues (Keogh
and Pettingill, 1983). Viruses of lactic acid streptococci belonging to group 2c and phage kh require
specific protein (a phage infection protein) for irreversible secondary binding with bacterial cell wall
(Monteville et al., 1994; Babu et al., 1995; Geller
et al., 1993; Valyasevi et al., 1991).
Receptors localized in capsular polysaccharides,
pili and flagella. Many bacteriophages are attracted
to bacterial pili, flagella, capsular and slime polysaccharides as receptors. Among viruses adsorbing to
flagella several agents have been reported including
phage P infecting representatives of Enterobacteriacae
family – Salmonella, Serratia, and E. coli, phage PBS7
attached to B. subtilis, B. pumilus, B. licheniformis,
phage PBP7 specific to B. pumilis, phage 7–7–1 infects
R. lupine (Shade et al., 1967; Lovett, 1972; Lotz et al.,
1977). The phages have the same mechanism of adsorption, where the virion is fixed to the distal part of
flagella via tail fibers. This adsorption stage is reversible; electron microscopic photos show that phage
attachment does not result in release of nucleic acid
from capside. Further on the virion moves closer to cell
surface ultimately binding irreversibly to the baseplate
of flagella.
Phages MAcM4 and MAcS2 infecting Asticcacaulis biprosthecum also specifically adsorb to flagella
via site connecting head and tail of the phage whereas
distal part of the tail remains free for adsorption to
the surface of bacterial cell (Pate et al., 1973). The
attached virion is able to move along flagella towards
cell and also may adsorb to the surface of neighbor cell.
Many bacteria have external protective layers in
the form of capsules or slime. Such layer may block
access of bacteriophage to receptor localized in the
cell wall or may be used for adsorption of phages,
particularly those which fail to attach to bacteria devoid of capsules (Chakrabarty et al., 1967; Park, 1956).
One of the bacteriophage receptors located in capsules of gram-negative bacteria is Vi-antigen typical for
representatives of Salmonella, Citrobacter and E. coli.
The polymer consists of residues of N-acetyl-Dgalactosaminuronic acid linked by "1,4-bond and partially O-acetylated (Luderitz et al., 1968). Studies on
interaction of phage II with isolated Vi-antigen demonstrated that virion adsorption was accompanied by
enzymatic cleavage of side acetyl groups while total
chain depolymerisation did not occur (Taylor, 1965;
1966). The enzyme catalytically governing this reaction is localized in phage tail. Deacetylated Vi-polysaccharide loses capacity of further phage binding, but
149
this property is recovered after reverse acetylation. It
should be noted that such interaction is reversible,
while components of bacterial cell wall are essential
for irreversible binding.
Adsorption of other phages to capsular polysaccharides is also associated with enzymatic activity but
in this case it is aimed at depolymerisation of main
chain. Enzymes displaying endoglucosidase activity
were characterized for phages of E. coli K29 and
phage of Klebsiella K11 (Stirm et al., 1971a; 1971b).
Virus K2 hydrolyzes capsule of Aerobacter aerogenesis
using glucane hydrolase (Yurewicz et al., 1971) splitting "1, 3-bond between galactose residues. A common feature of these phages is their similar virion
morphology and the interaction of phages with capsular polysaccharides is a reversible process. The capsule
acts as a receptor for initial phage attachment whereas
cell wall components are essential for irreversible
binding (Taylor, 1966; Stirm et al. 1971b).
Viruses of 2 types are present among bacteriophages that are able to adsorb to pili of RNA-containing viruses with isometric capsid and DNA-containing
viruses in the form of filaments. A peculiarity of such
phages is that they use as receptors only sex pili of
bacteria, able to adsorb several hundred phage virions.
Phages P17, M12, fr, Q$, f2, f4 infecting E. coli
are most thoroughly studied among RNA-containing
viruses. All above-mentioned phages composed of vast
amount of identical subunits are about 27 nm in diameter (Hohn and Hohn, 1970). The second capside component is protein A responsible for recognition and
adsorption of virion to pili (Roberts and Steitz, 1967).
This protein is available in virion as one copy and
upon RNA injection it penetrates into host cell with
nucleic acid (Steitz, 1968a; 1968b; Krahn et al., 1972).
DNA-containing filamentous phages recognizing
pili as receptors may be subdivided into 2 groups:
Ff and If phages adsorbing to terminal parts of F
and I pili, respectively (Meynell and Lawn, 1968;
Schlesinger, 1932). Unlike RNA-containing phages,
only few virions may adsorb to one pilus. Binding is
also mediated by protein A, similar to RNA phage
case (Meynell and Lawn, 1968).
Mechanisms of phage adsorption and penetration
Rate of adsorption is the value characteristic of each
phage-host pair and it may vary depending on concentration of phage/host. Since bacteriophages do not have
specific structures responsible for virion motion and,
consequently, they cannot move independently, the
adsorption process is the result of random phage-cell
collision described by active mass law (Schlesinger,
1932). It appears therefore that as concentration of virions and bacterial cells grows, the number of random
150
Rakhuba D.V. et al.
collisions tends to rise which, in turn, leads to higher
adsorption rate.
Rate of adsorption is also determined by a series
of diverse non-specific physical-chemical factors
(pH, temperature, presence in the media of certain
substances and ions) and depends on host physiological state and cultural conditions (Hershey et al.,
1994; Delbruck, 1940; Quiberoni and Reinheimer,
1998; Sillankorva et al., 2004).
Virion adsorption on host cell surface is usually
illustrated as the process consisting of 2 stages: reversible and irreversible binding. It should be noted
that molecular mechanisms of interaction at both
stages of adsorption are specific for different phagehost systems and they may vary significantly in representatives of diverse taxonomic groups.
As a rule, penetration of nucleic acid takes place
after irreversible adsorption phase. Mechanisms of
this process are specific for each phage, or phage
group. Electrochemical membrane potential, ATP
molecules, enzymatic splitting of peptidoglycan layer
or all three factors may be vital for penetration of genetic material inside the bacterial cell.
Processes of adsorption and phage penetration into
cells are investigated in most detail for viruses of
E. coli, namely T4, T5, T7 (Letellier et al., 2004).
Some findings are available on processes of adsorption
and penetration of viruses incorporating plasmatic
membrane. Below these mechanisms will be considered separately for each virus.
T4-like phages. Initial stage of adsorption for
T4-like bacteriophages consists in reversible attachment of long tail fibers to specific receptors on the
surface of outer membrane. It is necessary for successful infection that 3 or more of tail fibers could
adsorb to cell surface since they play a critical role in
triggering conformational changes of phage tail essential for DNA penetration into the cell (Crawford and
Goldberg, 1977; 1980; Arscott and Goldberg, 1976).
After phage gets attached to the cell via long fibers,
baseplate changes its shape and as a result it takes
stellar conformation. Finally 6 short fibers are generated and they irreversibly adsorb to heptose moiety in
LPS core (Riede et al., 1985, Montag et al., 1987).
Conformational alteration of baseplate simultaneously
launches contraction of tail sheaths so that inner hollow tube punctures bacterial outer membrane (Moody,
1973). To facilitate penetration through peptidoglycan layer, enzyme lysozyme – an integral part of baseplate protein gp5 is localized at the end of the tube.
X-ray spatial analysis of the complex has revealed
that domain responsible for peptidoglycan degradation is located at C-terminal part of spike-form structure (Kanamaru et al., 2002). Contact of this site with
phosphatidylglycerol of the inner membrane is a signal
for DNA transport along tail tube and its introduction
3
into the cell. Specific mechanism of DNA penetration
via inner membrane remains to be elucidated, yet it
is clear that phage tail does not penetrate through
inner membrane and the process requires electrochemical potential on the inner membrane (Labedan
and Goldberg, 1979).
Bacteriophage T7. Phage T7 infection results in
restructuring of tail proteins making up a cylinder
shape inside phage head. This structure consists of
3 protein gp16 copies, 12 protein gp15 copies and
18 protein gp 14 copies. In addition, phage head contains 2 other proteins playing a key role in DNA transport into the cell – gp13 and gp7.3. DNA molecule is
spiraled onto the cylinder formed within the capsid.
Initially, phage T7 interacts with bacterial LPSs via
tail fibers. As soon as such contact occurs, the signal
triggering irreversible virion binding is transmitted
into phage capsid. Phage tail, tail fibers and protein
gp13 are involved in signal transfer. Irreversible binding is associated with degradation of proteins gp13
and gp7.3 while proteins gp14–16 pass through phage
tail channel and shape the pathway across bacterial
cell wall (Molineux, 2001; 2005). N-terminal part of
gp16 is homologous to bacterial lytic enzyme transglycosylase making a major contribution in the restructuring of the peptidoglycan layer. Perhaps this domain
is responsible for penetration of formed tubular structure through peptidoglycan to the inner membrane.
DNA transport via the channel slows down when
the first 850 base pairs get into the cell. The reason is
protein gp16 serves as a special clip retarding rate
of DNA penetration. The essence of this mechanism is
that partial DNA uptake initiates transcription process
and produces inhibitor of cell restrictases. Slow penetration of nucleic acid allows to synthesize this inhibiting factor earlier than DNA sites sensitive to restrictases appear inside the cell (Molineux, 2001; 2005).
T5 and similar phages. Bacteriophage T5 includes hexagonal head 90 nm in size and long flexible tail around 200 nm. Protein FhuA localized in
cellular outer membrane and engaged in transport
of iron into the cell acts as a receptor for this phage.
Adsorption to such protein is energy-sparing and
irreversible, leading to DNA release in absence of other
factors (Letellier et al., 2004).
It was shown that besides irreversible adsorption,
T5 is able to bind reversibly to O-antigen of bacterial
LPS (Heller and Braun, 1979; 1982). Irreversible
binding accelerates DNA introduction into host cell
but it is not a crucial factor in adsorption process. This
conclusion was made after it was proven that loss of
tail fibers by the phage and lack of LPS O-chain in
bacteria would not affect plating efficiency.
The precise mechanism of DNA cellular uptake
has not been established so far. It is known that injection of genetic material proceeds in 2 stages. Introduc-
3
Receptors, adsorption and penetration of bacteriophage
tion of 8% DNA into cytoplasm causes a pause lasting
4 minutes (Lanni, 1965; 1968). Entered viral DNA
controls synthesis of proteins responsible for degradation of bacterial DNA and switching off its transcription. Later DNA transport is resumed and the rest
of nucleic acid is transferred inside bacterial cell.
Phages T1 and n80 use the same transport protein
FhuA as a receptor, although adsorption to it requires
energy. Electrochemical proton gradient generated on
inner membrane of bacterial cell by electron transport
chain is applied as energy source. Electrochemical potential is transmitted to outer membrane via mediation of protein TonB. Its N-terminal part is anchored
to cell inner membrane, while C-terminal interacts
with FhuA receptor. Specific mechanism of energy
transfer and mechanism of DNA transport into bacterial cell remains to be decoded.
Bacteriophages incorporating plasmatic membrane. Bacterial viruses structurally comprising lipid
bilayer attract vivid interest due to large diversity of
mechanisms engaged in viral infection of host cell. For
instance, morphologically identical DNA-containing
phages PRD1 and PM2 use different receptors for adsorption, have distinct cell penetration mechanisms and
infect different hosts. Bacteriophage PRD1 is characterized by a relatively broad host range, including
E. coli, P. aeruginosa, S. enteric, but it can infect only
strains carrying conjugative plasmids of N, P or W type
(Olsen et al., 1974). These plasmids encode bacteriophage receptor (Lyra et al., 1991). Capsid in the
form of an icosahedron is constituted from 24 copies
of protein P3, and each vertex is crowned with spikes
consisting of proteins P2, P5 and P31 (Benson et al.,
1999; Butcher et al., 1995; Mindich et al., 1982; Grahn
et al., 1999; Rydman et al., 1999). Membrane vesicle
surrounding double-stranded DNA is inside the capsid.
Protein content in the membrane is approximately 50%.
At the first stage of adsorption the phage is reversibly bound to cell receptor via protein P2 and as a result spiky protein complex (P2, P5, P31) and a part of
capsid proteins (P3) are released. Such modifications
produce a hole in capsid envelope (Rydman et al.,
1999). Further on phage membrane within the capsid
is subjected to structural regrouping, yielding tubular
tail penetrating into the bacterial cell via outer membrane and peptidoglycan layer (Lundstrom et al., 1979;
Bamford and Mindich, 1982). Two proteins possessing
lytic activity – P7 and P15 are localized in the newly
generated membrane tube. These proteins acting concertedly break down peptidoglycan at the penetration
site, generating small holes (Rydman and Bamford,
2000). Reaching internal membrane, bacteriophagederived tube fuses with it, releasing DNA into cytoplasm. The process is accompanied by massive extracellular secretion of potassium ions and ATP molecules
(Daugelavicius et al., 1997).
151
Phage PM2 also includes intracapsid membrane
vesicle surrounding double-stranded DNA molecule
(Espejo and Canelo, 1968). Penetration mechanism of
genetic material in host cell is not thoroughly investigated but available data indicate that it differs significantly from that in phage PRD1. Adsorption to host
cell surface is followed by capsid dissociation into
protein constituents. Increased membrane permeability
for lipophylic molecules of gramicidine B is also observed, pointing to potential fusion with bacteriophage
membrane (Kivela et al., 2004). Protein P7 possessing
lytic activity and probably playing an important role
in the process of penetration through peptidoglycan
layer was identified in membrane vesicle (Kivela et al.,
2004). Penetration of genetic material inside the cell
is associated with depolymerisation of the microbial
inner membrane.
RNA-containing bacteriophage n6 entering P. syringae cell, apart from nucleic acid, should inject
RNA-dependent-RNA-polymerase because the host
cell does not contain enzymes able to transcribe viral
RNA. Structural peculiarity of this phage is the
presence of outer lipid-protein envelope surrounding
a capsid with confined complex – RNA plus RNApolymerase (Bamford et al., 1976; Butcher et al.,
1997; Kenney et al., 1992; Vidaver et al., 1973;
Daugelavicius et al., 2005). Main receptors for phage
n6 are type IV pili (Bamford et al., 1976; Vidaver
et al., 1973) where phage is attached via protein P3
(Daugelavicius et al., 2005; Romantschuk and Bamford, 1985). Integral protein P6 localized in plasmatic
membrane of the virion initiates fusion of host outer
membrane and phage lipid envelope (Daugelavicius
et al., 2005; Bamford et al., 1987). As a result of such
membrane integration the virus capsid with contained
nucleic acid floods into the periplasmic space. Endopeptidase P5 localized in the capsid envelope splits
the peptidoglycan layer at the point of attack and the
virus nucleocapsid reaches the internal membrane of
the bacteria, (Daugelavicius et al., 2005., Caldentey
and Bamford, 1992; Mindich and Lehman, 1979).
According to electron microscopy observations, the
final stage of virus penetration into bacterial cell envisages generation of membrane vesicle incorporating phage nucleocapsid (Peisajovich and Shai, 2002).
The process is similar to viral endocytosis in humans
and animals (Smith and Helenius. 2004). Mechanisms
of disclosing vesicle in host cytoplasm and virion
decoating are not well established to date.
DNA-containing phage Bam35 infects cells of
gram-positive bacteria B. thuringiensis and contains
plasmatic membrane located within the capsid. The
mechanism of phage penetration into the cell is not
fully investigated. It is known that N-acetylmuramic
acid residue – a cell wall component – serves as
a receptor. Penetration through the peptidoglycan layer
152
Rakhuba D.V. et al.
is related to enzymes gp26 and gp30 localized in the
capsid envelope. The transport of genetic material
across the plasmatic membrane depends on presence
of bivalent cations in the media, whereas phage adsorption and degradation of peptidoglycan are not dependent (Gaidelyte et al., 2006).
Conclusions
The process of bacteriophage adsorption to a receptor on cell surface is the first stage in virus-host interaction. Adsorption phase defines phage-host specificity and mechanisms governing resistance of bacteria
to virus infection. The nature of receptor, aspects of
its chemical composition and spatial configuration,
structure of viral receptor-binding protein and specific
interaction mechanisms – all these factors play a key
role in shaping stable bacteriophage-host population.
In early studies on phage-host interactions it was
assumed to regard processes of adsorption and penetration of nucleic acid into bacterial cell separately from
each other as different stages of a virus life cycle. The
massive amount of data collected so far evidences a deep
correlation between virus adsorption and penetration
into bacterial cell. Irreversible adsorption stage virtually always initiates penetration of genetic material
inside host cell. It appears desirable therefore to consider the first phase of interaction between virus and
bacterial cell as a complex process comprising adsorption, structural alterations of virus and host cell wall
and transport of nucleic acid into the cell.
It should be stated that analysis of literature findings indicates a large diversity of bacteriophage-host
populations with respect to nature and structure of the
receptor, virus antireceptor and molecular mechanisms
of virion-cell interactions. It supports the conclusion
that investigation of structural-functional aspects and
interaction mechanisms for specific phage-host population is indispensable and doubts feasibility of creating general model embracing the whole spectrum of
existing bacterial viruses and their hosts.
Acknowledgments
This work was supported in part by the grant from the
Projects and Network Co-operation within the Visby Programmed
University Co-operation with Central-Eastern Europe (Si reference number: Dnr 01147/2007).
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Polish Journal of Microbiology
2010, Vol. 59, No 3, 157–160
ORIGINAL PAPER
Interactions between Borrelia burgdorferi and Mouse Fibroblasts
TOMASZ CHMIELEWSKI* and STANIS£AWA TYLEWSKA-WIERZBANOWSKA
Laboratory of Rickettsiae, Chlamydiae and Spirochetes, National Institute of Public Health
– National Institute of Hygiene, Warsaw, Poland
Received 18 June 2010, revised 15 July 2010, accepted 17 July 2010
Abstract
Borrelia burgdorferi spirochetes are an infectious agent of Lyme borreliosis. The aim of our studies was to investigate the fate of engulfed
B. burgdorferi cells in L-929 mouse fibroblasts and to observe development of intracellular infection in vitro after 2 and 48 h. Electron
microscopic studies reveal consecutive stages of B. burgdorferi spirochetes penetration to mouse fibroblasts in vitro. It has been observed,
as a first step attachment and engulfment of spirochetes followed by formation of vacuoles. After 48 hours of infection, vacuoles of
fibroblastic cells have been seen full of B. burgdorferi bacteria and latter they have been released from infected cells to extracellular space.
It can be the evidence that B. burgdorferi multiply intracellulary.
K e y w o r d s: Borrelia burgdorferi, fibroblasts, interaction
Introduction
Borrelia burgdorferi spirochetes are an infectious
agent of Lyme disease, also known as borreliosis,
which is the most common tick-borne disease in the
northern hemisphere. Early manifestations of infection
include fever, headache, fatigue, and a characteristic
skin rash called erythema migrans. Untreated, it can
cause late symptoms involving tissue of the joints,
heart, and nervous system (Stanek et al., 1996).
Microscopic studies indicate that the bacteria can
bind to the cell surface and enter the cytoplasm directly
after inducing local engulfment and fragmentation of
the plasma membrane. Several reports have described
interactions between B. burgdorferi bacteria and different host cells. It has been shown that the spirochetes can enter mammalian immune cells and other
cells as well as tick tissue. This probably allows the
pathogen to survive in host tissues, to infect them and
to escape the host defense (Hu et al., Linder et al.,
2001, Sigal, 1997, Szczepanski et al., 1990, Thomas
et al., 1989). However, there is no information on the
fate of B. burgdorferi spirochetes inside eukaryotic
cells and the way they leave the host cells.
Various modes of bacterial entry into the host cell
have been described as an essential pathogenic factors.
Legionella pneumophila uptake is completed by a process termed “bacteriopexis”, followed by engulfment
of the organisms with microvilli in association with
intracellular cytoplasmic filaments (Oldcham et al.,
1985). Another pathogen, Rickettsia prowazekii, can
enter endothelial cells via induced phagocytosis and
it is then released into the cell cytoplasm by disruption
of the phagosomal membrane (Walker, 1984). Transmission electron microscopy has shown that viable or
killed Candida organisms were attached to endothelial
cells, then enveloped by cell membrane and incorporated into the endothelial cells within phagosomes
(Rotrosen et al., 1985).
The ability of Borrelia burgdorferi to attach to and
invade human fibroblasts was investigated by confocal
and scanning electron microscopy. Scanning electron
microscopy has revealed that B. burgdorferi are tightly
attached to fibroblast monolayers after 24–48 h. Spirochetes were observed in the perinuclear region within
human fibroblasts by laser scanning confocal microscopy (Klempner et al., 1993).
The aim of our studies was to investigate the fate
of engulfed B. burgdorferi cells in mouse fibroblasts and to observe the development of intracellular infection in vitro in electron and fluorescence
microscopy.
* Corresponding author: T. Chmielewski, Laboratory of Rickettsiae, Chlamydiae and Spirochetes, National Institute of Public
Health – National Institute of Hygiene; 24 Chocimska Street, 00-791 Warsaw;, Poland phone/fax (+48) 22 4521250; e-mail:
[email protected]
158
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Chmielewski T. and Tylewska-Wierzbanowska S.
Experimental
Material and Methods
Borrelia afzelii VS461 strain (ATCC 51567) was
grown in BSK-H medium Complete (Sigma Aldrich,
St. Louis, USA) supplemented with 6% of rabbit
serum for 7 days at 35°C in 5% CO2 atmosphere. The
number of bacteria per 1 ml was counted in a Thoma
counting chamber.
Line L-929 (ATCC CCL-1, USA) were propagated
in Eagle’s minimum essential medium (MEM) with
L-glutamine and NaHCO3 (Biomed, Lublin, Poland),
supplemented with 4% of fetal calf serum (ATCC,
USA) at 37°C in 5% CO2 for 2 days. Cells were grown
in shell-vials on glass coverslips inside tubes with
screw caps (Sterilin, United Kingdom) until a confluent
monolayer was obtained.
Bottles containing cell line monolayer were inoculated with spirochetes culture containing 108 bacteria
per 1 ml. L929 cells infected with B. burgdorferi (initial density 106 organisms/ml medium) were incubated
for 2 and 48 hours at 35°C in 5% CO2 atmosphere.
Infected mouse fibroblasts were fixed with acetone,
washed three times with PBS and incubated 30 minutes with anti-B. burgdorferi human immune serum
at 37°C. Next they were washed three times with PBS
followed by incubation with rabbit anti-human immunoglobulins conjugated with FITC (DAKO, Denmark).
Immunofluorescence was observed in the fluorescence
microscope Eclipse E 400 (NIKON, Japan) at 500X
magnification.
All specimens were prepared according to standard
technics (Glauert, 1975). The cultures were washed
twice with PBS, fixed overnight with 2.5% glutaraldehyde. On the next day the cells were centrifuged at
750 g at 4°C and the pellet was washed with 2 ml
0.2 M sodium cacodylate buffer (pH 7.4) and re-centrifuged as above. After three washes, the cells were
Fig. 1. Attachment of spirochetes to the cytoplasmic membrane
of L-929 cell (X 30 000).
fixed for 2 hours in 1% OsO4 in 0.1 M sodium cacodylate buffer. The cells were then washed three times
in 0.1 M sodium cacodylate buffer and stained for
30 minutes by being resuspending in 1% aqeous uranyl acetate (Roth, Karlsruhe, Germany) solution. For
embedding, fibroblasts were centrifuged and then
dehydrated through a wash series in methyl alcohol
solutions (from 25% to 100%) and embedded in epoxy
resin and incubated overnight at 65°C. Transverse
thin sections were cut and transferred to copper mesh
300 grids (Polysciences, St. Goar, Germany), stained
with lead citrate and uranyl acetate and dried. The cells
were observed in a JOEL 100C electron microscope
(Japan) at magnifications from ×6000 to ×35000.
Results
Various stages of B. burgdorferi spirochetes infection in L-929 fibroblasts were observed. After two
hours of incubation spirochetes were seen outside of
the host cells. Their position suggested that most of
them were motile. The first observed interaction step
was attachment of the bacteria to the surface of the
fibroblasts. In fluorescence microscopy it has been
seen as adhesion to the cell surface. After two hours
of incubation the spirochetes were bound and entered
the mammalian cells. Most of them were bound
apically. This contact triggered the engulfment of the
bacteria in the cytoplasmatic host membrane (Fig. 1,
Fig. 2). This process initiated the cellular uptake of
single bacteria into phagocytic-like vacuoles. Within
two hours of incubation such bacterial cell, surrounded
by host cell membrane, were seen inside the fibroblast (Fig. 3). Some bacteria entered fibroblast cells
in a different way. Fibroblast pseudopods bent around
single spirochetes in the intercellular spaces. The
pseudopods had a characteristic hook-like form, turned
back to the fibroblasts membrane and slided along the
Fig. 2. Cell membrane penetration (X 30 000).
3
Interactions between Borrelia burgdorferi and mouse fibroblasts
159
Fig. 6. Translocation of spirochetes to the intracellular space
(X 6 700).
Fig. 3. Single B. burgdorferi bacteria in vacuole
in L-929 cytoplasm (X 33 000).
were located in the peripheral part of the host cell adjacent to the cell membrane. Strong immunofluorescence
of vacuoles and bacteria was observed in fluorescence
microscopy. The vacuoles contained 15 to 20 bacterial
cells as it has been seen on a cross sections. The number of spirochetes in the host vacuoles observed after
two days of infection indicated that B. burgdorferi
multiplied inside the fibroblasts (Fig. 5). Some of the
vacuoles were disrupted and B. burgdorferi spirochetes were released to the extracellular space (Fig. 6).
Discussion
Fig. 4. Endocytosis of B. bugdorferi spirochetes (X 27 000).
cell membrane. The observed manner of penetration
resembled endocytosis however characteristic for coiling phagocytosis long appendages wrapping the spirochetes were also observed (Fig. 4).
On the second day of infection the fibroblasts were
seen to be vacuolized. Many vacuoles with bacteria
Fig. 5. Multiplication of spirochetes within vacuoles (X4 000).
Electron microscopic studies revealed the consecutive stages of B. burgdorferi spirochetes penetration
into mouse fibroblasts in vitro. The first observed step
was the attachment and engulfment of the spirochetes,
followed by the formation of vacuoles and multiplication of bacteria inside vacuoles followed by release
from the infected cells to the extracellular space. After
48 hours of infection, vacuoles of fibroblastic cells
containing dozens of B. burgdorferi bacteria were seen.
This can be taken as evidence that B. burgdorferi multiply intracellularly similarly to Legionella pneumophila, Coxiella burnetii and other obligatory intracellular parasites (Baca et al., 1983, Oldcham et al.,
1985, Walker et al., 1984, Rotrosen et al., 1985).
Studies by Comstock et al., (1989) with electron
microscopy revealed that borreliae entered the endothelial cells and suggested that the organisms penetrated the host monolayers primarily by passing
through them. Attachment of spirochetes is time and
temperature dependent and pretreatment with heat, immune human serum or monoclonal antibodies to OspB
reduce the binding to the endothelial cells (Thomas
et al., 1989). Examination of spirochete-endothelial
interactions demonstrated the presence of spirochetes
in the intercellular junctions between endothelial cells
as well as beneath the monolayers. Scanning electron
microscopy identified a mechanism of transendothelial
160
Chmielewski T. and Tylewska-Wierzbanowska S.
migration whereby spirochetes pass between cells into
the amniotic membrane at areas where subendothelium
is exposed (Szczepanski et al., 1990).
Other study with Vero cells revealed that essential
for the attachment process is metabolic activities of the
spirochaete, not viability (ability to grow) (Hechemy
et al., 1992). After entry of untreated B. burgdorferi,
most of the spirochaetes were either free in the cytoplasm or tightly bound to the host membrane. In contrast, heat treated spirochaetes remained bound to host
membrane in large phagosome-like vesicles (Comstock
et al., 1989).
It seems that several eukaryotic cells provide
B. burgdorferi spirochetes with a protective environment contributing to their long-term survival (Peters
et al., 1997, Rittig et al., 1998, Rittig et al., 1992).
B. burgdorferi have been protected in fibroblasts for
at least 14 days of exposure to ceftriaxone In the
absence of fibroblasts, organisms did not survive.
They were not protected from ceftriaxone by glutaraldehyde-fixed fibroblasts or fibroblast lysate, suggesting that a living cell was required. The ability of the
organism to survive in the presence of fibroblasts was
not related to its infectivity (Georgilis et al., 1992).
Mouse keratinocytes, HEp-2 cells, and Vero cells
showed a similar protective effect. Doxycycline or
erythromycin were more effective in killing B. burgdorferi when they were grown in the presence of
eukaryotic cells (Brouqui et al., 1996). Our findings
show that in fibroblasts could occurs process of spirochetes multiplication.
Difficulties with the isolation of B. burgdorferi from
clinical material when cultured on artificial media also
indicate that spirochetes are very fastidious bacteria,
which require the presence of certain substances in
the host cells for their growth. These observations
allowed us to isolate several strains by inoculating cerebrospinal fluids, synovial fluids and blood of Lyme
borreliosis patients into cell line culture (Chmielewski
et al., 2003, Tylewska-Wierzbanowska et al., 1997).
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Polish Journal of Microbiology
2010, Vol. 59, No 3, 161–165
ORIGINAL PAPER
Clonal Analysis of Staphylococcus aureus Strains Isolated
in Obstetric-Gynaecological Hospital
EWA SZCZUKA1, ANNA SZUMA£A-K¥KOL2, ANNA SIUDA1 and ADAM KAZNOWSKI1*
1 Department
of Microbiology, Institute of Experimental Biology, Faculty of Biology, Adam Mickiewicz University,
Umultowska 89, 61-614 Poznañ, Poland
2 Hospital Laboratories, Obstetric-Gynaecological Clinical Hospital, Polna 33, 60-535 Poznañ, Poland
Received 1 December 2009, revised 16 June 2010, accepted 17 June 2010
Abstract
Epidemiological studies were carried out on 135 isolates of Staphylococcus aureus strains originating from medical staff, patients, and
hospital environment. Restriction fragment length polymorphism (RFLP) analysis revealed genetic diversity of S. aureus isolates. Some
clones were transmitted among nurses, doctors and patients. Our studies also demonstrate contamination of the hospital environment with
S. aureus strains and there is a possibility that the patients acquire staphylococci from the environment. Moreover, we found that many
medical staff workers were colonized with S. aureus and the transmission of these strains to patients is possible.
K e y w o r d s: Staphylococcus aureus, epidemiological studies, RFLP analysis
Introduction
Staphylococcus aureus has been recognised as
an important pathogen causing many infections such
as septicemia, pneumonia, wound infections, septic
arthritis, osteomyelitis and postsurgical toxic shock
syndrome (Kloos and Bannerman, 1999). On the
other hand, many healthy people are persistently or
intermittently colonized with S. aureus at their anterior nares. Approximately 20% of individuals almost
always carry one type of strain, 60% harbor S. aureus
intermittently. About 20% of people almost never
carry S. aureus. Colonization of human noses by
S. aureus appears to play a role in the epidemiology
and pathogenesis of infection (Kluytmans et al., 1997).
S. aureus is one of leading agents of nosocomial infections therefore for public health epidemiologists
and clinicians involved in patient management of prime
importance is understanding the dynamics of the
spread and transmission the of bacteria within a hospital, this being crucial for their control and eradication. Molecular typing approaches have been used
to a great advantage in identifying and monitoring
the local and international spread of S. aureus strains
(Štìpán et al., 2004).
The aim of the present study was to evaluate the
clonal composition of Staphylococcus aureus strains
isolated from specimens taken from patients, medical
staff and hospital environment at the ObstetricGynaecological Clinic Hospital in Poznañ, Poland.
Moreover, we wanted to elucidate the spread of clones
in different departments and the possible transmission
routes of these clones.
Experimental
Material and Methods
Bacterial strains. S. aureus strains were isolated
from clinical specimens obtained from neonates and
adult patients treated in different medical units in
a hospital in Poznañ. Several members of the healthcare
staff were screened for S. aureus nasal, throat and
hand carriage. Also specimens from medical equipment and hospital environment were taken. All strains
were identified as S. aureus by analysis of cell morphology, Gram stain, and catalase production, using
the latex coagulase test and the ID 32 Staph Kit
(bioMérieux, France). Methicillin susceptibility test
* Corresponding author: A. Kaznowski, Department of Microbiology, Faculty of Biology, A. Mickiewicz University,
ul. Umultowska 89, 61-614 Poznañ, Poland; phone (+48) 61 529 5937; fax (+48) 61 829 5590; e-mail: [email protected]
162
Szczuka E. et al.
3
Fig. 1. Dendrogram showing
genetic relatedness
of 135 strains of S. aureus
determined by analysis
of RFLP fingerprint patterns
using Dice similarity coefficient
and UPGMA cluster method.
3
163
Clonal analysis of S. aureus
was determined by disc diffusion method and the
results were interpreted in accordance to the criteria
of the Clinical Laboratory Standards. The presence of
the mecA gene was determined by PCR as described
previously (Geha et al., 1994).
RFLP analysis. Chromosomal DNA from the
S. aureus strains were extracted according to methods
described by Pitcher et al. (1989). A PCR was applied
to simultaneously amplified part of the hypervariable
region (HVR), a part of spa gene and a part of the coa
gene based on primers established by Wichelhaus
et al., 2001. The PCR product was incubated overnight with 10 units of HaeII restriction enzyme (MBI
Fermentas) at 37°C. The resulting fragments were
separated in 1.5% agarose gel. The DNA in gels were
documented with V.99 Bio-Print system (Vilber
Lourmat, Torcy, France). A computer analysis was
carried out using GelCompar II (version 3.0; Applied
Maths, Kortrijk, Belgium) software. Similarity between
fingerprints was calculated with the Dice coefficient.
Cluster analysis was performed using the unweighted
pair-group method with average linkages (UPGMA).
Results and Discussion
All strains included in this study were methicillin/
oxacillin sensitive phenotypically and did not harbour
the mecA gene. Our results demonstrated that many
clones of S. aureus were coexisting in one hospital.
This conclusion is based upon the results generated
by clonal analysis of 135 strains of S. aureus (Fig. 1).
We revealed 25 clusters at the 90% similarity level
(Table I). Strains within these clusters were considered to be genetically related. We identified three
major clones (cluster 8, 10, and 12), which included
28% strains of S. aureus. As shown in the dendrogram, there are five smaller clusters consisting from
six to four strains. In addition, we found a considerable number of minor clusters, each harboring three
or two strains. Minor clusters reached 36 strains
(29%). Moreover, we identified 32 single strains with
unique genotypes. The fact, that 25 clusters were
identified next to 32 unique genotypes indicating
a large genetic diversity among isolates of S. aureus
obtained from the hospital. Similar result was obtained
previously by Van Dijk et al., 2002, who described
genetic diversity among S. aureus isolates from a Dutch
Teaching Hospital.
Although S. aureus has been described as the normal flora of nasal carriage, several epidemiological
studies indicated that nasal carriage have increased
risk for staphylococcal infections especially in specific
group of patients (Archer and Climo 2001; KooistraSmid et al., 2004; Melless et al., 2004). Staphylococcus sp. infections are most commonly acquired from
Table I
Results of S. aureus clinical isolate typing
by using RFLP analysis
CluStrain No
ster
1 MPU S 68, 69
2 MPU S 43
MPU S 76
3 MPU S 62, 136
4 MPU S 8, 9
MPU S 52, 58, 56
MPU S 158
5 MPU S 25
MPU S 26
6 MPU S 55, 61
7 MPU S 73, 128
MPU S 114, 151
MPU S 129
8 MPU S 10, 126, 133,
152, 155
MPU S 27, 41
MPU S 118, 156, 119,
145, 148
MPU S 153, 160
MPU S 154
MPU S 157
MPU S 159
9 MPU S 13, 54
MPU S 131
10 MPU S 33, 39, 42, 83, 99
MPU S 35, 84
MPU S 37, 38
MPU S 40
MPU S 123
11 MPU S 106
MPU S 107
MPU S 110
12 MPU S 18, 48, 108
MPU S 34, 53
MPU S 60
MPU S 109
MPU S 111
13 MPU S 67, 91
14 MPU S 29
MPU S 121
MPU S 122, 124
15 MPU S 36
MPU S 46, 49
16 MPU S 77, 142
MPU S 132, 135
MPU S 134
MPU S 137
17 MPU S 31
MPU S 32
18 MPU S 94
MPU S 98
MPU S 113
19 MPU S 70, 72, 130
MPU S 79
MPU S 93
MPU S 120
Source of isolation
nasal swab of medical staff
clothes of medical staff
throat swab of medical staff
throat swab of medical staff
abscess of neonates
nasal swab of medical staff
hand basin
throat swab of medical staff
hands of medical staff
nasal swab of medical staff
throat swab of medical staff
wound of patient
patient’s bed
throat swab of medical staff
clothes of medical staff
nasal swab of medical staff
patient’s bed
hand basin
vagina of patient
throat swab of neonate
throat swab of medical staff
skin of neonate
clothes of medical staff
nasal swab of medical staff
hands of medical staff
skin of neonate
scale in delivery room
hand basin
abscess of patient
clothes of medical staff
nasal swab of medical staff
throat swab of medical staff
abscess of neonate
vagina of patient
hands of medical staff
patient’s bed
throat swab of medical staff
hand basin
patient’s bed
clothes of medical staff
skin of neonate
hand basin
patient’s bed
catheter of neonate
nasal swab of medical staff
nasal swab of medical staff
hands of medical staff
hand basin
patient’s bed
vagina of patient
nasal swabs of medical staff
hands of medical staff
patient’s bed
scale
164
3
Szczuka E. et al.
Table I continued
CluStrain No
ster
20 MPU S 86, 87
MPU S 89
21 MPU S 85, 92
22 MPU S 14
MPU S 19
23 MPU S 146
MPU S 150
24 MPU S 22, 80
25 MPU S 20
MPU S 23
Source of isolation
medical equipment
nasal swab of medical staff
nasal swabs of medical staff
skin of neonate
hands of medical staff
throat swab of medical staff
wound of neonate
nasal swab of medical staff
nasal swab of medical staff
conjunctive of neonate
patients’ own flora, however patients may become infected from other healthy carriers. It is worthy to note
that S. aureus carriers can contaminate their clothes
and their surroundings through air, dust etc. It is well
known that decolonization may reduce the risk of
S. aureus infections in carries and prevent transmission to other patients. Recently, Gilpin et al. (2010)
indicated that the standard decolonization protocol
did not result in long-term clearance of MRSA carriage for most patients. In this study we found that
many nurses and doctors harbored and/or were colonized with S. aureus strains. We also found strains of
S. aureus on nurses’ hands and clothes.
We found six clusters (4, 7, 8, 10, 11, 16) comprising strains isolated from patients, nurses and hospital
environment. It is difficult to determine exactly where
and to whom transmission of S. aureus occurred. It
is probably, that the cross-transmission of S. aureus
occurred via hands, which may be contaminated by
contact with colonized or infected body sites of medical staff or colonised or infected patient or with devises. Nurses’ and doctors’ hands could be contaminated by strains existing in the hospital environment.
For example, cluster 4 included strains of S. aureus
isolated from two neonates, three nurses and one strain
from environment. It is important to note that these
neonates suffered from skin infection. We also identified clusters (9, 12, 22, 23) that included only strains
isolated from patients and medical staff. For example
cluster 22 included one strain isolated from skin of
neonate and one isolate originated from nurse’s hands
working in delivery room. Therefore, we think that
the medical staff could be considered an important
vector in the chain of S. aureus transmission. Previously, it has been reported that strains isolated from
nurses’ hands could be regarded as the source of staphylococcal scaled skin syndrome (SSSS) of neonates
in a maternity unit in Paris (Helali et al., 2005).
Similary, Bertin et al., 2006, indicated that strains isolated from a healthcare worker suffering from otitis
externa and carrries Staphylococcus aureus could be
responsible for the outbreak of bloodstream infections
in a neonatal intensive care unit. Hand hygiene has
been recognized as the key to prevent transmission of
S. aureus strains and to reduce the nosocomial infections. Sroka et al. (2010) indicated that the increasing
consumption of hand antiseptics was associated with
a significant reduction of S. aureus rate.
We isolated strains from medical equipment, patients’ beds or hand wash basins. Previously, several
authors also demonstrated that hospital equipment and
environment could be the reservoirs of S. aureus
(Ohara et al., 1998; Embil et al., 2001; Hardly et al.,
2006; Sexton et al., 2006). In addition, it is noteworthy
that staphylococci can persist in clinical areas for a long
period of time (Sexton et al., 2006). Recently, Aldeyab
et al. (2009) indicated that environmental decontamination using detergents and hypochlorite was effective
in eliminating MRSA strains from hospital environment. We identified many clusters, which grouped
strains isolated from patients and hospital environment.
This suggests that patients acquired S. aureus from the
hospital environment. However, we can not exclude the
possibility that patients contaminate their surrounding
such as hospital beds, hand wash basins etc. We found
cluster 10, which included strains isolated from neonate, from scales used to weigh neonates after birth in
the delivery room. In this cluster we also found strains,
which were derived from cultures originated from
healthcare workers. It might be speculate that strains
obtained from scales were via contaminated nurse’s
hands transformed on childish skin. It is well known
that shared equipment in common places is an additional source of dissemination of S. aureus.
Our results illustrate the great genetic diversity
among S. aureus strains in a hospital. This study also
reveals contamination of hospital environment with
S. aureus strains and the need for more effective
cleaning of the hospital environment in order to eliminate reservoirs of these strains. We believed that crosstransmission events can be reduced by strict hand
hygiene and other prevention procedures.
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3
Polish Journal of Microbiology
2010, Vol. 59, No 3, 167–173
ORIGINAL PAPER
Simultaneous Detection and Differentiation of Pathogenic
and Nonpathogenic Leptospira spp. by Multiplex Real-Time PCR (TaqMan) assay
ORHAN BEDIR1, ABDULLAH KILIC1*, ERDINC ATABEK2, AHMET MERT KUSKUCU 1,
VEDAT TURHAN3 and A. CELAL BASUSTAOGLU1
1 Department
of Microbiology and Clinical Microbiology, Gulhane Military Medical Academy
and School of Medicine 06018, Ankara, Turkey
2 Central Veterinary Control and Research Institute, Etlik, Ankara, Turkey
3 Department of Infectious Diseases, Gulhane Military Medical Academy and School of Medicine,
Haydarpasa Training Hospital, Istanbul, Turkey
Received 3 August 2009, revised 1 June 2010, accepted 15 June 2010
Abstract
Leptospirosis, caused by pathogenic Leptospira, is one of the most important zoonoses in the world. Several molecular techniques have
been developed for detection and differentiation between pathogenic and saprophytic Leptospira spp. The aim of this study was to develop
a rapid and simple assay for specific detection and differentiation of pathogenic Leptospira spp. by multiplex real-time PCR (TaqMan)
assay using primers and probes targeting Leptospira genus specific 16S ribosomal RNA gene, the pathogen specific lig A/B genes and nonpathogen Leptospira biflexa specific 23S ribosomal RNA gene. Sixteen reference strains of Leptospira spp. including pathogenic and
nonpathogenic and ten other negative control bacterial strains were used in the study. While the 16S primers amplified target from both
pathogenic and non-pathogenic leptospires, the ligA/B and the 23S primers amplified target DNA from pathogenic and non-pathogenic
leptospires, respectively. The multiplex real-time PCR (TaqMan) assay detection limit, that is, the sensitivity was found approximately
1× 102 cells/ml for ligA/B gene and 23S ribosomal RNA gene, and 10 cells/ml 16S ribosomal RNA. The reaction efficiencies were 83–105%
with decision coefficients of more than 0.99 in all multiplex assays. The multiplex real-time PCR (TaqMan) assay yielded negative results
with the ten other control bacteria. In conclusion, the developed multiplex real-time PCR (TaqMan) assay is highly useful for early
diagnosis and differentiation between pathogenic and non-pathogenic leptospires in a reaction tube as having high sensitivity and specificity.
K e y w o r d s: Leptospira genus, leptospirosis, multiplex real-time PCR (TaqMan) assay, pathogenic Leptospira, saprophytic Leptospira
Introduction
Leptospirosis is caused by spirochetes of the genus
Leptospira (Guerra, 2009). The Leptospira genus has
been classified into 17 species by DNA-DNA hybridization. This genus is further divided into three groups
as pathogenic, nonpathogenic, and opportunistic/possibly pathogenic. The pathogenic leptospires include
eight species: Leptospira interrogans, Leptospira
kischneri, Leptospira borgpetersenii, Leptospira santarosai, Leptospira weilii, Leptospira alexanderi, Leptospira genomospecies 1 and Leptospira noguchii
(Brenner et al., 1999). The Leptospira genus has also
been classified on the basis of surface antigen patterns.
They are divided into at least 250 serotypes that have
major antigens in common and are combined into
24 serogroups (Dutta and Christopher, 2005).
Leptospirosis is identified as one of the emerging
infectious diseases and a major public health concern
worldwide. The organism affects virtually any mammal, including humans. Humans acquire the organisms through contact with contaminated soil, water,
vegetation or with the body fluids of animals harboring leptospires (Palaniappan et al., 2005). More than
500 000 severe leptospirosis cases have been reported
each year around the world (WHO, 1999). The rate of
leptospirosis cases varies depending on the climate
and animal reservoirs. The incidence of leptospirosis
is especially highest during the summer season with
heavy rains and floods (Xue et al., 2008).
Since the currently available microscopic agglutination test (MAT) that is a known gold standard does
not permit early diagnosis and other serologic methods
have low sensitivity, more rapid and sensitive methods
* Corresponding author: A. Kilic, Department of Microbiology and Clinical Microbiology, Gulhane Military Medical Academy,
School of Medicine, 06018, Ankara, Turkey; phone: (+90) 312-3043412; e-mail: [email protected]
168
Bedir O. et al.
are needed for detection of Leptospira as well as the
ability to distinguish pathogenic and nonpathogenic
Leptospira. Therefore, a number of molecular methods
such as conventional multiplex PCR (Kositanont et al.,
2007), real-time PCR (Slack et al., 2007; Levett et al.,
2005), nested-PCR (Bomfim et al., 2008), loop mediated isothermal amplification (LAMP) (Lin et al.,
2009), nested PCR-restriction fragment length polymorphism (RFLP) (Djadid et al., 2009) have been developed for specific detection of pathogenic Leptospira
spp. in diagnostic laboratories. The TaqMan real-time
PCR method has also been used for detection of
pathogenic Leptospira spp. based on specific target
sequences including the ribosomal 16S ribosomal
RNA gene, and the Leptospira immunoglobulin-like
protein A and B gene (lig A and ligB) (Palaniappan
2005). The lig A and ligB genes encode amino-terminal lipoprotein signal peptides followed by 10 or
11 big domain repeats. The lig genes are only detected
in pathogenic Leptospira spp. as a unique virulence
factor. Conversely, these genes are not found in nonpathogenic Leptospira spp. (Matsunaga et al., 2003).
The aim of this study was to develop a rapid and
simple assay for the specific detection and differentiation of pathogenic Leptospira spp. by multiplex
real-time PCR TaqMan method using primers and
probes specific for Leptospira genus 16S ribosomal
RNA gene, pathogen specific lig A/B genes and nonpathogen Leptospira biflexa specific 23S ribosomal
RNA gene.
Experimental
Materials and Methods
Leptospira reference strains and control bacterial strains. Sixteen reference strains of Leptospira
spp. including pathogenic and nonpathogenic were
obtained from the Etlik Central Veterinary Control
and Research Institute (ECVCRI), WHO Collaborating Center, Ankara, Turkey (Table I). All strains were
stored in Fletcher media (Difco, Detroit, MI, USA)
and then cultured in liquid Ellinghausen McCullough
Johnson Harris (EMJH) (Difco) media supplemented
with 10% serum of hemolysed rabbit blood at 30°C
for 7 days. Other control bacterial strains including
Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC
13883, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 27270, Salmonella typhimurium NCTC 12023, Legionella pneumophila, Neisseria gonorrhoeae NCTC 8375, Borrelia burgdorferi
strain B31, and Streptococcus pyogenes NCTC 12696
were selected from stock reference culture collection
in our laboratory. The isolates were stored at –70°C
3
in trypticase soy broth (Merck, Darmstadt, Germany)
supplemented with 15% glycerol before being tested.
Design of primers and probes. Oligonucleotide
primers and probes for multiplex real-time PCR
(TaqMan) assay were designed based on a particular
region of the ligA/B gene sequence (ligA/B-P1-5’cggttc tcacttctattcaa-3’, ligA/B-P2-5’-attgaagaatcggat
gagaa-3’, and ligA/B-Probe-Texas red-5’-atcctgtaaa
tcctt ctcttgcaaa-3’-Bhq-2) for pathogenic Leptospira
spp. (Genbank accession nos FJ030916, EF517920,
AF534640, AF368236, and AY221109), the 16S ribosomal RNA gene sequence (16S-P1-5’-tagtgaacgg
gatagatac-3’, 16S-P2-5’-ggtctacttaatccgttagg-3’, and
16S-Prob-Fam-5’-aatccacgccctaaacgttgtctac-3’-Bhq-1)
for Leptospira spp. genus (Genbank accession nos
FJ154560, FJ154600, FJ154577, FJ154571, FJ154569,
FJ154568, FJ154564, FJ154563, FJ154556, FJ154555,
and FJ154553), and the 23S ribosomal RNA gene
sequence (23S-P1-5’-acaatcttaccaaaccctatc-3’, 23SP2-5’-ttaccacttagcgtagattt-3’, and 23S-Prob-Joe-5’tccgaatactgtaacttgaagtactgca-3’-Bhq-1) for non-pathogenic L. biflexa (Genbank accession no CP000786) by
using oligo analysis and design program Oligaware 3.0
developed in our institution (Table II). BLAST program
was used to initially asses the ability of the primers
and probes to identify target sequences (Smythe et al.,
2002; Altschul et al., 1997). The primers and probes
were synthesized by Metabion International, Germany.
DNA extraction. DNA was extracted from the
samples by treatment with 1% SDS and 100 mg proteinase K (Sigma Chemicals, St. Louis, Missouri,
USA) in a buffer containing 50 mM Tris (pH 8.0),
50 mM EDTA, 100 µM NaCl. After 2 hours of incubation at 55°C in a waterbath, the DNA was purified by
repeated extraction with phenol/chloroform/isoamyl
alcohol (25:24:1). The DNA was concentrated by precipitation with 99% ethanol. The precipitate was collected by centrifugation, then dried and resuspended
in deioinized sterile water (Veloso et al., 2000).
Multiplex real-time PCR (TaqMan). The multiplex real-time PCR (TaqMan) method was performed
by using a 7500 ABI Prism Sequence Detector (Applied Biosystems, Foster City, Calif., USA). In brief,
2 ml of the extracted nucleic acid solution was added
to 23 µl of reaction mixture containing 0.8 µM of each
primer and 0.4 µM each fluorophore probe (final concentration), and mixed with 12,5 µl of TaqMan Universal PCR Master Mix (Applied Biosystems). The
TaqMan cycling conditions included a 10 min degradation of the preamplified templates at 95°C and then
40 cycles of denaturation at 95°C for 15 s and annealing and extension at 60°C for 60 s. All experiments
were repeated at least twice for testing the reproducibility of the assay.
Detection limits determination and assay validation. Pathogenic L. interrogans strain Wijnberg and
3
Detection and differentiation of pathogenic and nonpathogenic Leptospira spp.
169
Table I
Leptospira strains used in this study
Serovar
Genomospecies
Leptospira interrogans
Leptospira interrogans
Leptospira interrogans
Leptospira interrogans
Leptospira interrogans
Leptospira interrogans
Leptospira interrogans
Leptospira. interrogans
Leptospira interrogans
Leptospira interrogans
Leptospira interrogans
Leptospira borgpetersenii
Leptospira borgpetersenii
Leptospira borgpetersenii
Leptospira kirschneri
Leptospira biflexa
australis
autumnalis
bataviae
bratislava
hebdomadis
icterohaemorrhagiae
hardjo
canicola
icterohaemorrhagiae
pomona
pyrogenes
ballum
javanica
tarassovi
grippotyphosa
patoc
Strain
Ballico
Akiyami A
Swart
Jes Bratislava
Hebdomadis
Wijnberg
Hardjoprajitno
Hond Utrecht IV
RGA
Pomona
Salinem
Mus 127
Veldrat Batavia 46
Perepelitsin
Moskova V
Patoc I
Table II
Oligonucleotide sequence of the primers and probes used in this study
Target region
Oligo Name
Sequence (5'–3')
ligA/B gene for pathogenic ligA/B-P1 (Forward) 5’-cggttctcacttctattcaa-3’
Leptospira spp.
ligA/B-P2 (Reverse) 5’-attgaagaatcggatgagaa-3’
ligA/B-Prob
Texas Red-5’
-atcctgtaaatccttctcttgcaaa-3’-Bhq-2
23S rRNA gene for non23S-P1 (Forward)
5’-acaatcttaccaaaccctatc-3’
pathogenic Leptospira spp. 23S-P2(Reverse)
5’-ttaccacttagcgtagattt-3’
23S-Prob
Joe-5’-tccgaatactgtaacttgaagtactgca-3-Bhq-1
16S rRNA gene
16S-P1(Forward)
5’-tagtgaacgggattagatac-3’,
for Leptospira genus
16S-P2 (Reverse)
5’-ggtctacttaatccgttagg-3’,
16S-Prob
Fam-5’-aatccacgccctaaacgttgtctac-3’-Bhq-1
L. biflexa strain Patoc I were cultured in EMJH
(Difco) media to obtain a cell density of 1×108 cells/ml
for each of the target species, and then total DNA
was extracted. Serial 10-fold dilutions of extracted
DNA were prepared ranging from 1× 106 cells/ml to
1× 100 cells/ml by using sterile ddH2O. The threshold
cycle (Ct) value of each dilution was recorded. The
lowest number of leptospires detected visually was
defined as the detection limit of the assay.
Results
Multiplex real-time (TaqMan) PCR assay using
LigA/B, 16S, and 23S primers and probes. The sixteen standard strains of Lepotospira spp. were tested
for all genes. The multiplex real-time PCR (TaqMan)
assay was used to amplify specific leptosiral sequences
including ligA/B genes, 16S ribosomal RNA gene and
Genbank accession no
FJ030916, EF517920, AF534640,
AF368236, and AY221109
CP000786
FJ154560, FJ154600, FJ154577,
FJ154571, FJ154569, FJ154568,
FJ154564, FJ154563, FJ154556,
FJ154555, and FJ154553
23S ribosomal RNA gene simultaneously in a single
tube. The ligA/B primers and probe designed based on
particular region of ligA/B of pathogenic leptospires
gave a detectable product, Ct values ranged from 17 to
39 and averaged 24 for only all pathogenic leptospires,
whereas the 23S primers and probe designed based on
the particular region of 23S ribosomal RNA non-pathogenic leptospires gave a detectable product Ct values
ranged from 18 to 36 and averaged 23 for only nonpathogenic leptospires. The 16S primers and probe
designed from the conserved region of 16S ribosomal
RNA of Leptospira genus amplified genomic DNA
from both pathogenic and non-pathogenic leptospires.
Sensitivity and specificity of multiplex real-time
PCR (TaqMan) method. The reaction efficiency of
each assay was counted from the slopes of standard
curves. The reaction efficiencies were found to be
83–105% with decision coefficients of more than 0.99
in all multiplex assays. To determine assay sensitivity,
170
3
Bedir O. et al.
Fig. 1. Amplification curves and dilution end point standard curves of log10 genome equivalents versus CT cycle number.
The analytical sensitivity of this assay for ligA/B (A1-A2) and 23S ribosomal RNA (B1-B2) assays were approximately
100 genome per ml and 16S rRNA (C1-C2) assay was approximately 10 genome equivalents per ml.
Leptospiral DNA was adjusted by 10-fold serial dilution ranging from 1× 106 cell/ml to 1× 100 cells/ml
from both pathogenic Leptospira interrogans strain
Wijnberg and Leptospira biflexa strain Patoc I. Linear
dynamic range of multiplex real-time PCR (TaqMan)
assay was performed with triplicates of 10-fold dilutions of the purified genomic DNA extracted from
pathogenic and nonpathogenic leptospires, and was
observed constantly (Fig. 1). The standard curves obtained with 10-fold serially diluted genomic DNA
preparations were linear over seven orders of magnitude for targeting sequence. The assay detection limit,
that is the sensitivity, was found to be approximately
1× 102 cells/ml for ligA/B gene and 23S ribosomal
RNA gene, and 10 cells/ml 16S ribosomal RNA. To
determine the assay specificity of multiplex real-time
PCR (TaqMan) assay, other control bacteria were tested
with ligA/B, 23S and 16S primer and probes. The multiplex real-time PCR (TaqMan) assay yielded negative results with the ten other control bacteria. The
ligA/B, the 16S, and 23S primers and probes were
specific for pathogenic leptospires, Leptspira genus,
and nonpathogenic leptospira, respectively (Fig. 2).
Discussion
Leptospirosis is known to be an emerging zoonotic
disease caused by the genus Leptospira worldwide
(Guerra, 2009). Since there is no national Leptospira
3
Detection and differentiation of pathogenic and nonpathogenic Leptospira spp.
171
Fig. 2. Multiplex amplification results with Leptospira
interrogans strain Wijnberg, Leptospira biflexa strain Patoc I
and mix DNA samples. In 10 fold dilutions 4 times.
reference center in Turkey, reliable data about the incidence of leptospirosis are not available. Twelve
Turkish cases were reported from south region of Turkey in 1997 (Saltoglu et al., 1997). In 2005, three cases
of anicteric leptospirosis with mild and sever complications, a case of icteric leptospirosis with resistant
immune hemolytic anemia, and four cases of anicteric leptospirosis with mania and psychosis were reported from the central Anatolia and northwest region
of Turkey (Erdinc et al., 2006; Solmazgul et al., 2005;
Semiz et al., 2005). Recently a Turkish leptospirosis
case complicated with cerebral venous thrombosis
was reported from the northwest region of Turkey
(Turhan et al., 2006).
A spectrum of human leptospirosis is recognized
ranging from subclinical infection to a severe syndrome of multiorgan infection such as severe vasculitis
and intravascular coagulation (Guerra, 2009). However, the symptoms of leptospirosis at the prodromal
stage are almost indistinguishable from various other
bacterial and viral febrile infections such as salmonellosis and dengue virus infection (Srimanote et al.,
2008). In addition, the lack of reliable techniques for
rapid diagnosis of leptospirosis may cause delay in
treatment of patients and lethal sequela (Djadid
et al., 2009). In diagnosis of leptospirosis, serological
methods such as MAT and enzyme-linked immunosorbent assay (ELISA) based on the immunogenic
response of the host and culture method to observe
the organisms from clinical samples are usually per-
formed in many laboratories. MAT is used to detect
leptospiral antibody from the patient sera using live
organisms as sources of antigens. Since antileptospiral
antibody becomes detectable only after 8–10 days from
the onset of illness, MAT cannot provide early diagnosis of leptospirosis (Smythe et al., 2002). In addition
to MAT, other serologic tests detecting leptospires
from clinical samples are not appreciable, especially
during the early phase of the infection (Palaniappan
et al., 2005; Srimanote et al., 2008). Leptospira is
a fastidious microorganism and difficult to grow in
culture medium. Moreover, culture which requires
a special medium and at least two weeks to yield the
organisms, cannot provide an early diagnosis. Thus,
these techniques are used mainly for epidemiological
purposes (Palaniappan et al., 2005; Srimanote et al.,
2008). Therefore, there is a need for a reliable and
accurate method for detection and differentiation of
leptospires from clinical samples. The multiplex realtime PCR (TaqMan) assay is a rapid, sensitive and
specific assay for detection of leptospires during early
stage of infection (Palaniappan et al., 2005; Smythe
et al., 2002; Slack et al., 2006). This assay has also
been used as a diagnostic tool for the discrimination
of pathogenic and non-pathogenic leptospires using
particular gene sequence (Smythe et al., 2002).
In this study, we developed a multiplex real-time
PCR (TaqMan) assay to detect infection and the same
time distinguish pathogenic leptospires from nonpathogenic leptospires in same reaction tube. The
172
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Bedir O. et al.
ligA/B, 23S, 16S primers and probes were used for specific detection of pathogenic, non-pathogenic leptospires and leptospira genus. This assay was able to detect rapidly leptospira genus and distinguish pathogenic
leptospires from non-pathogenic ones. The detection
limit of the assay was 100 leptospira cells/ml for pathogenic, non-pathogenic leptospires, and 10 cells/ml for
leptospira genus. While the 16S primers amplified the
target from both pathogenic and non-pathogenic leptospires, ligA/B amplified target DNA only from pathogenic leptospires and the 23S primers were specific
for non-pathogenic ones. Thus, this assay may enable
the recognition and discrimination between pathogenic and non-pathogenic, environmental contaminant
leptospires in the same reaction tube.
A number of gene sequences have been used to
detect and distinguish pathogenic leptospires from
non-pathogenic ones. Woo et al. developed a realtime PCR (TaqMan) method using 23S rRNA gene
sequence for identification of pathogenic Leptospira
(Woo et al., 1996). Levett et al. evaluated a highly
sensitive and specific real-time PCR (TaqMan)
method for lipL32 gene, which is a probable virulence
gene, for detection of pathogenic leptospires (Levett
et al., 2005). Slack et al. used DNA gyrase subunit
B gene for identification of pathogenic Leptospira
spp. When compared to the 16S rRNA gene, the gyrB
gene showed greater nucleotide/evolutionary divergence allowing superior identification (Slack et al.,
2006). Kawabata et al. used flab gene and suggested
that PCR-RFLP was an efficient tool for rapid detection and identification of species of Leptospira from
clinical specimens (Kawabata et al., 2001). Djadid
et al. developed a nested PCR-RFLP assay using 16S
rRNA as a rapid and specific available technique for
differentiate pathogenic and non-pathogenic Leptospira
spp. in the early stage of infection (Djadid et al.,
2009). In this study, the ligA/B primers were used in
a multiplex real-time (TaqMan) PCR assay to detect
and distinguish pathogenic Leptospira serovar. The lig
genes encode surface proteins containing immunoglobulin-like repeat predicted to play a role in adhesion
to host tissues. Previous studies have demonstrated
that pathogenic leptospires contain these genes, while
they are absent from the non-pathogenic saprophyte
(Xue et al., 2008). Diagnostic methods such as PCR
and ELISA targeting lig genes have been recently developed to discrimination pathogenic and non-pathogenic leptospires (Palaniappan et al., 2005; Srimanote
et al., 2008). Palaniappan et al. developed a real-time
PCR (TaqMan) assay using lig1/lig2 primers targeting the conserved region of ligA and B as a sensitive
and rapid tool for early diagnosis of leptospirosis
(Palaniappan et al., 2005). One of the most significant advantages of our multiplex real-time PCR
(TaqMan) assay is that both pathogenic and non-patho-
genic leptospires were detected at the same cycling
conditions allowing all three reactions to be performed in a single PCR tube simultaneously. We also
analyzed leptospires based on the genus in the same
reaction tube as an internal control. To our knowledge, this is the first study detecting three leptospire
gene sequences in the same reaction tube. The efficiency of each assay ranged between 83 and 105%;
the accepted ranges for PCR efficiency are generally
between 80.0 and 110.0%.
In conclusion, the developed multiplex real-time
PCR (TaqMan) assay targeting ligA/B gene, 16S ribosomal RNA and 23S ribosomal RNA sequences is
highly useful for early diagnosis of leptospirosis and
differentiation between pathogenic and non-pathogenic leptospires in the same reaction tube with high
sensitivity and specificity.
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Polish Journal of Microbiology
2010, Vol. 59, No 3, 175–178
ORIGINAL PAPER
Methanogenic Diversity Studies within the Rumen of Surti Buffaloes Based
on Methyl Coenzyme M Reductase A (mcrA) Genes Point to Methanobacteriales
KRISHNA M. SINGH1, PARESH R. PANDYA2, SUBHASH PARNERKAR2, AJAI K. TRIPATHI1, UMED RAMANI1,
PRAKASH G. KORINGA1, DHARAMSHI N. RANK3, CHAITANYA G. JOSHI1 and RAMESH K. KOTHARI4
1 Department
of Animal Biotechnology, College of Veterinary Science and A.H.,
Anand Agricultural University, Anand (388 001), Gujarat, India
2 Animal Nutrition Research Station, College of Veterinary Science and A.H.,
Anand Agricultural University, Anand (388 001), Gujarat, India
3 Department of Animal Genetics & Breeding, College of Veterinary Science and A.H.,
Anand Agricultural University, Anand (388 001), Gujarat, India
4 Department of Microbiology, Christ College, Rajkot, Gujarat, India
Received 3 April 2010, revised 21 June 2010, accepted 12 July 2010
Abstract
Methane emissions from ruminant livestock are considered to be one of the more potent forms of greenhouse gases contributing to global
warming. Many strategies to reduce emissions are targeting the methanogens that inhabit the rumen, but such an approach can only be
successful if it targets all the major groups of ruminant methanogens. Therefore, basic knowledge of the diversity of these microbes in
breeds of buffalo is required. Therefore, the methanogenic community in the rumen of Surti buffaloes was analyzed by PCR amplification,
cloning, and sequencing of methyl coenzyme M reductase (mcrA) gene. A total of 76 clones were identified, revealing 14 different
sequences (phylotypes). All 14 sequences were similar to methanogens belonging to the order Methanobacteriales. Within
Methanobacteriales, 12 clones (6 OTUs) were similar to Methanosphaera stadtmanae and the remaining 8 phylotypes (64 clones) were
similar to unclassified Methanobacteriales. Overall, members of the Methanobacteriales dominated the mcrA clone library in the rumen
of Surti buffalo. Further studies and effective strategies can be made to inhibit the growth of Methanobacteriales to reduce methane
emission from the rumen which would help in preventing global warming.
K e y w o r d s: methyl coenzyme-M reductase, PCR, ruminant methanogens
Introduction
The rumen is characterized by high microbial
population density and complexity of micro-ecological
interactions. Methane is biologically produced by the
metabolism of a diverse group of methanogenic microorganisms, methanogens, which are phylogenetically
placed exclusively as members of the domain archaea.
They inhabit typical anaerobic environments, such as
wetlands, sediments, geothermal springs and the digestive tracts of mammals (Garcia et al., 2000). Methane
is an important greenhouse gas which significantly
contributes to global warming. Livestock is a major
anthropogenic source of methane emission from agriculture and contributes about 18% of global greenhouse gas (GHG) emissions, and as much as 37% of
anthropogenic methane, mostly from enteric fermenta-
tion by ruminants (FAO, 2006). Livestock rearing has
been an integral part of the agricultural system in
India. Currently, India possesses the world’s largest
livestock population of 485 million, which accounts for
13% of the global livestock population (MOA, 2003).
It has 57% of the world’s buffalo and 16% of the cattle
population. The contribution of methane emission in
India by buffalo is 42% (Chhabra et al., 2009).
Several groups have reported the monitoring of
methanogen populations from environmental samples
through targeting of the 16S ribosomal gene (Yu et al.,
2005; Stewart et al., 2006; Wright et al., 2007). While
researchers have traditionally used the 16S rRNA gene
for phylogenetic diversity, many researches are now
addressing the diversity of the methanogenic archaea
by studying sequence divergence within the methyl coenzyme-M reductase subunit A (mcrA) gene (Lueders
* Corresponding author: Ramesh K. Kothari /Krishna M. Singh, Department of Microbiology, Christ College, Rajkot, Gujarat,
India; phone 02692 261201; fax 02692 261201; e-mail: [email protected], e-mail: [email protected]
176
Singh K.M. et al.
et al., 2001; Luton et al., 2002; Hallam et al., 2003;
Tatsuoka et al., 2004; Rastogi et al., 2008). Methyl coenzyme-M reductase is ubiquitous to methanogens and
is crucial to the terminal step of methanogenesis where
it is involved in the reduction of the methyl group
bound to coenzyme-M. There is no report concerning
mcrA genes from buffalo rumen, therefore, we examined the community of methanogens using comparative sequence analysis of the mcrA amplified from total DNA extracted from rumen fluid of Surti buffaloes.
Experimental
Materials and Methods
Sampling and DNA extraction. The experiment
was carried out on 3 adult Surti buffaloes reared at
the Department of Animal Nutrition, College of Veterinary Science and A.H., Anand. All the animals were
maintained under uniform feeding regime (I.C.A.R.,
1998) for minimum 30 days. The diet consisted of
green fodder (Sorghum and NB21), dry mature pasture
grass (Dicanthium annulatum) and compound concentrate mixture (20% CP, 65% TDN). The animals
were offered 10 kg green, ad-lib dry grass and 2.5 kg
of concentrate mixture daily. Approximately 500 ml
of liquor was collected via a stomach tube located in
the mid part of the rumen and connected to a vacuum
pump at 0, 2, 4 and 6 hrs post feeding (Khampa et al.,
2006). About 100 ml liquor was passed through four
layers of cheese cloth to remove particulate matter.
Remaining liquor was stored at –80°C for further
study. Total DNA (each hrs) was extracted separately
by using a commercially available kit according to
the manufacturer’s instructions (QIAGEN Stool kit;
QIAGEN, CA). The individual DNA was used as
a template in PCR to amplify mcrA gene.
PCR primers and amplification. The mcrA primers used were ME1 (5’-AGCMATGCARATHGGWA
TGTC-3-’) and ME2 (5’-ATCATKGCRTAGTTDGG
RTAGT-3’) (Hales et al., 1996), subsequently mcrA
gene were amplified (760 bp) by PCR using metagenomic DNA and Master mix (Fermentas, USA).
A total of 25 µl of reaction mixture consisted of
10 pmol of each primer, 30 ng of template DNA,
12.5 µl of Master mix (Fermentas, USA). The PCR
amplification was carried out as follows: 1 cycle at
95°C for 3 min, 35 cycles of 95°C for 30 s, 60°C for
1 min, 72°C for 1 min and a final elongation at 72°
for 10 min by using thermal Cycler (ABI, USA). The
anticipated product of approximately 760 bp was
cleaned separately using a Qiagen DNA Gel Extraction Kits (QIAGEN, CA) in accordance with the
directions of the manufacturer and pooled the purified
PCR products in equimolar concentration.
3
Cloning and sequencing. The purified PCR products were cloned in InstaTA cloning kit (Fermentas,
USA) as per the instructions of the manufacturer. The
recombinant plasmids then were extracted by the
Qiagen mini-prep plasmids extraction kit (QIAGEN,
CA). Sequencing was performed for all the clones in
the library with an ABI Prism 310 Genetic analyser
(Applied Biosystems Inc., CA) using BigDye Terminator (version 3.1) at Animal Biotechnology laboratory, AAU, Anand, Gujarat, India.
Sequence analyses and phylogenetic tree construction. All reference sequences were obtained
from the Genbank/EMBL/DDBJ (Benson et al.,
2007). Sequences (~600 bp) from the current study
were analysed by the CHECK_CHIMERA program
(Maidak et al., 2001) to remove any chimeric clone.
The similarity searches for sequences were carried out
by BLAST (http://www.ncbi.nlm.nih.gov/ BLAST/
Blast.cgi (Madden et al., 1996) and alignment was
done using CLUSTAL W (http://www.ebi.ac.uk/
Tools/clustalw2/index.html (Thompson et al., 1994).
Ambiguously and incorrectly aligned positions were
aligned manually. The distance matrix was calculated
using the PRODIST program included in PHYLIP
(Felsenstein, 1985) and used to assign sequences
in various operational taxonomic units (OTUs) or
phylotypes by DOTUR (Schloss and Handelsman,
2005) and total of 14 OTUs were generated. The
percentage of good coverage was calculated as
[1 – (n/N)] X 100, where ‘n’ is the number of single
clone OTUs and ‘N’ is the library size. Phylogenetic
tree was constructed by the neighbour joining method
using MEGA 4.0 (Tamura et al., 2007). Bootstrap
re-sampling analysis for 1000 replicates was performed to estimate the confidence of tree topologies.
The prefix mcrA was used to denote OTU identified and nucleotide sequences have been deposited in
the Genbank database under the accession numbers
GQ120890-GQ120965.
Fig. 1. Ethidium bromide-stained agarose gel showing PCR products
(670 bp) amplified from DNAs extracted from the three rumen samples.
Template DNAs are as follows: lane 1, sample1; lane 2, sample 2; lane 3;
sample 3 and lane 4, shows DNA size marker.
3
Sequencing of mcrA genes of ruminant methanogens
Results
Methanogen-specific DNA fragments were amplified from DNA extracted from the Surti rumen fluid by
PCR with primers targeting mcrA genes. The amplified
fragments from three rumen fluids, which were approx.
760 bp, are shown in Fig. 1. All the clones were subjected to sequence analysis followed by online homology search, Genbank which implements the BLAST
algorithm (Madden et al., 1996). None of clone (76)
from our library was assigned any genera/species.
Because the similarity values of our sequences were
too low to assign them to particular taxa with a reasonable degree of confidence. In our library 14 sequences
(phylotypes) were generated. Phylogenetic analysis
was performed to clarify their taxonomic position. The
phylogenetic placements of the deduced DNA sequences are shown in Fig. 2. The good coverage of
mcrA library was 94.73% in present study. This level
of coverage showed that the mcrA sequences identified
in library represent the majority of methanogen diversity. The sequences obtained in the present study were
placed in the single cluster Methanobacteriales.
177
Table I
Analysis of mcrA gene phylotypes diversity retrieved from
the rumen fluid of Surti buffaloes
Items
Library size (N)
OTUs b
Single clone OTU c (n)
Good’s coverage d (%)
Clone distribution
(i) Methanobacteriales
a Methanosphaera stadtmanae
b. Unidentified Methanobactriales
c. Unknown methanogen
a
16S r DNA library
76
14
4
94.73
14 OTUs (76 clones)
6 OTUs (12 clones)
7 OTUs (63 clones)
1 OTU (01 clone)
a
Number of clones analyzed from library; b OTUs based on mcrA gene
sequences; c OTUs containing only single clone; d The higher percentage
coverage means more diversity is captured
Within the Methanobacteriales, 63 clones (7 OTUs)
belonged to the unidentified Methanobacteriales and
12 clones (6 OTUs) sequences were found to be the
closest to Methanosphaera stadtmanae. Of the clones
isolated from rumen sample, only one clone represented unknown methanogens (Table I).
Fig. 2. The evolutionary history was inferred using the Maximum Parsimony method. The bootstrap consensus tree inferred from 1000 replicates
is taken to represent the evolutionary history of the taxa analyzed. The percentage of replicate trees in which the associated taxa clustered together
in the bootstrap test (1000 replicates) are shown next to the branches. Phylogenetic analyses were conducted in MEGA4. (UNI = u nidentified and
MSS = Methanosphaera stadtmanae).
178
Singh K.M. et al.
Discussion
Of 14 phylotypes, 13 sequences (Phylotypes) of the
DNA were obtained from the 76 clones in the present
study; these sequences were placed in the same cluster,
which was relatively close to unidentified Methanobacteriales (63 clone) and M. stadtmanae (12 clones),
in phylogenetic placements (Fig. 2). Whitford et al.
(2001) also found several sequences that were related
to Methanosphaera stadtmanae. Wright et al. (2007)
also found a sequence (ON-CAN.13) in cattle from
Ontario that was 95.8% similar to that of Methanosphaera stadtmanae and 99.8% similar to that of
their clone ARC29. Methanogens similar to Methanosphaera stadtmanae have also been reported in pasture-fed dairy cattle (Skillman et al., 2006) The DNA
sequences of mcrA genes, isolated in this study,
showed similarities with unidentified methanobacteriales and to M. stadtmanae. It has been known that
mcrA genes could be used as phylogenetic tool for the
specific detection and the identification of methanogenes, because the phylogeny of the mcrA genes and
16S rDNA from the recognized orders of methanogens
clearly had strong similarity (Luton et al., 2002).
Our results show that rumen of Surti buffaloes
contains one of the essential and diagnostic genes of
the methanogenic pathway. The identification of these
genes provides a means to identify cluster/group on
the basis of mcrA sequence. Moreover, identification
of rumen associated mcrA groups defines a functional
genomic link between methanogenic and putative
reverse methanogenic archaea. Specific questions relating to methanogenic protein function in buffalo
rumen await further genomic, biochemical, structural,
and proteomic analysis.
Acknowledgements
Financial support provided by the Department of Biotechnology Govt. of India, New Delhi to conduct the study reported here
is acknowledged with respect and gratitude.
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FAO, FAOSTAT. 2006. Online Statistical Service, Food and
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Garcia J.L., B.K.C. Patel and B. Olliver. 2000. Taxaonomic,
phylogenic, and ecological diversity of methanogenic Archaea.
Anaerobe 6: 205–226.
Hales B.A., C. Edwards, D.A. Ritchie, G. Hall, R.W. Pickup
and J.R. Saunders. 1996. Isolation and identification of metha-
3
nogen specific DNA from blanket bog peat by PCR amplification
and sequence analysis. Appl. Environ. Microbiol. 62, 668–675.
Hallam S.J., P.R. Girguis, C.M. Preston, P.M. Richardson and
E.F. DeLong. 2003. Identification of methyl coenzyme M reductase A (mcrA) genes associated with methane-oxidizing archaea.
Appl. Environ. Microbiol. 69: 5483–5491.
ICAR. 1998. Nutrient requirements of livestock and poultry.
Indian Council of Agricultural Research, New Delhi, India.
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microbial protein synthesis in lactating dairy cows raised under
tropical condition. Asian- Aust. J. Anim. Sci. 19: 837–844.
Lueders T., K.J. Chin, R. Conrad and M .Friedrich. 2001.
Molecular analyses of methyl-coenzyme M reductase alpha-subunit (mcrA) genes in rice field soil and enrichment cultures reveal
the methanogenic phenotype of a novel archaeal lineage. Environ.
Microbiol. 3: 194–204.
Luton P.E., J.M. Wayne, R.J. Sharp and P.W. Riley. 2002. The
mcrA gene as an alternative to 16S rRNA in the phylogenetic
analysis of methanogen populations in landfill. Microbiology 148:
3521–3530.
Madden T.L., R.L .Tatusov and J. Zhan. 1996. Application of
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RDP-II (Ribosomal Database Project). Nucl. Acids Res. 29: 173–174.
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Rastogi G., D.R. Ranade, T.Y. Yeole, A.K. Gupta, M.S. Patole
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Polish Journal of Microbiology
2010, Vol. 59, No 3, 179–183
ORIGINAL PAPER
Optimisation of Synthetic Medium Composition for Levorin Biosynthesis
by Streptomyces levoris 99/23 and Investigation
of its Accumulation Dynamics Using Mathematical Modelling Methods
VESELIN S. STANCHEV1*, LUBKA Y. KOZHUHAROVA2, BORIANA Y. ZHEKOVA3
and VELIZAR K. GOCHEV 4
1 Department
of Automatics, Information and Control Systems, University of Food Technologies, Plovdiv, Bulgaria
2 Department of Biotechnology, University of Food Technologies, Plovdiv, Bulgaria
3 Department of Biochemistry and Molecular Biology, University of Food Technologies, Plovdiv, Bulgaria
4 Department of Biochemistry and Microbiology, P. Hilendarski University, Plovdiv, Bulgaria
Received 3 April 2010, accepted 25 May 2010
Abstract
The composition of a synthetic culture medium for levorin biosynthesis by Streptomyces levoris 99/23 was optimised using mathematical
modelling methods. The optimal concentrations of the medium components were established by means of an optimum composition design
at three factor variation levels. An adequate regression model was obtained. Levorin biosynthesis by Streptomyces levoris 99/23 in the
optimised synthetic medium was over 38% higher than in the initial medium. The antibiotic biosynthesis dynamics in the optimised culture
medium was studied by means of a non-linear differential equation system. The resultant model was valid.
K e y w o r d s: Streptomyces levoris 99/23, biosynthesis dynamics, mathematical modelling, optimisation of levorin biosynthesis
Introduction
Levorin is an antifungal preparation widely used
in medicine. The antibiotic biosynthesis mechanism
and the characteristics of its producers in a physiological aspect are studied mainly by means of synthetic
culture media (Belousova et al., 1970; Jakovleva,
1980). These media have strain-specific compositions
which are determined experimentally. Culture medium
optimisation in a quantitative and qualitative aspect
using mathematical modelling methods is insufficiently
studied. Orthogonal Latin rectangles were used by
Jakovleva (1980), and linear models were applied by
Gotchev et al. (2002). There are individual reports on
biosynthesis description using neural networks (XianFa et al., 2000). From a practical point of view, however, they are more suitable for process control rather
than investigation since kinetic constant values are
most often hidden either in the architecture or in the
weight coefficients of the neural network.
This paper aimed to determine the optimal concentrations of the synthetic culture medium components
for levorin biosynthesis by Streptomyces levoris 99/23
and study levorin accumulation dynamics using mathematical modelling methods.
Experimental
Materials and Methods
Microorganism. A Streptomyces levoris 99/23
strain stored in a lyophilised form in the Biotechnology Department’s collection at UFT was used as
a levorin producer (Kozhuharova et al., 2002). The
culture was maintained on a medium described by
Kozhuharova et al. (2008).
Media and cultivation conditions. The initial nutrient medium for S. levoris 99/23 cultivation, which
was subject to optimization, had the following composition (%): glucose 1.5; starch 2; (NH4)2SO4 0.6;
KH2PO4 0.005; KCl 0.1; MgSO4 0.25; CaCO3 0.3.
After pH adjustment to 7.2, and sterilization at 121°C
for 30 min, the nutrient medium was inoculated with
* Corresponding author: V.S. Stanchev, 26 Maritza Boulevard, 4002 Plovdiv, Bulgaria; phone: (+359) 32603898; fax: (+359)
32644102; e-mail: [email protected]
180
2% (v/v) spore inoculum containing 2.109 cfu/ml.
Strain cultivation and levorin biosynthesis were carried
out in 500 ml Erlenmayer flasks containing 50 ml of
each nutrient medium at a temperature of 28°C, on
a rotary shaker (220 min–1) for 96 h.
Mathematical modelling. The optimal concentrations of medium components were determined using
optimal composition design with three variation levels
of the factors (Koleva et al., 2005; Mason et al.,
2003). Such an approach enables generation of nonlinear regression models with a minimum number of
experiments:
Ymod = b0 +
3
Stanchev V.S. et al.
k
Σ
i=1
k
k–1 k
Σ bii.xi2 + iΣ= 1 j=Σ2 bij.xj.xj
i=1
bi.xi +
(1)
where: Ymod is the predicted response, bi, bij and bii are
coefficients accounting for the effect of each factor (xi),
of their interrelations (xi.xj), and those to the square of
two (xi2) respectively, and k is the number of factors.
The experimental data statistical processing and
the results analysis were performed using Anova
(Microsoft Excel 2003).
The process dynamics was studied by submerged
cultivation of the strain in the optimised culture medium. The input parameter values were approximated
by third order spline functions (Mathews and Fink,
2001). The calculation and optimisation procedures
were performed within the Eureka software environment (The Software Eureka 2000). The graphic presentation was based on Microsoft Excel 2003 and
Sigma Plot 9.0.
Assays. Levorin concentration in the culture medium was analysed according to the spectrophotometric method suggested by Bob et al. (1978) and expressed in mg/ml. One antibiotic activity unit (IU)
corresponds to 0.04 µg of levorin (Bolshakova et al.,
1989). Reducing sugars (substrate) concentration was
determined by the dinitrosalicylic acid method
(Miller, 1959). The biomass quantity was determined
after drying at 105°C to constant weight. pH was measured potentiometrically.
Results and Discussion
Following a series of single-factor experiments, the
variation interval for the basic culture medium components was determined. The real and coded values
of the independent variables are shown in Table I. The
remaining constituents were fixed at the following
levels (%): KCl 0.1; MgSO4 0.25; CaCO3 0.3.
The experimental data was formed as the mean
value of the results of six parallel experiments. The design matrix, the experimental results (Yexp) and model
values (Ymod) obtained by equation (2) are presented
in Table II.
Table I
Real and coded values of independent variables
Coded value
Factor (%)
–1
0
+1
X1: glucose
1.0
1.5
2.0
X2: starch
1.0
2.0
3.0
X3: (NH4)2SO4
0.4
0.6
0.8
X4: KH2PO4
0.001
0.0055
0.01
Table II
Optimum composition design for 4 factors and three levels
of their variation
No
X1
X2
X3
X4
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
1
1
1
1
1
1
1
1
–1
–1
–1
–1
–1
–1
–1
–1
–1
1
0
0
0
0
0
0
1
1
1
1
–1
–1
–1
–1
1
1
1
1
–1
–1
–1
–1
0
0
–1
1
0
0
0
0
1
1
–1
–1
1
1
–1
–1
1
1
–1
–1
1
1
–1
–1
0
0
0
0
–1
1
0
0
1
–1
1
–1
1
–1
1
–1
1
–1
1
–1
1
–1
1
–1
0
0
0
0
0
0
–1
1
Yexp (mg/ml) Ymod (mg/ml)
0.667
0.724
0.785
0.576
1.242
0.581
0.860
0.529
0.718
0.726
0.851
0.573
0.894
0.572
0.588
0.527
0.592
0.691
0.903
0.812
0.714
0.700
0.635
0.620
0.597
0.748
0.737
0.603
1.135
0.581
0.806
0.537
0.781
0.751
0.921
0.605
0.950
0.578
0.622
0.535
0.693
0.693
0.820
0.820
0.739
0.646
0.691
0.590
The analytical expression of the regression equation obtained is:
Ymod = 0.6928 +0.0472.X3+ 0.1007.X4 – 0.0467.X1.X2
– 0.0459.X2.X3 –0.0595.X2.X4– 0.0454.X1.X2.X4
– 0.0714.X2.X3.X4+ 0.1276.X22 – 0.1024.X42 (2)
The model was adequate at confidence level
" = 0.05 and degrees of freedom < = 9 (Table III).
The analysis of (2) revealed several considerations.
The two carbon sources (X1, X2) were not present
on their own in the model but the coefficient before
X22 in (2) had higher positive value. Its effect in Ymod
in relation to b0 was 18.4%. This showed that
S. levoris 99/23 preferred a carbon source with a relatively high molecular mass. On the other hand, X1
3
Mathematical modeling for levorin biosynthesis optimisation
181
Table III
Statistical analysis results according to Anova
Parameter
Df
SS
MS
F
Significance F
Regression
Residual
Total
9
14
23
0.5176539
0.0788534
0.5965073
0.057517098
0.005632389
10.21185
9.32311E-05
Df – degree of freedom; SS – sum square; MS – mean square; F – Fisher coefficient
participated in two terms of (2) with total weight of
13.3% in relation to b0. Since the effect of both factors in Ymod is commensurate, there is sufficient
ground to believe that they are in optimum correlation ensuring the absolute extremum of (2).
X4 had a pronounced individual influence in the
model (15%, which is comparable to that of X22).
KH2PO4 was a source of phosphorus, an important
element for the S. levoris growth and levorin biosynthesis regulator. In this respect, the results obtained
are in agreement with the theoretical formulations and
data reported by other authors (Belousova et al., 1970).
The nitrogen source can be seen as having no significant effect on levorin biosynthesis.
The conditions maximising (2) were found by
means of a gradient optimisation method (The Software Eureka 2000):
max
Y mod
= 1.135 mg/ml, at: X1 = + 1; X2 = – 1;
(3)
X3 = + 1; X4 = + 1
The response function (2), in graphic form, with
variation of X1 and X2 within the limits set in Table I
and optimum values of X3 and X4 (+1), is presented
on Fig. 1.
The hypothesis of equality of the mathematical exmax
pectation of the experiment results Yexp
under the
optimal conditions with that of the predicted
max
Ymod
= 1.135 was checked (Mason et al., 2003). The
max
values (mg/ml) were as follows: Yexp,
1 = 1.20;
max
max
max
max
Y exp, 2 = 1.22; Yexp, 3 = 1.16; Yexp, 4 = 1.15; Yexp,
5 = 1.10;
max
max
max
Yexp, 6 = 1.12; Yexp, 7 = 1.06; Yexp, 8 = 1.00. For degrees of
freedom< = 7 and confidence level "= 0.05, tcrit.= 2.365
(Student’s table). Since tcalc.= 0.535<tcrit., there was no
max and Y max
statistically significant difference betweenY exp
mod
A significant increase in levorin biosynthesis was
detected with the optimised medium. The yield
achieved was with 38% higher in comparison to the
yield with the initial medium.
The dynamics of levorin biosynthesis by S. levoris
99/23 with the optimised medium was modelled by
means of a system of the following non-linear differential equations:
dX(t) = µ(t) X(t)
dt
(4)
1 dX(t)
dS(t)
=–
–Y2X(t)
Y1 dt
dt
dP(t)
dX(t)
= "1
+ "2 X(t)
dt
dt
S(t)
– $X(t)
µ(t) = µm
ks + S(t)
(5)
(6)
(7)
X(t), S(t), and P(t) are the biomass concentrations, substrate concentration, and levorin concentration respectively when the process operates in the periodic mode,
Y1 is an economic coefficient, Y2 is related to the rate
of substrate assimilation by the cells in a stationary
phase of the process, ks is a saturation constant, µm is
the maximum specific growth rate, $ is the decay coefficient, "1 is the coefficient of substrate transformation
into a metabolism product, and "2 is levorin accumulation rate in the stationary phase of the process.
Using (7), the microbial population growth was
modelled in the presence of substrate limitation – modified Verhulst law.
The experimental data were formed by using the
mean value of the results from six parallel experiments on the process dynamics. The lag phase time
(24 h) was excluded from the data set.
The kinetic constant values in the model were determined using an optimisation procedure minimising
the following criterion:
Fig. 1. Ymod = f (X1, X2) at an optimum value of X3 and X4 + 1.
182
J=
Stanchev V.S. et al.
n
[(Xexp, i – Xmod, i)2 + (Sexp, i – Smod, i)2
Σ
i=1
3
+
+ (Pexp, i – Pmod, i)2] → min
(8)
where exp, i and mod, i denote the process parameters according to the experimental data and models
(4–7), and n is the number of observations.
With this setup, the numerical values of the kinetic constants for the model were determined to be as
follows:
µm = 0.05583 h–1; ks = 0.97 mg/ml;
$ = 0.01054 ml/mg.h; Y1 = 0.233;
Y2 = 0.0077 h–1; "1 = 0.169; "2 = 0.00117 h–1
The process dynamics is presented graphically in
Fig. 2, Fig. 3 and Fig. 4. There was a good coincidence
between the experimental and model results. P(t)
reached a maximum equal to 1.08 mg/ml at t =131.5 h
(Fig. 4). This value was obtained after approximation
of the analytically calculated P(t) data according to (6),
with a second-order spline function and maximisation
of P(t), within the 108–144 h time interval and degree
of freedom t. No check of the experiment reproduci-
Fig. 2. Growth dynamics of S. levoris 99/23:
(◆) experimental data; (¨) model data.
Fig. 3. Dynamics of substrate assimilation by S. levoris 99/23:
(◆) experimental data; (¨) model data
Fig. 4. Dynamics of levorin biosynthesis by S. levoris 99/23:
(◆) experimental data; (¨) model data.
bility was run since it generally coincided with that of
the studies on the process statics.
The kinetic constants can be interpreted in the following manner in a biotechnological aspect.
Under the experimental conditions, the value of
decay coefficient $, was 5.3 fold lower than the value
of µm. At time point t = 131.5 h, the available biomass
exceeded the initial biomass by 10.7 folds.
Over 23% of the substrate was assimilated for biomass accumulation (Y1), and around 18.5% was used
for maintaining the life activity in the stationary phase
of the process. Since the values mentioned were close,
it could be considered that approximately the same
substrate amount was spent for both purposes.
The main antibiotic quantity was synthesised in the
process stationary phase, and it was 6 fold higher than
the value in the exponential phase, at a priori specified stationary phase duration of 36 h.
The ratio "1/("2.)t) for )t = 36 h, in the stationary
phase was 1:6. This came as another proof that levorin
biosynthesis took place mainly at this stage of the microbial population growth.
At the end of the process, over 15.6% of the substrate remained unassimilated (Fig. 3). This was an
indication for the presence of a critical value in respect
to S0 at which, the function would reach its maximum
along with the complete substrate utilisation. Such an
effect was not registered under the conditions of the
experiment.
The material balance of the process came to the
following considerations. The substrate consumption
for biomass accumulation in the exponential growth
phase of S. levoris 99/23 was about 23.3%, and the
value for life activity during the process stationary
phase was about 18.5%. About 15.6% of the substrate
was unassimilated at the end of the biotechnological
process. For antibiotic synthesis in the exponential
growth phase about 16.9% of the substrate was used,
and the corresponding value for the stationary phase
3
Mathematical modeling for levorin biosynthesis optimisation
was 27.7%. Total carbon source consumption was determined to be 102%.
We consider that the results obtained provide an
objective idea of the material balance in the system
taking into account the subjective, methodological and
instrumental error in the experimental data analysis.
The dynamics model of the levorin biosynthesis
by S. levoris 99/23 was valid. It described in detail
even the lysis processes at the end of the stationary
phase. In proof of this statement, the experimentmodel error dispersion values are presented for all
observation points as follows: F2x=0.0025; F2s=0.26;
F2p=0.0005. The main share in criterion (8) is mainly
attributed to S since its natural values are 100 and
more times higher than those of X and P.
Conclusion. As a result of the optimisation of the
nutrient medium for levorin biosynthesis by S. levoris
99/23 using mathematical modelling methods, the optimal composition of the medium was determined.
The yield achieved with this medium was 38% higher
in comparison to the initial one.
The dynamics of the antibiotic biosynthesis by
S. levoris 99/23 was studied by means of a non-linear
differential equation system. The kinetic constant
values were calculated for Verhulst model, describing
the presence of substrate limitation. A valid model
of the dynamics of levorin biosynthesis by S. levoris
99/23 was obtained and the material balance of the
process was assayed.
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3
Polish Journal of Microbiology
2010, Vol. 59, No 3, 185–190
ORIGINAL PAPER
Chromate Reduction by Cell-Free Extract of Bacillus firmus KUCr1
GOPI BALLAV SAU, SWAGATA CHATTERJEE and SAMIR KUMAR MUKHERJEE*
Department of Microbiology, University of Kalyani, Kalyani 741235, India
Received 9 February 2010, revised 10 June 2010, accepted 20 June 2010
Abstract
Microbial enzymatic reduction of a toxic form of chromium [Cr(VI)] has been considered as an effective method for bioremediation of this
metal. This study reports on the in vitro reduction of Cr(VI) using cell-free extracts from a Cr(VI) reducing Bacillus firmus KUCr1 strain.
Chromium reductase was found to be constitutive and its activity was observed both in soluble cell fractions (S12 and S150) and membrane
cell fraction (P150). The reductase activity of S12 fraction was found to be optimal at 40 µM Cr(VI) with enzyme concentration equivalent
to 0.493 mg protein/ml. Enzyme activity was dependent on NADH or NADPH as electron donor; optimal temperature and pH for better
enzyme activity were 70°C and 5.6, respectively. The Km value of the reductase was 58.33 µM chromate having a Vmax of 11.42 µM/min/mg
protein. The metabolic inhibitor like sodium azide inhibited reductase activity of membrane fraction of the cell-free extract. Metal ions like
Cu2+, Co2+, Ni2+ and As3+ stimulated the enzyme but others, such as Ag+, Hg2+, Zn2+, Mn2+, Cd2+ and Pb2+, inhibited Cr(VI) reductase activity.
K e y w o r d s: Bacillus firmus, cell-free extract, chromium reductase
Introduction
Application of Cr-resistant bacteria to detoxify
Cr(VI) has been considered as an effective bioremediation method (Ohtake and Silver, 1994; Shakoori
et al., 1999; Ganguli and Tripathi, 2002; Cheung and
Gu, 2007; Sau et al., 2008). Bioremediation of Cr(VI)
can occur by enzymatic reduction of Cr(VI) to Cr(III)
via Cr(V) and Cr(IV) intermediates (Camargo et al.,
2003; Xu et al., 2004; Xu et al., 2005; Pal et al., 2005;
Cheung et al., 2006) or through immobilization (Losi
et al., 1994). In the course of aerobic reduction, the
cytosolic fractions of most chromium resistant bacteria exhibit Cr(VI) reductase activity (Campos et al.,
1995; Cervantes et al., 2001), while under anaerobic
conditions, CrO42– is used as a terminal electron acceptor and is reduced in the cell membrane (McLean
and Beveridge, 2001).
Requirement of electron supply is obvious during
the reaction process, considering the process a redox
reaction. The enzyme responsible for Cr(VI) reduction has not yet been well characterized, though there
are reports on reduction kinetics of Cr(VI) reduction by cell-free extracts (CFE) with varied results
(Ishibashi et al., 1990; Suzuki et al., 1992; McLean
and Beveridge, 2001; Park et al., 2000; Camargo et al.,
2003; Pal et al., 2005). Studies using partially purified Cr(VI) reductase from Pseudomonas ambigua
G-1 (Suzuki et al., 1992) and P. putida MK1 (Park
et al., 2000) have indicated Cr(VI) reduction using
NADH or NADPH as a source of electrons. In Bacillus sp. ES29, Cr(VI) reductase was found in the
soluble fraction of CFE, which utilizes NADH as
electron donor (Camargo et al., 2003).
Cr(VI) reducing Bacillus firmus KUCr1 has been
reported and its potential to reduce Cr(VI) using
whole cells under culture condition was documented
earlier (Sau et al., 2008). This study reports on the
reduction of Cr(VI) by CFE of this strain, effects of
different electron donors, metal ions, inhibitors, pH
and temperature on its Cr(VI) reducing activity, and
cellular localization of Cr(VI) reductase.
Experimental
Materials and Methods
Microorganism and growth condition. A Cr(VI)
resistant Bacillus firmus KUCr1 (NCBI GenBank 16S
rDNA sequence Accession No EU784699) was earlier reported (Sau et al., 2008) and used in this study.
* Corresponding author: S.K. Mukherjee, Department of Microbiology, University of Kalyani, Kalyani 741235, India; phone:
(+91) 3325827315; fax: (+91) 3325828282; e-mail: [email protected]
186
3
Sau G.B. et al.
Cells were grown aerobically in PYG medium (peptone 10 g/l; yeast extract 5 g/l; glucose 3 g/l; pH 7.2)
supplemented with or without (control) 0.1 mM Cr(VI)
as K2CrO 4 under continuous shaking on a rotary
shaker at 35°C for 24 h.
Preparation of cell-free extract (CFE) and cellular fractions. Cell-free extract (CFE) and cellular fractions were prepared following McLean and Beveridge
(2001) with little modification. Cells grown in PYG
medium were harvested at the exponential phase,
washed three times in 0.2 M phosphate buffer (pH 7.2)
by centrifugation (4000× g at 4°C), resuspended in the
same buffer to have a 10 ml suspension and kept in an
ice bath. Cells were disrupted with an ultrasonicator
(Hielscher Ultrasound Technology, UP50H). Power
was applied five times in 1min pulses with 100%
amplitude. After sonication the suspension was centrifuged at 12 000 × g for 10 min at 4°C to obtain
a soluble fraction (S12). Five ml of S12 fraction was
centrifuged at 150 000× g for 90 min at 4°C to obtain
S150 fraction. The pellet fraction was washed twice
with 0.2 M phosphate buffer (pH 7.2) to remove
soluble chromium reductase, if any and was resuspended in same buffer (5 ml) to obtain membrane
fraction (P150). Equivalent volume of S12, S150 and P150
fraction were assayed for studying the cellular localization of Cr(VI) reductase enzyme.
Cr(VI) reductase assay. Cr(VI) reductase activity of CFE was assayed following the procedure of
Park et al. (2000). The reaction mixture (1 ml) for
the enzyme assay contained 0.2 mM K2CrO4, 0.2 mM
NADH and 400 µl of CFE in 0.2 M phosphate buffer
(pH 7.2). The reaction was stopped by adding 0.5 ml
of 20% trichloroacetic acid (Horitsu et al., 1987).
Reduction of Cr(VI) was measured by estimating
the decrease in Cr(VI) in the reaction mixture after
30 min of incubation at 35°C and quantified colorimetrically using 1,5-diphenylcarbazide as the complexing reagent (Urone, 1955). To eliminate the abiotic reduction of Cr(VI), if any, a control set was
prepared through out the course of the study without
putting any biotic component including bacterial cell.
Whenever required, abiotic reduction of Cr(VI) has
been subtracted from the total reduction to get the
exact influence on chromium reduction by the biotic
factors. Amount of protein in the CFE was estimated
by the folin-phenol method (Lowry et al., 1951)
using bovine serum albumin as the standard. One unit
of Cr(VI) reductase activity was defined as the
amount of enzyme which decreased l.0 µM Cr(VI)
per min at 35°C. The effect of pH and temperature
on Cr(VI) reductase were measured at different pH
values (4 to 10.6) of the reaction mixture at 35°C
and at different reaction temperature (20 to 100°C) at
pH 7.2 respectively.
Results and Discussions
The reductase activities of the soluble fraction (S12)
from both the induced and uninduced cells of B. firmus
KUCr1 were almost similar with regard to time course
(Fig. 1). This study revealed that the chromate reductase in this strain is constitutive, which supports earlier
reports on the enzymatic reduction of Cr(VI) under
aerobic conditions (Bopp and Ehrlich, 1988; Campos
et al., 1995; Wang and Xiao, 1995; McLean and
Beveridge, 2001; Pal et al., 2005), though an inducible
reductase in the soluble fraction of CFE of Ochrobactrum sp. was reported (Thacker and Datta, 2005).
As the reductase activity was found to be constitutive, thus for further experiments CFE (S12) was prepared from cells grown in Cr(VI)-free medium for
characterization of chromate reductase activity.
The effect of initial concentration of Cr(VI) on
reductase activity of S12 fraction was determined at
a concentration range of 0 to 80 µM of Cr(VI). Specific activity increased with increasing initial concentration of chromate up to 40 µM, after that it slowed
down but reduction continued (Fig. 2a) with enzyme
equivalent to 0.493 mg protein/ml. The saturation
kinetics of Cr(VI) reduction of S12 fraction fit with
the linearized Lineweaver-Burk plot, and the apparent Michaelis-Menten constant (Km) was found at
58.33 µM chromate and Vmax was 11.42 µM per
min/mg protein (Fig. 2b). The Km and Vmax values differed from the enzyme activity of the CFE of Bacillus
sp. ES 29 (Camargo et al., 2003), B. subtilis (Garbisu
et al., 1998), B. Sphaericus AND 303 (Pal et al., 2005),
P. putida (Ishibashi et al., 1990; McLean and Beveridge
Fig. 1. Cr(VI) reduction by the extracts (S12) from both induced
[0.1 mM Cr(VI) in the medium] and non-induced cells of
B. firmus KUCr1. The reaction mixture contained 0.2 mM Cr(VI)
and 0.2 mM NADH as electron donor in 0.2 M phosphate buffer
(pH 7.2) and the reaction temperature was 35°C.
3
187
Chromate reduction by B. firmus cell-free extract
Fig. 3. Effect of pH on chromate reductase activity (S 12)
of B. firmus KUCr1 at 35°C for 30 min of incubation.
Fig. 2. Kinetics of Cr(VI) reduction of cell-free extract (S12) of
B. firmus KUCr1 at different Cr(VI) concentrations. Reaction
time was 30 min at 35°C (a). Linearized Lineweaver-Burk plot
for Cr(VI) reduction of cell-free extract (S12) (b).
2001; Park et al., 2000) and in P. ambigua G-1 (Suzuki
et al., 1992). Lower K m value of Cr(VI) reductase
suggests higher affinity for the substrate, at least with
what was found with the cell-free extract in this strain.
The effect of pH on reductase activity was assessed
at a pH range of 4.0 to 10.6 using 0.2 M of citrate
buffer (pH 4.0 to 5.6), 0.2 M of phosphate buffer (pH
6.0 to 7.6), 0.2 M of tris-HCl buffer (pH 8.4 to 8.8)
and 0.2 M of glycine-NaOH buffer (pH 9.2 to 10.6)
separately. The reductase activity achieved a maximum at pH 5.6 (Fig. 3). Similarly, the optimum temperature for highest Cr(VI) reduction was found at
70°C (Fig. 4). These results varied from other earlier
reports with B. sphaericus AND 303, where they were
30°C and 6.0 respectively (Pal et al., 2005) and with
P. putida MK1 (Park et al., 2000) or P. ambigua G-1
(Suzuki et al., 1992), the optimal temperature and pH
being 80°C, 50°C, and pH 5.0, 8.6 respectively.
The effect of electron donors, inhibitors and metal
ions on chromate reduction by CFE (S12) of B. firmus
KUCr1 was determined. 0.2 mM each of NADH,
NADPH, glutathion, D-glucose, and D-fructose were
Fig. 4. Effect of temperature on chromate reductase activity (S12)
of B. firmus KUCr1. The assay was conducted in 0.2 M phosphate
buffer (pH 7.2) for 30 min.
used as electron donors. Among the electron donors
used, only NADH showed a significant effect on chromate reductase activity (72% activity over control).
NADPH gave 32% less activity than NADH (Table I).
Table I
Effect of electron donor on Cr(VI) reductase activity
in cell-free extracts (S12) of B. firmus KUCr1
Electron donor
(0.2 mM)
None (control)
NADH
NADPH
Glutathione
D-glucose
D-fructose
a
b
Specific activitya
(U/mg protein)
2.40 (± 0.01)
8.65 (± 0.06)
5.87 (± 0.08)
2.53 (± 0.02)
2.89 (± 0.01)
2.70 (± 0.07)
Relative specific
activityb (%)
27.74
100.00
67.86
29.24
33.41
31.21
Data are the mean of three replications plus standard error. The reaction mixture contained 0.2 mM Cr(VI) in 0.2 M phosphate buffer
(pH 7.2) and was incubated for 30 min at 35°C.
{(Specific activity) / (specific activity)NADH } X 100
188
3
Sau G.B. et al.
Fig. 5. Effect of azide (NaN3, 0.2 mM)) and some selected metals
(0.2 mM) on Cr(VI) reductase activity in the cell-free extracts
(S12) of B. firmus KUCr1. Data are the mean of three replications
with error bars.
In the presence of glutathion, D-glucose, and D-fructose,
the non-enzymatic reductants of Cr(VI), the activity of
the reductase was almost equal to that of the control.
The cell-free enzyme of B. firmus KUCr1 required
NADH or NADPH as an electron donor for better
enzymatic Cr(VI) reduction. The reductase became
sharply more active in the presence of NADH than
NADPH, suggesting the requirement of a cofactor for
catalytic activity. NADH dependent Cr(VI) reduction
was also advocated by several researchers earlier in
Bacillus (Garbisu et al., 1998; Camargo et al., 2003)
and in Pseudomonas (Suzuki et al., 1992; Park et al.,
2000). Our study also supports the earlier observations on the role of nonenzymatic reductants like glutathione, D-glucose and D-fructose on Cr(VI) reduction by CFE (Branca et al., 1990; Shi and Dalal, 1990;
Suzuki et al., 1992).
Cr(VI) reduction by CFE was significantly inhibited in the presence of 0.2 mM sodium azide (Fig. 5).
The metal cations, Ag+, Zn2+, Cd2+, Pb2+, Mn2+, and
Hg2+ inhibited reductase activity by more than 50%
over the control in the reaction mixture (Fig. 5). However, Co2+, Ni2+, As3+ and Cu2+ stimulated the activity
of CFE. The order of stimulation by these metal cations in reductase activity was found to be As3+ > Ni2+
> Co2+ > Cu2+.
In this study, Cr(VI) reductase activity was found
to be inhibited by a respiratory inhibitor, sodium azide
(0.2 mM). Though chromate reductase in CFE was
reported to be unaffected by azide in Escherichia coli
ATCC 33456 (Shen and Wang, 1993), Bacillus sp.
ES29 (Camargo et al., 2003) and in B. megaterium
TKW3 (Cheung et al., 2006) by 1.0 mM NaN3. However, inhibition of Cr(VI) reduction due to NaN 3 in
live cells of a Bacillus subtilis strain was reported
(Garbisu et al., 1998). Inhibition of a cytoplasmic
membrane associated Cr(VI) reductase by azide was
also reported in Shewanella putrefaciens MR-1 (Myers
et al., 2000). The interference of azide in Cr(VI)
reduction in this study and views from earlier reports
suggest the possible coexistence of Cr(VI) reductase
in the cytosol and membrane as well. However, further investigations are required to elucidate this particular feature.
The cations Hg2+ and Ag + (0.2 mM) inhibited reductase activity. The noncompetitive inhibitory effect
of Hg2+ and Ag+ on Cr(VI) reduction in P. putida was
reported earlier (Ishibashi et al., 1990). The inhibitory effect of Hg2+ is expected, because of its affinity
for ligands containing thiol (-SH) group of a variety
of enzymatic proteins. Enzymatic reduction of Cr(VI)
by CFE of B. firmus KUCr1 was stimulated by Cu2+,
As3+, Ni2+ and Co2+ at 0.2 mM concentration separately, though Pal et al. (2005) reported the inhibition
of Cr(VI) reductase activity of B. sphaericus AND
303 by Ni2+ and Co2+ at 100 µM concentration. The
reductase activity was found to be unaffected by As3+
in P. putida MK1 (Park et al., 2000). Stimulation of
enzyme activity by Cu2+ might be due to its nature as
a prosthetic group of many reductase enzymes and also
indirectly involved in the protection of chromate reductase from O2, for oxygen-sensitive enzyme (Ettinger
1984; Camargo et al., 2003). Abe et al. (2001) reported
that Cu2+ acting as electron-transport protection or
acting as a single electron redox center and as a shuttle
for electron between protein subunits. Zn2+, Cd2+,
Pb2+ and Mn2+ inhibited the reductase activity at
0.2 mM. Inhibitory effects by Cd2+ and Zn2+ support
the earlier views (Park et al., 2000; Pal et al., 2005;
Desai et al., 2008). On the contrary, Camargo et al.
(2003) showed slight stimulatory effect of Mn2+ on
Cr(VI) reductase in Bacillus sp. ES 29. These variations seem to be due to the different functional nature
of the Cr(VI) reductase in B. firmus KUCr1, which
warrants further investigation.
Cr(VI) reductase activities by different cellular
fractions are presented in Table II. In the presence
of electron donor (0.2 mM NADH) S150 fraction
showed higher activity than fractions S12 and P150.
Table II
Localization of Cr(VI) reductase in cell fractions
of B. firmus KUCr1 and its catalytic activity
Cellular
fractiona
a
b
c
% Cr(VI)
reductionb
Specific activityc
(U/mg protein)
S12
14.21
9.12 (± 0.03)
S150
15.66
14.68 (± 0.04)
P150
6.98
7.34 (± 0.04)
S stands for soluble cellular fractions and P stands for membrane fractions (see Materials and Methods).
Reaction mixture contained 0.2 mM Cr(VI) in phosphate buffer (pH
7.2) and was incubated for 30 min at 35°C.
Data are the mean of three repetitions plus standard error.
3
Chromate reduction by B. firmus cell-free extract
The percentage of Cr(VI) reduction by fraction P150
is significantly less compared to other fractions, but
the specific activity signifies its catalytic function,
suggesting the occurrence of some membrane-bound
protein responsible for Cr(VI) reduction. In Bacillus
QC1-2 (Campos et al., 1995), B. sphaericus AND 303
(Pal et al., 2005) and P. putida (Ishibashi et al., 1990;
Park et al., 2000) the chromium reductase activities
were reported to be associated with the cytosolic and
soluble fractions. In Enterobactor cloacae (Wang
et al., 1990), S. putrefaciens MR-1 (Myers et al.,
2000), B. megaterium TKW3 (Cheung et al., 2006)
and P. fluorescences (Bopp and Ehrlich, 1988) Cr(VI)
reductase appears to be membrane associated. Lovley
and Phillips (1994) reported that both the soluble and
membrane fractions reduced chromate in Desulfovibrio vulgaris but soluble protein fraction reduced
Cr(VI) faster than the membrane fraction did. In
Bacillus cereus S-6 (Iftikhar et al., 2007) Cr(VI)
reduction occur both in cytosolic and membrane fraction of this strain but percentage of reduction in cytosolic fraction is higher than membrane fraction. It
seems that KUCr1 harbors Cr(VI) reductase constitutively both in membrane and cytosol. Thus further investigations to visualize the exact pathway of Cr(VI)
reduction in this particular strain are required.
Enzyme inhibition by respiratory inhibitors like
NaN3 and Cr(VI) reductase activity by the P150 fraction suggest the co-existence of a membrane-bound,
respiratory-chain-linked reductase activity in this
strain; study with purified proteins of both the cytosolic and membrane fractions would provide more
knowledge about the exact pathway of Cr(VI) in this
particular strain. Moreover, the feature of higher temperature optima of Cr(VI) reductase of the cytosolic
soluble fraction in KUCr1 suggests the possibility to
clone the gene into a thermophilic bacteria, so that it
could offer a decisive advantage in chromium bioremediation under conditionally elevated temperature
or in a bioreactor system using immobilized enzyme.
Acknowledgement
This work was supported by the grant received from the University of Kalyani, India.
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Polish Journal of Microbiology
2010, Vol. 59, No 3, 191–200
ORIGINAL PAPER
Occurrence and Characterization of Colletotrichum dematium (Fr.) Grove
ZOFIA MACHOWICZ-STEFANIAK
Department of Phytopathology, University of Life Sciences Lublin, Poland
Received 9 November 2009, revised 20 January 2009, accepted 1 March 2010
Abstract
Colletotrichum dematium was isolated from caraway for the first time in Poland in 2005. Isolations of this fungus were repeated in 2006
and 2007. The cultures of fungus were obtained from superficially disinfected leaves, root necks, roots, stems and umbels. The isolates
were identified on culture media: PDA and malt agar with addition of pieces of caraway stems and on the base of macro and microscopic
structures. Studies on the biotic effect between C. dematium and other species of phyllosphere fungi of caraway showed that the majority
of the studied species limited the growth and development of C. dematium, but the size of the limiting effect was different. The species
from Trichoderma and Gliocladium genera were the most effective against C. dematium, causing degeneration and lysis of hyphae and
precluded the formation of the pathogen’s acervuli and conidia. C. dematium in dual culture with E. purpurascens, A. radicina, S. sclerotiorum,
B. cinerea and R. solani produced an inhibition zone which indicated its capacity for antibiosis.
K e y w o r d s: Colletotrichum dematium, biotic activity, caraway isolates, phylosphere fungi
Introduction
Fungi from the genus Colletotrichum occur in all
climatic zones and cause diseases of various plant
species, especially in hot and moderate climates. They
can be polyphagic but some of them are pathogens of
only one species of host plant (Frencel et al., 1997;
Sutton, 1980). Till the middle of the 20th century, over
1000 species of these fungi were described basing on
the morphological structures of conidia, setoses and
host plant (Von Arx, 1957). As a result of studying
these fungi on artificial culture media in the laboratory,
the number of species was reduced to 40 (Sutton,
1980). At present, the numerous existing species of the
genus Colletotrichum are gathered in one common
species on the basis of genetic diversity (Frencel et al.,
1997). The following species belong to the commonly
occurring pathogenic species: C. gloeosporioides
(Penz.) Sacc. (teleomorph: Glomerella cingulata
(Stonem.) Spauld. et Schrenk), C. lindemuthianum
(Sacc. et Magn.) Br. et Cav., C. atramentarium (Berk.
et Br.) Taubenh., C. acutatum Simmonds, C. lini
(Manns) Bolley, C. orbiculare (Berk. et Mont.) Arx and
others (Sutton, 1980; Farr et al., 1995; Frencel et al.,
1997; Gärber and Schenk, 2001).
C. dematium, the genus typical species, is a saprotroph and colonizes various plant species as a second-
ary pathogen and pathogenic strains of this fungus
cause plant diseases (Von Arx, 1957; Sutton, 1980;
Farr et al., 1995). C. dematium f. circinans cause
anthracnose of onion cultivated in various climatic
regions in the world (Sutton, 1980). The occurrence
of C. dematium was ascertained in India on various
pea-plant cultivars and in South Africa on stems and
pods of cowpea (Smith et al., 1999; Shinde et al.,
2003). The species was isolated from dying plants of
Catharanthus roseus (L.) G. Don in Florida in 1991
and positive results of artificial infection of plants
were obtained (McMillan and Graves, 1996). C. dematium caused anthracnose of spinach on some farms
in Australia and the harmfulness of this fungus for
various spinach cultivars was confirmed by pathogenicity tests (Washington et al., 2006). In Japan, the
harmfulness of C. dematium for Japanese radish was
confirmed by pathogenicity tests (Smith et al., 1999).
Anthracnose caused by C. dematium was discovered
on various cultivars of strawberry in India, which fact
was confirmed by pathogenicity tests (Singh et al.,
2003). C. dematium occurred on mulberry in Japan
and on various species of plants from the Amaryllidaceae family (Yoshida and Shirata, 1999; BonillaBernal et al., 2003). In post-culture liquids of C. dematium f. sp. epilobii, the pathogenic species for willow
herb (Epilobium angustifolium L.), the presence of
* Corresponding author: Z. Machowicz-Stefaniak, Department of Phytopathology, University of Life Sciences Lublin, Poland;
e-mail: [email protected]
192
3
Machowicz-Stefaniak Z.
secondary metabolites with phytotoxic and zootoxic
abilities was found (Abou-Zaid et al., 1997; Mendiratta
et al., 2005).
As a result of many-years’ studies conducted on
diseases of herb plants C. dematium was isolated for
the first time on caraway in Poland in 2005. Therefore,
attention to the occurrence of this fungus in the next
years of studies was directed and macroscopic and
microscopic features of isolates of the pathogen and
the biotic effects between C. dematium and other species of phyllosphere fungi of caraway were studied.
Material and Methods
The studied material consisted of isolates of Colletotrichum dematium (Fr.) Grove obtained from the
organs of one-year-old and two-years-old plants of
caraway cultivated in the Lublin region in the years
2005–2007 (Table I). The artificial culture method
and malt agar medium were used for the isolation of
this fungus (Machowicz-Stefaniak and Zalewska,
2008). One- spore cultures of 15 isolates of fungus:
K117, K123, K425, K426, K510, K514, K612, K625,
K626, K 628, K630, K631, K633, K651, K657 were
chosen randomly from our professional collection. For
identification, the isolates were cultured on malt agar
medium with an addition 50 g/dm 3 of 2–3 mm pieces
of caraway stems and on PDA medium (Difco), in
a thermostat, at the temperature 24°C, in dark conditions for 14 days (Machowicz-Stefaniak, 2009). The
character of cultures, the color of the averse and the
reverse, the formation and morphology of fungus
acevuli and conidia were studied at this time. To determine the structures mentioned above, the measurements of 150 acervuli (15 isolates per 10 acervuli) and
600 conidia (15 isolates per 40 spores) were made.
Moreover, the size of setoses and appressoria of the
studied isolates was determined. The photos of the
above mentioned morphological elements were taken
using light and scanning – SEM microscope. To identify the studied isolates, the descriptions of Von Arx
(1957), Pidoplièko (1977) and Sutton (1980) were used.
To study the biotic effects of fungi, 3 isolates of
C. dematium: K425, K426, K625 and 22 isolates
of fungi species mentioned in Table III were taken.
Those isolates were chosen randomly from our professional collection of fungi gathered in the years
2001–2008, as a result of a study on diseases of caraway (Machowicz-Stefaniak and Zalewska, 2004; 2008;
Machowicz-Stefaniak, 2009).
Because of the lack of information concerning the
biotic relation between C. dematium and other fungi,
the maximum number of fungal species was taken for
this study, irrespective of the frequency of their isolation from caraway (Machowicz-Stefaniak and Zalew-
Table I
Occurrence of C. dematium on aboveground organs
of caraway (Carum carvi L.) in 2005–2007
Organs
Years
leavs
stems
base of stems
roots
umbels
2005
++
++
+++
+++
+++
Participation of izolates
2006
2007
++
++
+
+++
+
–
++
++
++
+++
+ – frequency of occurrence < 5%
++ – frequency of occurrence from 5 to 10%
+++ – frequency of occurrence > 10%
ska, 2004; 2008; Machowicz-Stefaniak, 2009). The
species Gliocladium catenulatum, G. fimbriatum,
G. roseum and Trichoderma viride were taken from
other cultivated plants, because they were not isolated
from caraway.
The study of biotic effects was carried out using
the biotic series method on PDA (Difco) medium,
which was elaborated for soil fungi community firstly
(Mañka, 1974; Mañka, 1995). This method was
adapted for fungi colonizing the phyllosphere of
plants (Mañka, 1995; Machowicz-Stefaniak, 1998;
Król and Machowicz-Stefaniak, 2008). The two-organism cultures consisting of C. dematium and one of
the fungi representing the community component
were studied on PDA medium in Petri dishes, according to the method by Mañka (1974) and Mañka
(1995). The dishes with medium on which the mycelium of a single fungal species was placed constituted
the control. For each combination, i.e. for C. dematium with a species of fungus representing the community component and control, 4 replications were
made. The biotic effect of the fungi in dual cultures
was evaluated after 12 days of common growth, but
in the case of Gliocladium spp. after 24 days, at 23°C,
in dispersion light, based on an eight-degree scale.
One colony being surrounded by other species, the
occurrence of the inhibition zone between them and
the reduction of the colony size were taken into account while the IBEs were evaluated (Mañka, 1995).
If the colony of C. dematium was overgrown by other
species of fungi, the appearance of mycelium and
conidia of the studied fungus were evaluated. The biotic effect of fungi representing the phyllosphere of
caraway on C. dematium was estimated as an individual biotic effect (IBE). The size of IBE consisted
of the arithmetic sum of values for surrounding of
the colony, the inhibition zone and the reduction
of colony size. The size of IBE indicates the effect
of one isolate of the community species on pathogen
growth (Mañka, 1974). Positive IBE indicates suppressive effect on pathogen growth, negative – indi-
3
193
Grove of Colletotrichum dematium (Fr.) from caraway
Fig. 2. Acervuli and setose of C. dematium in light photo
microscope (Photo E. Zalewska).
Fig. 1. 14-day-old colony of C. dematium isolate K 425 on the
PDA medium, at 25°C (Photo E. Zalewska).
cate slack of suppressive effect on pathogen growth,
while the value of the effect may be „0” indicating
neutral influence (Mañka, 1974; Mañka, 1995).
Results
The isolates of C. dematium were obtained for the
first time in 2005 from the leaves, the neck of roots
and the roots of 0.66% of caraway seedlings. The isolations of fungi repeated in 2006 and 2007, respectively from 1.2% and 2.16% of plants in the second
year of cultivation. The cultures of fungus were
obtained most often from the stems, the umbels and
the roots with different frequency in different years
(Table I). C. dematium was isolated from the parts
of plants with nonspecific etiological and disease
lesions, which can indicate the presence of the pathogen in the plant tissues. On the other hand, among the
fungi isolated from plants on malt agar medium there
were isolates coloring the medium violet and forming
dark gray, velvet mycelium with numerous brown appressoria an the top and crossing hyphae, the organs
characteristic of Colletotrichum genera. However,
acervuli and spores of fungi were obtained scarcely
on malt agar medium with an addition of pieces of
caraway stems and on PDA medium.
The one-spore-cultures of this fungus chosen for
the subsequent studies were cultivated on the above
mentioned two artificial media for 14 days with their
size ranging from 3.5 to 5.5 cm. The colonies were
loose and slightly fluffy in the beginning but later the
hyphae became more and more thickened and formed
a compact surface. The colour of the mycelium was
gray or dark gray. The reverse of the colonies was
pink to violet on PDA medium with a visible diffusion of the dye to the culture medium (Fig. 1). On the
malt agar medium the reverse was beige-yellow. At
the beginning of day 6 of cultivation, acervuli were
formed on PDA medium and after 10 days on malt
agar medium with an addition of caraway stems
pieces. Numerous acervuli were observed after 14 days
of the cultivation, and covered the entire surface of
the colony but sometimes they were formed in sectors. Acervuli were slightly immersed in the medium,
almost black, lenticular, flat or pulvinate with sufficiently visible and high rends. The diameter of acervuli was 495.44× 371.48 µm (Table II). All around
the ostiole and on the surface of acervuli numerous
setoses were formed. (Fig. 2, 3). The setoses were
dark brown or almost black, generally septate (Fig. 2),
unpliant, smooth, tapered to the apices, their size
ranging from 36.05 to 202.99 µm in length and at the
Table II
Size (µm) of morphological structures of C. dematium on PDA medium (mean for 15 isolates)
Author
acervuli (µm)
setose (µm)
conidia (µm)
appressoria
Own measurements
Sutton 1980
Pidoplièko 1977
Von Arx 1957
495.44 × 371.48
36.05–202.99 × 3.83–5.74
7.66–15.28 × 5.74–13.37
8–14.5 × 6.5–8
250
150 × 4
100–600
17.19–24.83 × 3.82–5.72
19.5–24 × 2–2.5 (–3.5)
25 × 5
18–30 × 3–4.5
194
3
Machowicz-Stefaniak Z.
(a)
(b)
Fig. 3. Acervuli and setose of C. dematium (a), setose (b) in SEM (Photo M. Wróbel).
base from 3.83 to 5.74 µm in width (Table II). Thick,
almost black, shining drops resulting from the large
number of conidia were emerged from the mature
acervuli. The conidia were hyaline, aseptate, smooth,
falcate or fusiform, and they had acute apices (Fig. 4).
A visible gutate breaking the light was observed
in the middle of the conidia. The size of the studied
isolates conidia was 17.19–24.83× 3.2–5.72 µm on
PDA, and 18.5–26.74× 2.88–3.7 µm on malt agar with
an addition of caraway stem pieces (Table II).
At the end of hyphae or in the middle of them abundant appressoria occurred. Unlike the vegetative
hyphae, they were brown or almost black, irregular
in shape, single or gathered and their size was
7.66–15.28×5.74–13.37 µm (Fig. 5). The formation
of sclerotia was not observed in the studied isolates.
Among the 22 tested species of phyllosphere fungi,
the majority, i.e. 18 species limited the growth and development of two isolates of Colletotrichum dematium
– K425 and K426, and 17 species of isolate K625,
Table III
Biotic effect of fungi isolated from caraway (Carum carvi L.) on Colletotrichum dematium
Fungal isolates
Alternaria alternata (Fr.) Keissler (K 461)
Alternaria radicina Meier, Drechsler et Eddy (K1723)
Botrytis cinerea Pers. (K 1777)
Cladosporium cladosporioides (Fres.) de Vries (K 518)
Epicoccum purpurascens Ehrenberg (K 1696)
Fusarium avenaceum (Fr.) Sacc. (K 56)
Fusarium culmorum (W.G.Smith) Sacc. (K 284)
Fusarium equiseti (Corda) Sacc. (K304)
Fusarium oxysporum Schlecht (K 271)
Fusarium sporotrichioides Sherb (K 465)
Phoma exigua Desm. var exigua (K 1503)
Phomopsis diachenii Sacc. (K 255)
Rhizoctonia solani Kühn (K 1561)
Septoria carvi Syd. (K 1833)
Sclerotinia sclerotiorum (Lib.) de Barry (K 2313)
Stemphylium botryosum Wallv. (K 296)
Gliocladium catenulatum Gilman et Abbott (L 4940)
Gliocladium fimbriatum Gilman et Abbott (W 76)
Gliocladium roseum Bainier (L 830)
Trichoderma harzianum Rifai (K 428)
Trichioderma koningii Oud. (K 437)
Trichoderma viride Pers. et Gray (W 1222)
Individual biotic effect – IBE
after 12 days C. dematium isolates
K 425
–1
+1
+4
–2
+1
+4
+5
+5
+5
+7
+3
+6
+5
–6
+5
–4
+2
+2
+2
+8
+7
+7
K 426
–1
+1
+3
–2
+1
+4
+5
+4
+5
+6
+3
+6
+5
–6
+5
–3
+2
+2
+3
+8
+7
+7
K 625
0
+1
+4
–2
–2
+4
+4
+4
+5
+7
+3
+5
+5
–6
+5
–3
+2
+2
+3
+8
+8
+7
3
Grove of Colletotrichum dematium (Fr.) from caraway
(a)
195
(b)
Fig. 4. Conidia of C. dematium in light photo microscope (magnification × 500) (a) (Photo E. Zalewska), SEM (b) (Photo M. Wróbel).
Fig. 5. Appressoria of C. dematium on PDA medium
(magnification × 500) (Photo E. Zalewska).
which indicates the positive individual biotic effects
observed after 12 days of dual growth (Table III).
The species of fungi from Trichoderma genus
limited the growth of the studied isolates of C. dematium to the highest degree because their individual
biotic effect was + 8 for T. harzianum and + 7 for
T. viride and T. koningii (Table III, Fig. 6). The studied species from Trichoderma genus caused lyses and
degeneration of C. dematium hyphae (Fig. 7). The
observation showed that the colonies of each studied
Trichoderma species totally overgrew the inoculums
of C. dematium and made the growth and sporulation
of the pathogen impossible. In the slides of 12-daysold colonies of C. dematium and T. harzianum and
of C. dematium with T. viride the concentration and
disintegration of cytoplasm in C. dematium hyphae
were observed. Moreover, after 12 days of dual
growth of the pathogen with T. koningii deformed
hyphae of C. dematium with a big concentration of
melanine were observed (Fig. 7).
The fungi from the genera Gliocladium slightly
limited the growth of C. dematium in the first day of
common growth. G. catenulatum and G. fimbriatum
grew on 1/3 and G. roseum on 1 of the surface of the
pathogen’s colony after 12 days of dual growth
(Fig. 8). In the slides chains of miss-shapen, dark hyphae of C. dematium and numerous hyphae of the
pathogen being subject to lysis were observed
(Fig. 9). Moreover, it was noticed that after 18 days
of dual growth micoparasitic fungi practically overgrew the whole surface of the pathogen, i.e. 7/8 of the
pathogen’s colony and after 24 days the whole surface of C. dematium colony was overgrown by them
causing total lyses of the pathogen»s hyphae.
The growth of C. dematium colony was strongly
limited by P. diachenii as its IBE was + 6 and in
the case of isolate K625 IBE was + 5 (Table III).
Among fungi of the Fusarium genus the growth
of the pathogen was limited more strongly by F. sporotrichioides species and its IBE was + 7 (Table III).
On the other hand, F. avenaceum, F. culmorum,
F. equiseti, F. oxysporum, Rhizoctonia solanii, Sclerotinia sclerotiorum and Botrytis cinerea slightly
limited the growth of C. dematium colony in comparison to F. sporotrichioides (Table III). Moreover, it
was observed that C. dematium in dual growth with
S. sclerotiorum, B. cinerea and R. solani formed an
inhibition zone (Fig. 10).
The fungi Alternaria radicina and Epicoccum purpurascens inhibited the growth of C. dematium colonies in a small degree and their IBEs were +1. However, isolate K625 C. dematium limited the growth of
Epicoccum purpurascens, which was shown by the
negative IBE value (Table III). Moreover, the fungus
C. dematium limited the growth of Septoria carvi
giving IBE-6, Cladosporium cladosporioides-2, Alternaria alternata-1, Epicoccum purpurascens and Stemphylium botryosum from – 3 to – 4 (Table III).
196
(a)
Machowicz-Stefaniak Z.
3
(b)
(c)
Fig. 6. C. dematium isolate K 425 (left) and T. harzianum (right)
after twelve days of dual growth (a), individual growth of
C. dematium (b) and T. harzianum (c) (Photo E. Zalewska).
(a)
(b)
Fig. 7. Degeneration of C. dematium hyphae isolate K 425 caused by T. harzianum (magnification x 500) (Photo E. Zalewska).
3
Grove of Colletotrichum dematium (Fr.) from caraway
(a)
197
(b)
(c)
Fig. 8. C. dematium isolate K 425 (left) and Gliocladium catenulatum (right) after twelve days of dual growth (a), individual growth
of C. dematium (b) and G. catenulatum (c) (Photo E. Zalewska).
Fig. 9. Degeneration of C. dematium isolate K 425 caused by
G. catenulatum, condense of cytoplasma (a), degeneration of the
hyphae (b) (magnification × 750) (Photo E. Zalewska).
198
Machowicz-Stefaniak Z.
(a)
3
(b)
(c)
Fig. 10. C. dematium isolate K 425 (left) and Sclerotinia sclerotiorum (right) after twelve days of dual growth (a), individual growth
of C. dematium (b) and S. sclerotiorum (c) (Photo E. Zalewska).
Discussion
Including the studied isolates in the species
Colletotrichum dematium was possible on the basis
of macroscopic and microscopic features of their
colony and on the basis of morphology and size of
acervuli and conidia. The above-mentioned features
were compared to those shown by Von Arx (1957),
Pidoplièko (1977) and Sutton (1980). Small differences, especially in the size (length and weight) of
conidia are a consequence of differentiation of culture medium on which the fungus grows. The dependence of the growth and development of numerous
fungi species from the culture conditions and from
the composition of culture medium was indicated by
a lot of authors (Uecker 1988, Sutton 1980). The
straight growth of studied isolates as well as the ability to produce the acervuli and conidia just after a few
days old colonies on PDA medium suggested that
medium is favorable to the culture and identification
of C. dematium. The production of a numerous number of acervuli by C. dematium on PDA was indicated
earlier by Azad et al. (2005).
The presence of a numerous and high setoses occurring all around the ostiole of acervuli and many
brown appressoria should be recognized as a favorable and characteristic micromorphological feature
causing the identification of the studied fungus easier,
which was indicated earlier by Von Arx (1957). The
latter organs, i.e. appressoria could be significant in
pathogenesis because they could attach hyphae of the
pathogen to the surface of plants (Von Arx, 1957). The
3
Grove of Colletotrichum dematium (Fr.) from caraway
high tinctorial power of the culture medium characteristic of all the studied isolated of C. dematium
points to the possibility of this fungus producing secondary metabolites.
The detection of C. dematium in Poland for the
first time on caraway plants increased the number of
host plant to this fungus. The present studies showed
that a lot of caraway phyllosphere fungi limited the
growth of C. dematium and the positive values of IBE
indicate it, but the size of limiting possibilities was
different. Among the fungi inhibiting the growth of
C. dematium there were the species from the genera
Trichoderma and Gliocladium. These fungi are
known for their antagonistic influence against pathogenic fungi (Fokkema, 1993; Machowicz-Stefaniak,
1998; Król and Machowicz-Stefaniak, 2008). T. harzianum, T. koningii and T. viride should be recognized
as the most effective antagonists for C. dematium,
which was indicated by complete overgrowth and destruction of the pathogen colonies by these antagonists just after a few days of dual growth. Similarly,
these special high antagonistic abilities of Trichoderma spp., were indicated earlier for other pathogens
of caraway like Septoria carvi and Phomopsis diachenii (Machowicz-Stefaniak et al., 2008; MachowiczStefaniak, 2009). It is probably possible thanks to the
emission of constitutional enzymes and those produced as an effect of contact with the pathogen, as
well as the ability to form toxic metabolites and the
ability to mycoparasite (Fokkema, 1993). Thanks to
these abilities, Trichoderma spp. are used in the production of biopreparates (Cohen et al., 1996). A significantly slower antagonistic effect of Gliocladium
spp., (unlike that of Trichderma spp.) towards
C. dematium and other pathogenic fungi results from
antibiosis and the ability to mycoparasite in the lack
of competitive abilities (Fokkema, 1993; MachowiczStefaniak et al., 2008; Machowicz-Stefaniak, 2009).
Therefore, full antagonistic activity of Gliocladium
spp. to C. dematium was not shown until 20 days in
vitro. On the other hand, recently Epicoccum purpurascens have been recognized as a fungus strongly inhibiting the growth of different microorganisms thanks
to the possibilities to produce siderophores, flavipine
and Epicorazine B (Frederick et al., 1981; Mallea et al.,
1991; Fokkema, 1993) In the present studies the fungus showed only slight possibilities of inhibiting the
growth of C. dematium. On the other hand, the production of inhibition zone by one of the pathogen’s studied isolates in a dual culture with E. purpurascens confirmed the ability of C. dematium to produce secondary
metabolites, which were detected in ethyl-acetyl extract
of culture liquids pathogenically forms of C. dematium
(Abou-Zaid et al., 1997, Mendirata et al., 2005).
The inhibiting effect of C. dematium isolates to
A. alternata, C. cladosporioides, S. carvi and S. botry-
199
osum also seems to confirm the ability of C. dematium
for antibiose. Fast-growing phytopathogenical fungi
possessing a big enzymatic ability and used in the
present studies, i.e. S. sclerotiorum, B. cinerea and
R. solani only partly limited the growth of C. dematium because the last mentioned species produced an
inhibition zone during the common growth with each
of the above mentioned species. The inhibition zone
formed by C. dematium was not observed in dual
growth with the species of genera Fusarium. The last
mentioned species and especially F. sporotrichioides
suppressed the colonies of C. dematium from the culture medium. It was probably possible thanks to the
big power of their growth and the ability to produce
secondary metabolites (Kiecana and Perkowski, 1998).
Taking into account the inhibiting effect of the
studied phyllosphere fungi towards C. dematium, it
seems that the species of Trichoderma and Gliocladium
genera could be recognized as positive antagonistic
fungi. In perspective, these species may be used in
biological control of C. dematium. The other studied
species of fungi, despite only partly limiting C. dematium growth, are dangerous pathogens of many cultivated plant and their occurrence in culture plant’s
phyllosphere is undesirable.
Comparing the results of the present studies with
the results of similar studies conducted for other pathogens of caraway like Septoria carvi (MachowiczStefaniak et al., 2008) and Phomopsis diachenii
(Machowicz-Stefaniak, 2009) it is possible to notice
that they have numerous antagonists among phyllosphere fungi. That is why their isolation from plant
tissues on artificial media may be difficult. Among
the three above-mentioned pathogenic fungi S. carvi
showed the worst antagonistic activity (MachowiczStefaniak et al., 2008). It seems that isolates of
P. diachenii have better competitive abilities than
C. dematium (Machowicz-Stefaniak, 2009). On the
other hand, C. dematium have high abilities for antibiosis. On the basis of the present results and the data
in literature we can suggest the need to study the
secondary metabolites of fungi not only in the aspect
of their phytotoxicy and zootoxicy but also in the
direction of knowing about their mechanisms of antibiosis. It is interesting if it results from secretion of
antibiotic, lytic enzymes or from excessive acidification or alkalization of the medium (Fokkema, 1993).
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Polish Journal of Microbiology
2010, Vol. 59, No 3, 201–205
ORIGINAL PAPER
Cytotoxic Activity of Serratia marcescens Clinical Isolates
SYLWIA KRZYMIÑSKA, MARTA RACZKOWSKA and ADAM KAZNOWSKI*
Department of Microbiology, Faculty of Biology, A. Mickiewicz University, Poznañ, Poland
Received 3 March 2010, revised 27 May 2010, accepted 1 June 2010
Abstract
Twenty Serratia marcescens isolates from clinical specimens were examined for their cytotoxic activity on four cell lines (HEp-2, Vero,
CHO, J774). Most of the isolates were found to be cytotoxic to CHO (70%), Vero (75%) and HEp-2 cells (90%). CHO cells were the most
sensitive to cell-free supernatants, followed by HEp-2 and Vero cells. Two strains produced cytotonic toxins which caused elongation of
CHO cells. Moreover, twelve isolates (60%) revealed cytotoxic potential to macrophage cell line J774. The results indicate that these
bacteria may destroy phagocytes and epithelial cells, which may lead to spread within the host.
K e y w o r d s: Serratia marcescens, cytotoxicity
Introduction
Strains of Serratia marcescens have been recognized as an important nosocomial pathogen causing
a variety of diseases, including respiratory and urinary
tract infections, meningitis, peritonitis and bacteremia.
The bacteria are becoming increasingly important
cause of many outbreaks and endemic nosocomial
infections, particularly among newborns and patients
submitted to invasive procedures (Buffet-Batoillon
et al., 2009; Friedman et al., 2008; Grimont and
Grimont, 2006). Although S. marcescens is the common Serratia species causing nosocomial infections,
little is known about the factors impacting their pathogenicity and virulence. The possible mechanism of the
pathogenesis is complex and multifactorial, with the
involvement of a number of putative virulence factors
whose role in development of disease is not clear. The
first step of pathogenesis is colonization of epithelial
cells. After adhesion to the cells strains produce many
potential virulence factors, including extracellular toxins which are probably the most common mechanism
of pathogenicity. Some S. marcescens isolates produce
hemolysin, which represents the prototype of a family
of pore forming toxins with hemolytic and cytotoxic
activity (Hertle, 2005). Strains of S. marcescens have
been reported to produce many extracellular proteins,
including proteinases, lipases, nucleases, chitinases,
lecithinases, which may directly contribute to cellular
cytotoxicity by exerting their damaging effects upon
host cells (Grimont and Grimont, 2006).
The major defense mechanism of host nonspecific
immunity represents cell-mediated killing. Phagocytic
cells such as macrophages and neutrophils contribute
to the primary line of innate defense against bacterial
pathogens by providing their removal and destruction
at the level of the epithelial barrier. Therefore, many
bacterial pathogens have developed specific strategies
to suppress the effective antimicrobial immune response of macrophages to avoid the innate immune
defense of the host (Navarre and Zychlinsky, 2000).
S. marcescens may infect numerous sites of the host
body, including lungs and respiratory epithelia, muscle
and soft tissues, therefore in the current study, we have
examined the cytotoxic activity of human isolates to
different mammalian epithelial cell lines (Vero, CHO,
HEp-2). Moreover, we investigated the effect of S. marcescens cell-free filtrates on macrophage cell line J774.
Experimental
Materials and Methods
Bacterial strains. A total of 20 isolates (listed in
Table I) identified as Serratia marcescens by biochemical test kit (API20E, bioMerieux) were analyzed.
They were originated from specimens of hospitalized
* Corresponding author: A. Kaznowski, Department of Microbiology, Faculty of Biology, A. Mickiewicz University,
ul. Umultowska 89, 61-614 Poznañ, Poland; phone (+48) 61 529 5937; fax (+48) 61 829 5590; e-mail: [email protected]
202
3
Krzymiñska S. et al.
Table I
Serratia marcescens strains used in the study
Source of origin
(number of strains)
Ulceration (4)
Urine (4)
Postoperative
wound (3)
Catheter (1)
Feces (1)
Blood (1)
Aspirate (2)
Pus: pharynx, ear,
abscess, drain (4)
Isolates No
MPU S1, MPU S4, MPU S7, MPU S23
MPU S3, MPU S12, MPU S18, MPU S21
MPU S6, MPU S9, MPU S11
MPU S20
MPU S15
MPU S22
MPU S2, MPU S10,
MPU S5, MPU S14, MPU S19, MPU S13
patients and belonged to Bacterial Culture Collection
of Department of Microbiology Poznañ University
(MPU). Seven isolates originated from wounds (postoperative and ulcerations), 4 from secretions (2 from
aspirates and 1 from ear and pharynx), 4 from urine,
1 from blood, 2 from catheter and drain, 1 from fecal
specimen. The isolates were maintained at –75°C in
trypticase soy broth (TSB, Difco) containing 50%
(vol/vol) glycerol.
Cell cultures. Human epidermoid carcinoma cells
from the larynx (HEp-2), Chinese hamster ovary cells
(CHO) and African monkey kidney (Vero) were cultured in Eagle Minimum Essential Medium (EMEM,
Sigma) with 5% fetal calf serum (FCS, Sigma) containing 2 mM glutamine, 50 IU of penicillin per milliliter, streptomycin (100 µg/ml) and nystatin (1 mg/ml).
The murine macrophage cell line, J774 was maintained
in growth medium (GM), containing RPMI 1640
supplemented with 10% heat-inactivated fetal calf
serum, gentamicin (5 µg/ml) and 2 mM L-glutamine
(Krzymiñska et al., 2009) The cells in number 1× 104
cells per well seeded with 100 ml of suspension and
incubated at 37°C in an atmosphere with 5% CO2.
Preparation of bacterial cell-free supernatants.
For preparing bacterial filtrates, the strains were cultured in brain heart infusion broth (BHI, Difco) at
37°C. The cultures were incubated on Luria-Bertani
medium (LB, Difco) in a shaking incubator with agitation at 300 rpm at 37°C for 24 h. After centrifugation
at 3000 rpm for 20 min, the supernatants were sterilized through 0.22 mm-pore size membrane filters
Millex-GV (Millipore). Sterile culture supernatants
were heated at 56°C for 20 min to destroy activity of
heat-labile toxins (Carbonell et al., 1997).
Cytotoxic and cytotonic activity to epithelial
cells. The assay was performed according to Krzymiñska et al. (2009). Twofold serial dilutions (from 1:2
to 1:512) of culture filtrates in phosphate buffered
saline (PBS, Biomed) added to the wells of tissue culture plate containing confluent Vero and CHO mono-
layers and incubated for 24 hours at 37°C. As negative controls the wells received non-pathogenic E. coli
K12C600 filtrates. Cytotonic activity revealed as elongation of CHO cells. The cytotoxic titer of each isolate was calculated by determining the reciprocal of
the highest dilution of culture filtrates which produced
a cytopathic effect. The results were observed under
an inverted microscope. The results were presented
as mean titers from two experiments in triplicate.
Cytotoxic activity to murine macrophage J774
cells by Neutral Red retention assay. Neutral Red
(NR) is a biomarker of cellular stress and supravital
dye taken up in the lysosomes of viable cells (Maleri
et al., 2008). The assay was performed in microtitration plates with a method by Carbonell et al. (1997)
with slightly modifications. Macrophage monolayer
was incubated with bacterial culture filtrates at 37°C
for 24 h. As a negative control, the cells were infected
with non-pathogenic E. coli K12C600 filtrate. Next,
the medium was removed and the cultures were
washed with PBS and 200 µl of NR (50 µg/ml) was
added to each well and incubated for 3 h at 37°C. After
incubation the dye solution was aspirated, cells were
rinsed in PBS before being fixed with formalin in
calcium chloride solution (40% formaldehyde, 10%
anhydrous calcium chloride) which was next removed,
and incorporated dye was eluted from the cells by
adding ethanol/acetic acid mixture (50% ethanol and
1% acetic acid). The absorbance at 540 nm was measured using a plate reader. All absorbance values were
corrected against blank wells which contained growth
medium alone which were processed as described
above. Cell viability was determined by comparing
the absorbance values obtained from the control wells
(taken as 100% viability).
Results
Cytotoxic and cytotonic activity to epithelial
cells. Cytotoxic potential of S. marcescens isolates was
studied on three different cell lines, including Vero,
CHO and HEp-2 cells. Microscopic examination of
the cells following incubation with cell-free supernatants revealed a number of changes: rounding and
shrinking of cells, followed by detachment, loss of
cytoplasmic extension, disorganization of cell monolayer (Fig. 1B, E, G.). Cytotoxicity to CHO cells
(Fig. 1B) was induced by 14 strains (70%) with cytotoxic titers ranging from 1 to 128. Eighteen of 20 (90%)
S. marcescens strains were found to be cytotoxic to
HEp-2 cells (Fig. 1E) with cytotoxic titers ranging
from 1 to 32. The highest cytotoxic activity revealed
10 isolates (from MPU S1 to MPU S7, MPU S10,
MPU S11, MPU S22). Fifteen strains (75%) were cytotoxic to Vero cells (Fig. 1G) with lower cytotoxic titer
3
203
Cytotoxicity of Serratia marcescens
A
C
B
D
E
F
G
Fig. 1. Cytotoxic effects of S. marcescens culture filtrates to CHO (B), HEp-2 (E) and Vero cells (G).
Cytotonic activity of S. marcescens MPU S15 to CHO cells (C). A, D, F – uninfected cells. Magnification, ×100.
(1–32). Preheating (56°C for 20 min) of the supernatants caused a decrease in cytotoxic activity to CHO,
Vero and HEp-2 cells (Table II). Mean cytotoxicity
titers for individual cell lines taking all isolates into
account were in the 3.55–22.7 range (Table III). CHO
cells expressed the highest sensitivity with mean titer
22.7 ± 0.11 whereas the least sensitive were Vero cells
(mean titer = 3.6 ± 0.07). Cytotonic activity identified
as elongation of CHO cells (Fig. 1C) was observed for
2 isolates (MPU S3, MPU S15). Non pathogenic strain
E. coli K-12 C200 was not cytotoxic to epithelial cells.
Cytotoxic activity of S. marcescens strains to
murine macrophages. Twelve strains (60%) were
cytotoxic to murine J774 cells after 24-hour infection,
as measured by NR assay (Table IV). The maximum
cytotoxicity (52–68%) was observed in macrophages
infected with two strains (MPU S21, MPU S22), the
lowest cytotoxic activity was demonstrated by seven
strains (35%). Cytotoxic activity to J774 cells was not
inhibited after preheating of the supernatants. E. coli
K-12 C200 strain, the negative control, was not cytotoxic to murine macrophages.
Table II
Cytotoxic activity of culture supernatants of S. marcescens strains
Mean
titers1
64 – 128
8 – 32
1–4
0
1
2
CHO
Vero
HEp-2
Non treated Heat inactivated Non treated Heat inactivated Non treated Heat inactivated
supernatant
supernatant
supernatant
supernatant
supernatant
supernatant
3 (15)2
6 (30)
5 (25)
6 (30)
0
1 (5)
7 (35)
12 (60)
0
2 (10)
13 (65)
5 (25)
0
1 (5)
8 (40)
11 (55)
0
9 (45)
9 (45)
2 (10)
0
2 (10)
10 (50)
8 (40)
Mean of the reciprocal of the highest dilution yielding rounding, detachment and destruction of 50% of CHO, Vero or
HEp-2 cells.
Number (and percentage) of strains that revealed cytotoxic activity.
204
Krzymiñska S. et al.
Table III
Mean and standard error (SE) of cytotoxicity titers
for different cell lines
Mean ± SE
Cell line
22.7 ± 0.11
10.8 ± 0.08
3.6 ± 0.07
CHO
HEp-2
Vero
Table IV
Cytotoxic activity of S. marcescens culture supernatants
to murine J774 macrophages
Cytototoxicity
range1
0
7.8 – 19.2
21.6 – 29.4
52.3 – 68.1
1
2
Number of cytotoxic isolates (%)
Non treated
Heat inactivated
supernatant
supernatant
8 (40)2
9 (45)
7 (35)
7 (35)
3 (15)
2 (10)
2 (10)
2 (10)
The percentage of cytotoxicity was determined 24 h after infection by
NR assay,
Number and (percentage) of strains revealing cytotoxic activity.
Discussion
Serratia marcescens is increasingly recognized as
a cause of morbidity in nosocomial units. It has been
considered as an etiologic agent in all kinds of infection in humans. However, the exact mechanism of
pathogenicity has not been sufficiently understood.
The analysis of incidence of cytotoxic activity in
S. marcescens isolates revealed that most strains produced cytotoxic toxins, which were noticed in the
case of CHO (70%), Vero (75%) and HEp-2 cells
(90%). The highest cytotoxic activity was detected in
strains isolated from ulceration (3), postoperative
wounds (2) and one each from urine, blood, aspirate
and pus. Previously, Carbonell et al. (1997) demonstrated that culture filtrates from 22% S. marcescens
isolates were cytotoxic to Vero and HeLa cells. Marty
et al. (2002) confirmed earlier reports that the strains
are cytotoxic to mammalian cells. Toxin production
by S. marcescens strains is still not clearly defined.
Marty et al. (2002) examined the 56-kDa metalloprotease of S. marcescens strains and found it to be the
most potent cytotoxic factor. They suggested that the
enzyme may possess a binding site for specific host
proteins that are internalized by an endocytic mechanism into host cells. Carbonell et al. (2004) isolated
a cytotoxic enterotoxin from a clinical isolate of
S. marcescens which was highly cytotoxic to CHO
cells but did not reveal hemolytic activity, suggesting
that the cytotoxin is distinct from S. marcescens hemolysins. In consecutive reports, Carbonell et al. (2003)
observed that the cytotoxic toxin was bound to the
CHO cell surface, without being internalized and was
3
able to trigger changes in the intracellular metabolism
of the cells and to induce cell death by apoptosis. In
the past several years there have been reports about the
family of Serratia-type pore forming toxins (Hertle,
2005). The hemolysin (ShlA) represents the prototype
of a new type of cytolysins which are distinct from
E. coli type "-hemolysins, staphylococcal "-toxins or
other related toxins. ShlA pore formation in nucleated eukaryotic cells and erythrocytes results in cell
lysis. The toxin additionally brings about cytoskeleton
rearrangement and apoptosis.
The S. marcescens culture filtrates incubated at
56°C for 20 min revealed a decrease in cytotoxic activity to CHO (86% filtrates), HEp-2 (90% filtrates) and
to Vero cells (43%). The strains probably produce heatlabile cytotoxins. Carbonell et al. (2003) showed that
a monolayer of Vero cells lost 30% cell viability when
the filtrates were heated to 60°C, and no cytopathic
effect was observed after incubation at 70°C.
In order to choose a suitable target cell line, we
compared the sensitivity of three epithelial cells to the
cytotoxic activity of clinical isolates. In the present
study CHO cells appeared to be the most sensitive to
the toxic effect of S. marcescens culture filtrates, followed by HEp-2 and Vero cells. The results indicate
that CHO cells could be used as a model to study the
exact mechanism of action of cytotoxic factors. Carbonell et al. (2003) also found that CHO and HEp-2
cells are highly sensitive to S. marcescens cytotoxin.
Interestingly, two isolates – MPU S3 and MPU S15,
appeared to be positive for cytotonic activity, which
revealed as elongation of CHO cells. Singh et al.
(1997) noticed that 4 of 6 S. marcescens strains isolated from food produced cytotonic toxins. There is
no evidence of cytotonic toxins production by strains
originating from human specimens. Cytotonic and
heat-labile enterotoxins were produced, respectively,
by Vibrio cholerae and E. coli strains. The toxins activated adenylate cyclase, which caused an increase in
intracellular cAMP concentration, inducing morphological alterations in CHO cells and producing an imbalance in electrolyte movement in epithelial cells,
resulting in abundant net fluid loss from the intestine
(Sanchez et al., 2005). The high cAMP concentration
impairs host cells functions, such as phagocytosis and
oxidative ability (Moss et al., 2000). Moss et al.
(2000) reported that Bordetella pertusis produced an
adenylate cyclase-hemolysin (AcHly) toxin which
caused increased cAMP level. An increase in the intracellular concentration of cAMP leads to apoptosis
of mammalian cells.
Phagocytes, either resident in tissues or circulating
in blood contribute to the primary line of innate defense against bacterial pathogens by providing their
removal and destruction at epithelial barrier level
(Navarre and Zychlinsky, 2000). Some bacterial ente-
3
Cytotoxicity of Serratia marcescens
ropathogens have developed strategies for avoiding
antimicrobial effects of phagocytes and have evolved
mechanisms which kill the immune cells. In this study
we have demonstrated that 60% of isolates were cytotoxic to murine macrophages J774 cells. The highest
activity was observed for strains isolated from blood
and urine. The results suggest that the cytotoxic activity of these bacteria may be an important mechanism
for evasion of host immune response during infection,
which may induce inflammation and development of
fatal bacteremia in weakened patients.
The results of the study demonstrate that S. marcescens clinical isolates reveal cytotoxic activity that may
modulate the properties of host epithelial cells. Moreover, we have observed the incidence of an antihost
strategy based on the elimination of host immune cells.
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Polish Journal of Microbiology
2010, Vol. 59, No 3, 207–212
ORIGINAL PAPER
Antibiotic Susceptibility and Genotype Patterns
of Escherichia coli, Klebsiella pneuomoniae and Pseudomonas aeruginosa
Isolated from Urinary Tract Infected Patients
M.I. ABOU-DOBARA1*, M.A. DEYAB1, E.M. ELSAWY2 and H.H. MOHAMED2
1 Faculty
of Science (Damietta), Damietta Branch, Mansoura University and
and Nephrology Center, Mansoura University, Egypt
2 Urology
Received 13 October 2010, revised 1 May 2010, accepted 15 May 2010
Abstract
Thirty nine isolates of Escherichia coli, twenty two isolates of Klebsiella pneumoniae and sixteen isolates of Pseudomonas aeruginosa
isolated from urinary tract infected patients were analyzed by antimicrobial susceptibility typing and random amplified polymorphic DNA
(RAPD)-PCR. Antibiotic susceptibility testing was carried out by microdilution and E Test methods. From the antibiotic susceptibility, ten
patterns were recorded (four for E. coli, three for K. pneumoniae and three for P. aeruginosa respectively). Furthermore, genotyping
showed seventeen RAPD patterns (seven for E. coli, five for K. pneumoniae and five for P. aeruginosa respectively). In this study,
differentiation of strains of E. coli, K. pneumoniae and P. aeruginosa from nosocomial infection was possible with the use of RAPD.
K e y w o r d s: Escherichia coli, Klebsiella pneumoniae, Pseudomonas areuginosa, antibiotic susceptibility,
urinary tract infection
Introduction
Urinary tract infections (UTIs) are most prevalent
among geriatric and critically ill patients and occur
more commonly after urinary catheterization (Turck
and Stamm, 1981; Centers for Disease Control, 1983).
Many bacterial species are associated with this infection, including Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa (Gales et al., 2000;
Gupta et al., 2007). Multi-drug resistance of antimicrobial classes is common among the uropathogenic
bacteria such as Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa (Yuksel et al.,
2006; Foda, 2008).
Among whole genome fingerprinting PCR methods, random amplified polymorphic DNA (RAPD)
(Williams et al., 1990) is used for demonstrating
differences between bacteria. RAPD can be used for
typing of organisms without previous knowledge
of DNA sequences. The use of a single primer leads
to amplification of several DNA fragments randomly
distributed throughout the genome. The primers used
in RAPD are short, usually 8–12 mers, with random
sequence composition (Lübeck and Hoorfar, 2003).
RAPD has received considerable attention in recent
years as a molecular typing method due to its simplicity, sensitivity, flexibility and relatively low cost
(Welsh and McClelland, 1990; Williams et al., 1990;
Belkum, 1994). The ability of RAPD to type a wide
variety of bacteria strains in a short time suggests that
it will be a useful molecular epidemiological tool
(Gori et al., 1996; Hilton and Penn, 1998; Bosi et al.,
1999; Patton et al., 2001). Typing helps in the identification of environmental sources as well as indicating whether transmission of strains has occurred
between patients (Kerr et al., 1995). It also reveals
whether strains emerging after antibiotic therapy are
variants of the original or newly acquired strains (Chit
and Chew, 1993).
Knowledge of the spread of specific strains is of
epidemiological importance in order to monitor the
broad spreading of both their pathogenicity and multiresistance (Lopes et al., 2005). The aim of this study
was to determine antibiotic susceptibility and to apply
RAPD typing method for characterization of the isolated bacterial pathogens.
* Corresponding author: M.I. Abou-Dobara, Botany Dept., Faculty of Science, New Damietta, Damietta P.O. Box 34517, Egypt;
e-mail: [email protected] or [email protected]
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Abou-Dobara M.I. et al.
Experimental
Material and Methods
Collection and transport of the samples. A total of
77 clinical samples were collected from urine samples
in sterile tightly locked containers from different hospitalized patients in Urology and Nephrology Center,
Mansoura University. The patients were not treated
with any of the eight antibiotics tested (Table I).
Table I
Antibiotic types retrieved in this studya
Antibiotic
IP
AK
PT
XL
NI
CT
NX
TS
a
b
MICb
S
≤4
≤ 16
≤ 16
≤8
≤ 32
≤8
≤4
≤2
I
8
32
32
16
64
16
8
–
Conc. Range [µg/ml]
R
≥ 16
≥ 64
≥ 128
≥ 32
≥ 128
≥ 64
≥ 16
≥4
Broth
1 – 16
2 – 64
8 – 128
4 – 32
16 – 128
8 – 64
4 – 16
2–4
E test
0.002 – 32
0.016 – 256
0.016 – 256
0.016 – 256
0.032 – 512
0.016 – 256
0.016 – 256
0.002 – 32
Abbreviations: IP, Imipenem; AK, Amikacin; PT, Piperacillin/tazobactom; XL. Amoxicillin/clavulanate; NI, Nitrofurantoin; CT, Cefotaxime; NX, Norfloxacin; TS, Trimethoprim-sulfamethoxazole;
Susceptibility was performed as described by National Committee
for Clinical Laboratory Standards [NCCLS, 2006], which was used to
categorize strains as susceptible or sensitive (S), intermediate (I) or
resistant (R).
Isolation of the bacterial isolates. The collected
samples were inoculated into sterile Petri dishes containing ready prepared Cled agar and blood agar media
and incubated at 35°C for 18–24 hours. After the incubation period, the plates were examined for growing
bacterial colonies. The isolated colonies were subcultured and purified for characterization.
Identification of the different isolates. The isolated bacteria were identified according to Bergey’s
Manual of Determinative Bacteriology (Brenner, 1986;
Ørskov, 1986a and b; Palleroni, 1986) and confirmation of species identification was carried out by using
automated Microscan (DADE BEHRING, USA).
Antibiotics sensitivity tests. The antimicrobial
susceptibility tests of the isolated bacteria were carried
out using the following methods:
1 – Broth Microdilution MIC Method. A sterile
plastic tray containing various concentrations of antimicrobial agents (Table I) was inoculated with a standardized number of test bacteria in Mueller Hinton
broth. After overnight incubation at 35°C, the minimal inhibitory concentrations (MIC) were determined
and interpreted as susceptible, intermediate, or resistant (Table I).
2 – E Test. The system comprises a E test strip with
predefined antimicrobic gradients (Table I), to deter-
3
mine the Minimum Inhibitory Concentration (MIC),
in µg/ml of individual agent against microorganisms
as tested on agar media (Mueller Hinton Agar). The
inoculated media were incubated at 35–37°C for
18–24 hours. After the incubation period the MIC values were recorded at the point of intersection between
the inhibition ellipse edge and the E test strip (Table I).
Genotyping of the bacterial isolates
Isolation of the bacterial DNA. Bacterial colonies
were removed and suspended in 1 ml distilled water,
then centrifuged for 10 min at 5000× g. DNA was
extracted using High Pure PCR Template Purification
Kit, Germany) as follow: The bacterial pellets were
suspended in 200 µl phosphate buffered saline. 15 µl
lysozyme was added and incubated for 15 min at
37°C. Subsequently 200 µl binding buffer and 40 µl
proteinase K were added, mixed immediately and incubated for 10 min at 72°C, then 100 µl isopropanol
was added to precipitate DNA. The filter tubes and the
collection tubes were combined and the samples were
pipetted, and then centrifuged for 1 min at 5000× g.
The upper reservoir was washed twice with 500 µl
washing buffer and centrifuged for 1 min at 5000× g.
200 µl of prewarmed (70°C) elution buffer was added
and the tubes were centrifuged for 1 min at 500× g.
Randomly Amplified Polymorphic DNA (RAPD)
Fingerprinting (Williams et al., 1990). RAPD was
carried out with some modification. The PCR mixture
was composed of 10× PCR buffer: 100 mM Tris-HCl,
500 mM KCl, 15 mM MgCl2, 0.01% (w/v) gelatin,
pH 8.3.2 mM of each dGTP, dATP, dCTP, and dTTP
was added. Taq DNA polymerases: Taq DNA polymerase (5 U/µL; Sigma). Template DNA: 10 to 25 ng/µl
stock solution containing good-quality, protein-free,
DNA can be resuspended in high-quality sterile, deionized water or TE (Tris-EDTA) pH 8.0. RNase (20 ng
per 1 ng of DNA). The following primer (OPA-02 5'TG
CCGAGCTG3') was used in this study at 25 pmol/µl.
The amplifications were done in thermal cycler
(PerkinElmer model 9700) programmed for the first
five cycles to denature for 1 min at 94°C, anneal for
2 min at 27°C followed by primer extension for 2 min
at 72°C. Then, a program for 45 cycles of 1 min denaturation at 94°C, 2 min of annealing at 32°C and 2 min
primer extension at 72°C followed by a final extension
period for 15 min at 72°C, was run.
Gel preparation and sample loading (Maniatis
et al., 1982). A 0.7% agarose was prepared in 1× TBE
and mixed with 0.5 ug/ml of ethidium bromide.
The gel was transferred to electrophoresis cell with
1× TBE buffer. Each sample (20 µl) was mixed with
4 ul loading dye and loaded into the gel and 1 µl
DNA marker ØX 174 Hae III was loaded into one
well of the gel.
3
E. coli, K. pneumoniae, P. aeruginosa isolates from urinary tract
Electrophoresis and detection. 80 volts for 2 hours
as 7.5 v/cm of the gel was applied. The gel was visualized using UV transilluminator and photographed
by Polaroid film in Polaroid camera with 4 seconds
exposure time.
Results
Characterization and identification of the isolated bacteria. Seventy-seven isolated bacterial samples were divided into three groups namely; group 1
(thirty-nine isolates were identified as Escherichia
coli), group 2 (twenty-two isolates were identified
as Klebsiella pneumoniae) and group 3 (sixteen isolates were identified as Pseudomonas aeruginosa)
according to their colony morphology, colony smell,
Gram stain response, shapes, pigmentation and biochemical properties.
E. coli was the most frequent bacterium isolated
(50%) followed by K. pneumoniae (29 %) and P. aeruginosa (21%). Imipenem, amikacin and piperacillin/
tazobactam were the most commonly used drugs for
the treatment of E. coli with 100% effectivity. Also
imipenem and amikacin were effective against K. pneumoniae and P. aeruginosa as the effectiveness of both
agents on them was 100%.
Amoxicillin/clavulanate, had a lower effect against
E. coli (72%). Nitrofurantion, cefotaxime, norfloxacin
and trimethoprim/sulfamethoxazole had a low effect
against E. coli (46%, 26%, 26% and 26% respectively). Amoxicillin/clavulanate, nitrofurantion, cefotaxime and norfloxacin had a low and similar effect
on K. pneumoniae (27%). Cefotaxime and norfloxacin
also showed low-level effectiveness against P. aeruginosa (50%). In addition, P. aeruginosa were completely resistant to nitrofurantion. Finally, trimetho-
209
prim/sulfamethoxazole had the lowest effect among the
tested antibiotics with K. pneumoniae and P. aeruginosa being resistant to them.
From the antibiotic susceptibility, ten patterns
were recorded (four for E. coli, three for K. pneumoniae and three for P. aeruginosa respectively).
For isolates of E. coli, the first pattern was resistant
to amoxicillin/clavulanat, nitrofurantion, cefotaxime,
norofloxacin, and trimethoprim/sulfamethoxazole. The
second pattern was resistant to nitrofurantion, cefotaxime, norofloxacin, and trimethoprim/sulfamethoxazole.
Pattern two was resistant to cefotaxime, norfloxacin,
and trimethoprim/sulfamethoxazole. Finally, pattern
four was susceptible to all the tested antibiotics.
The isolates of K. pneumoniae were distributed
into three patterns. The first pattern was resistant to
piperacillin /tazobactama, amoxicillin/clavulanat, nitrofurantion, cefotaxime, norfloxacin, and trimethoprim/
sulfamethoxazole. Pattern two was resistant to amoxicillin/clavulanate, nitrofurantion, cefotaxime, norfloxacin, and trimethoprim/sulfamethoxazole. Pattern
three was resistant to trimethoprim/sulfamethoxazole.
On the other hand, P. aeruginosa isolates were
separated into three patterns. The first one was resistant to amikacin, piperacillin/tazobactam, nitrofurantion, cefotaxime, norofloxacin and trimethoprim/
sulfamethoxazole. The second was resistant to nitrofurantion, cefotaxime, norfloxacin and trimethoprim/
sulfamethoxazole. Finally pattern three was resistant
to nitrofurantion and trimethoprim/ sulfamethoxazole.
RAPD-PCR analysis revealed different genotypes
for all the identified bacteria. E. coli had a different
RAPD pattern (Fig. 1), as did K. pneumoniae and
P. aeruginosa (Fig. 1 and 2). RAPD technique allowed
the amplification of many bands in all the isolated
bacteria. There was a difference in intensity of bands
within the same pattern or between the different
Fig. 1. Effect of different antibiotics on Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa.
210
3
Abou-Dobara M.I. et al.
P. aeruginosa. All isolates were found to have a band
of 1078 bp. Three patterns of P. aeruginosa resulting
from the antibiotic susceptibility testing were divided
into five distinct patterns in RAPD analysis (Fig. 2).
The RAPD patterns of different isolates of E. coli,
K. pneumoniae and P. aeruginosa from Urology and
Nephrology Center, Mansoura University may suggest
that those isolates constitute a clonal lineage.
Fig. 2. Polymerase Chain Reaction-Amplified DNA of a selection
of isolates of E. coli and Klebsiella pneumoniae showing random
amplification of polymorphic DNA assay fingerprints, separated
by gel electrophoresis and detected by ethidium bromide staining.
Lane 1: DNA molecular weight marker of Hae III digested phage øx174;
Lanes 2 to 8 E. coli DNAs; Lane 9 and 10, K. pneumoniae DNAs;
Lane 11 DNA ladders.
patterns. However, many isolates were found to be
identical in genotype-displayed variability in antibiotic susceptibility pattern.
For E. coli isolates, the amplification of eight
bands ranging in size from 280 to about 1078 bp
occurred. All isolates were found to have a band of
975 bp. The four patterns resulting from the antibiotic
susceptibility testing of E. coli were divided into
seven patterns in RAPD analysis (Fig. 1).
On the other hand, eleven bands ranging in size
from 360 to about 1900 bp were amplified in the isolates of K. pneumoniae. There was no common band
in all the isolates. Three patterns of K. pneumoniae
resulting from the antibiotic susceptibility testing
were divided into five distinct patterns in RAPD
method (Fig. 1 and 2).
Furthermore, nine bands ranging in size form 502
to about 1840 were amplified in the isolates of
Fig. 3. Polymerase Chain Reaction-Amplified DNA of a selection
of isolates of Klebsiella pneumoniae and Pseudomonas
aeruginosa showing random amplification of polymorphic DNA
assay fingerprints, separated by gel electrophoresis and detected
by ethidium bromide staining.
Lane 1: DNA molecular weight marker of Hae III digested phage
øx174; Lanes 2 to 4 K. pneumoniae DNAs; Lane 5 and 9 P. aeruginosa
DNAs; Lane 10 DNA ladders.
Discussion
Most of the clinical isolates of E. coli, Klebsiella
pneumoniae and Pseudomonas aeruginosa in this
study were susceptible to imipenem and amikacin
with 100% susceptibility rate and the degree of resistance to the other tested multiple antibiotics varied
according to the antibiotics. Foda (2008) recorded that
meronem and amikacin were highly active towards
E. coli, Klebsiella pneumoniae, Klebsiella oxytoca,
Proteus mirabilis and Pseudomonas aeruginosa. He
also reported that the susceptibility rate of urinary isolates was 76.19% for meronem followed by amikacin
(70.27%). Das et al. (2006) showed that the susceptibility rate of urinary isolates was the highest for
amikacin (87.2%).
The simplicity and wide applicability of the RAPD
method is dependent on the use of short nucleotide
primers, which are not related to known DNA sequences of the target organism. They are designed
within constraints including (i) a length of not less than
nine nucleotide residues, (ii) a GC content of > 50%
and (iii) a lack of palindromic sequences (William
et al., 1990). These primers used in PCRs have been
able to efficiently detect DNA polymorphisms and
identify interstrain variations in an increasing number
of species (Bingen et al., 1993a; 1993b). Genetic mapping and determination of the degree of relatedness
between strains have been performed with validation
by ribotyping (Williams et al., 1993). The banding
pattern derived in this process allows the identification of similar strains by a method significantly less
complicated and time consuming than ribotyping.
When directly compared in the analysis of bacterial
sample outbreak in a maternity unit, RAPD and
ribotyping were equivalent in their abilities to discriminate between strains (Bingen et al., 1993a). It is of
paramount importance that reaction conditions, including DNA template concentration, annealing temperature, and other PCR mixture concentration are strictly
standardized to avoid artifactural variation in RAPD
patterns (Ellsworth et al., 1993).
While RAPD gives information regarding similarity between isolates, the application of PCR based
techniques has a revolutionary impact on the diagnosis of infectious diseases. The most commonly used
3
E. coli, K. pneumoniae, P. aeruginosa isolates from urinary tract
molecular genetic fingerprinting technique by RAPD
revealed more genetic differences among avian bacterial strain than amplified fragment length polymorphism (AFLP) analysis (Gomes et al., 2005). In recent
years with the advent of molecular DNA techniques,
several arbitrary primer based RAPD-PCR techniques
have been used for delineating the bacteria according
to their genetic relatedness (Muzurier and Wernas,
1992; Eisen et al., 1995; Lin et al., 1996). Earlier researchers were of the opinion that RAPD
was the best method for detecting genetic differences
with respect to its speed and ability to type a wide
variety of bacterial species and suggested it would be
an increasingly useful molecular epidemiologic tool.
In the past, dendrogram-based analysis of the RAPD
profiles of various bacteria allowed understanding the
genetic relationship between isolates grouped into
several clusters. These phylogenetic studies successfully showed the predominance of a single epidemic
strain that was transmitted between hosts and its persistence over a period of time (Gomes et al., 2005).
In this study the four patterns resulting from the
antibiotic susceptibility testing of E. coli were divided
into seven patterns in the DNA method. There is a difference in intensity of bands within the same pattern
or between the different patterns, i.e. DNA method
identified additional heterogeneity among the related
strains. Seven patterns of E. coli generated by DNA
based method differed by the presence or absences
of one or two single DNA fragment when compared
one with another. Sometimes smearing is observed
when multiple DNA fragments, which differ slightly
in length, are visible.
Eisen et al. (1995) studied multi-resistant Klebsiella
pneumoniae strains by typing the isolates phenotypically and with random amplified polymorphic
DNA analysis (RAPD) and plasmid analysis and they
showed the predominance of a single epidemic strain
that was transmitted between patients in the Newborn
Services Unit. Lopes et al. (2005) found that 26 RAPD
genotypes among studied 30 K. pneumoniae and they
demonstrated the high discriminatory power of RAPD.
RAPD analysis in their research indicated that pathogenic K. pneumoniae strains comprise a genetically
high variable group of organisms. In this study, three
patterns of K. pnuomoniae resulting from antibiotic
susceptibility testing were divided into five patterns
in the RAPD analysis. Lai et al. (2000) reported that
a pathogenic K. pneumoniae strain was highly heterogeneous, based on the distribution of different nucleotide sequences. The high number of serotypes in
this species (Orskov and Orskov, 1984) could ahlso
explain the relevant degree of genetic diversity highlighted by RAPD.
Kerr et al. (1995) applied RAPD to 10 cases of
pneumoniae associated with sputum culture of Pseudo-
211
monas aeruginosa and they suggested that a single
strain of P. aeruginosa, isolated from 10 ICU patients,
was responsible for this outbreak of pneumonia. Nazik
et al. (2007) studied the typing of the Pseudomonas
aeruginosa isolates recovered from cystic fibrosis
(CF) patients by random amplified polymorphic DNA
(RAPD)-PCR and to determine the antibiotic susceptibility of these strains. Their study revealed that most
the P. aeruginosa isolates with dissimilar colony morphology or antibiotic susceptibility isolated from these
CF patients were of the same genotype, but colonization or infection with only one genotype is, however,
not a rule. These results were also recorded in other earlier studies (Sener et al., 2001; Horrevorts et al., 1990).
In this study, three patterns of P. aeruginosa resulting from antibiotic susceptibility testing were divided
into five patterns in the DNA method, that is the DNA
method identified additional heterogeneity among the
related strains. Khalifa et al. (2010) reported that
genotyping of Pseudomonas aeruginosa isolated from
clinical samples showed 83 RAPD types and they also
recorded that the isolates showing the same serotype
could show different genotypes. In addition, by using
RAPD, Trautmann et al. (2006) showed that isolates
of Pseudomonas aeruginosa from patients showed
a similar distribution of genotypes.
In conclusion, in this study, epidemiological typing
of 39 Escherichia coli, 22 Klebsiella pneumoniae and
16 Pseudomonas aeruginosa clinical samples was carried out by phenotypic and molecular methods. RAPD
technique detected genetic heterogeneity in different
strains of the studied bacteria. This can be useful to
understand the distribution of these pathogens in nosocomial infections.
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Polish Journal of Microbiology
2010, Vol. 59, No 3, 213–216
SHORT COMMUNICATION
rDNA-Based Genotyping of Clinical Isolates of Candida albicans
URSZULA NAWROT, MAGDALENA PAJ¥CZKOWSKA, KATARZYNA W£ODARCZYK, IZABELA MECLER
Department of Microbiology, Medical University of Wroclaw, Poland
Received 25 January 2010, revised 2 July 2010, accepted 7 July 2010
Abstract
The study presents an analysis of the restriction pattern of rDNA fragments of 95 C. albicans isolates previously classified on the basis of
the presence of the intron in rDNA into genotypes A (62 isolates), B (28), and C (5). Most isolates (61) with genotype A were classified as
“subtype a” and one as “subtype d” (Karahan and Akar; 2005). No differences were observed in the restriction patterns of the tested
genotype B isolates. Similarly, most genotype C strains (4/5) showed the same restriction pattern. The results indicate low subtyping
variations of the analyzed isolates, which is in contrast to published data obtained from a Turkish collection of yeasts.
K e y w o r d s: Candida albicans, group I self-splicing intron, rDNA genotyping, RFLP
Candida albicans is the most frequent fungal
pathogen, being the causative agent of both superficial and deep-seated and life-threatening mycoses.
Many molecular methods have been employed in
investigating species evolution and epidemiology. One
of them is the genotyping developed by Mercure et al.
(1993) and McCullough et al. (1999a) based on the
presence or absence of the self-splicing group I intron
in the large subunit (CaLSU) of rRNA genes. Strains
can be differentiated into three genotypes: genotype A
without the intron, genotype B harboring the intron,
and genotype C possessing LSU rDNA with and without the intron in a single genome. In the last decade,
several authors reported the distribution of the A, B,
and C genotypes in different groups of clinical C. albicans isolates (Tamura et al., 2001, Millar et al., 2002;
Karahan, 2004; Karahan and Akar, 2005; Qi et al.,
2005; Millar et al., 2005; Girish Kumar et al., 2006,
Nawrot et al., 2004; Nawrot et al., 2008). Although the
data obtained by the particular authors differ in detail,
genotype A has been reported as prevalent in most
groups of clinical isolates, including those regarded
as invasive. It is supposed that intron-containing
genotypes can be eliminated because of their high susceptibility to some drugs, for example 5-fluorocytosine,
pentamidine, and bleomycin, interfering with the selfsplicing process (Mercure et al., 1993; Zhang et al.,
2002; Jayaguru and Raghunathan, 2007). On the other
hand, some authors observed increased occurrence of
genotype C (McCullough et al., 1999b; Gurbuz and
Kaleli, 2010). Recently, Karahan and Akar (2005)
found significant differences in the LSU rDNA
sequences of genotype A isolates and constructed an
RFLP-based method for differentiating genotype A
into eight subtypes. In this communication we present
the results of the RFLP analysis of LSU rDNA of
C. albicans isolates, which is complementary to our
previous study on the distribution of CaLSU among
C. albicans isolated from blood and the respiratory
tract (Nawrot et al., 2008).
The study was performed on 95 isolates of C. albicans, including 55 blood isolates obtained from BCCM/
IHEM (30 genotype A, 20 B, and 5 C) and 40 isolates
(32 genotype A and 8 B) from the laboratory collection of Wroc³aw Medical University and originating
from different clinical samples (22 from sputum or
pharyngeal swabs, 7 from blood, and 8 from body
fluids or pus). Genomic DNA of the tested yeasts
was extracted using the CTAB method according to
O’Donnell et al. (1997). The PCR assay was performed
with the primer pair CA-INT-L (5’-ATA AGG GAA
GTC GGC AAA ATA GAT CCG TAA-3’) and CAINT-R (5’CCT TGG CTG TGG TTT CGC TAG ATA
GTA GAT-3’), described previously by McCullough
et al. (1999b). DNA samples were denatured at 94°C
for 3 min before 30 cycles of 94°C for 1 min, 65°C
* Corresponding author: U. Nawrot, Department of Microbiology, Medical University of Wroclaw, Cha³ubinskiego 4, 50-368
Wroc³aw, Poland; e-mail: [email protected]
214
Nawrot U. et al.
3
A)
B)
Fig. 1. Electrophoretic gel image of PCR products digested with Hae III (A)
and MspI (B). M-QX DNA Size Marker FX 17.
Lines 1–5 strains with genotype C (IHEM19482, 19076, 19265, 19491, 19608), line 6 genotype B (IHEM 19651),
line 7 genotype A subtype d (1228, laboratory collection), lines 8 and 9 genotype A subtype a (IHEM 19069, 19144).
Performed with the help of Biocalculator software (Qiagen).
for 1 min, and 72°C for 4 min, with a final extension
at 72°C for 4 min following the last cycle. The PCR
products were digested, separately overnight with the
enzymes Hae III (BsuRI) and MspI (HpaII, Fermentas),
in accordance with the protocol of Karahan and Akar
(2005). The undigested and digested PCR products
were analyzed by electrophoresis in 4% agarose gel
and visualized in UV after staining with EtBr. The
selected DNA samples were additionally analyzed
by capillary electrophoresis in the QIAxcel system
(Qiagen) using a QIAxcel DNA High Resolution Kit,
the QX Alignment Marker 15-bp/3-kb, and the QX
DNA Size Marker FX 174. The tests were performed with the OM500 method and the results were
analyzed by Biocalculator software and presented as
both simulated bands on gel images and peaks in
electrophoregrams.
PCR with the primer pair CA-INT-L/CA-INT-R
and DNA of genotype A resulted in a single PCR
product of ~ 460 bp (McCullough et al., 1999b),
3
Short communication
215
Fig. 2. A diagram presenting the restriction sites of a fragment of sequence DQ465844 (mether line) and the hypothetical sequence
in which the intron of DQ465844 was replaced by the X74272 sequence (performed with the help of SeqBilder, Lasergene software).
which can be slightly different in particular subtypes
(Karahan and Akar, 2005). The strains of genotype A
tested in this study give PCR products of typical size,
except for one isolate (no 1228, laboratory collection),
which gave a larger product (~470 bp). Most isolates
(61/62, 98%) of genotype A showed the same RFLP
pattern, namely three fragments of 294, 92, and 72 bp,
after digestion with Hae III and two fragments of 289
and 171 bp after digestion with MspI (Figs. 1 A and
B). This result corresponds well with “subtype a” described by Karahan and Akar (2005), which was characterized by three bands of 296, 93, and 71 bp after
digestion with Hae III. Isolate no 1228 showed a different restriction pattern, consisting of two bands of
~ 400 and 72 bp with enzyme Hae III and three bands
(290, 93, and 84 bp) with MspI, which is in accordance with the subtype classified as “subtype d” by
Karahan and Akar.
In their study, Karahan and Akar tested 144 genotype A isolates obtained from different clinical samples
from three Turkish hospitals and 52% of them were
identified as “subtype a”, whereas the other strains
were distributed among seven different subtypes. In
a recently published paper, Gurbuz and Kaleli (2010)
found 84 (84.8%) “subtype a” samples among 99
genotype A isolates. The genotype A isolates tested
in this study were highly homogenous (98% with
“subtype a”). Analysis of the results obtained by us
and by other authors, indicates that the level of diversity of LSU rDNA varies in particular C. albicans
populations and may be geographically related. It is
interesting that “subtype d” identified in this study,
as well as 9 isolates with “subtype d” described by
Karahan and Akar (2005), were obtained from blood,
which may suggest a high invasiveness of “subtype d”.
A future study performed with a higher number of
invasive and non-invasive isolates can be helpful
in verifying such an hypothesis.
In this study we also performed the RFLP analysis
of the PCR products obtained for genotypes B and C.
The public PubMed database (http://www.ncbi.nlm.
nih.gov/sites/entrez?db=pubmed) contains only two sequences (accession nos. DQ465844 and DQ465845)
of LSU of C. albicans genotype B which include
the DNA fragment flanked by the CA-INT-L and
CA-INT-R primers. The restriction analysis of these
sequences performed with the use of SeqBuilder
Lasergene software indicated the same pattern with
MspI (four bands of 310, 225, 169, and 131 bp) and
two different patterns with Hae III (398, 242, 93, 71,
31 bp for DQ465844, and 248, 242, 182, 93, and 71 bp
for DQ465845). This finding suggests sequence diversity in the analyzed LSU fragment of genotype B and
the usefulness of the Hae III enzyme in its testing.
The results obtained experimentally differed from those
predicted on the basis of an analysis of the reference
sequences. In our study, all the investigated strains
with genotype B showed the same PCR product size
(~840 bp) and the same restriction pattern with enzymes Hae III (six bands of 251, 239, 101, 90, 68, and
35 bp) and MspI (540, 170, and 130 bp). It is worth
noting that there was some discrepancy between the
results obtained by capillary electrophoresis (the above
pattern) and classical gel electrophoresis, which cannot easily distinguish the bands of ~239, 90, and 35 bp
after digestion with Hae III (data not shown). What
is interesting is that replacing the intron present in
DQ465844 by the sequence of the intron from another
strain (accession no X74272.1) resulted in changing
the restriction pattern to one more similar to our finding for genotype B, namely seven bands of 247, 242,
103, 93, 72, 32, and 20 bp for Hae III and three bands
of 539, 170, and 131 bp for MspI (Fig 1B). An analysis of the DQ465844 and DQ465845 sequences
performed after excluding the intron indicated their
high similarity (99.8%) to “subtype a”. This suggests
that the strains with genotype B tested in this study
can also be related to “subtype a” and their intron to
the X74272.1 sequence.
Genotype C generates two amplicons, one of
~460 bp and one of ~ 840 bp, so the PCR-RFLP result
could reflect the sequence diversity of the two products. The five strains of genotype C tested in this
study displayed the typical sizes of the PCR products
and the same restriction pattern with MspI (four bands
of 580, 290, 170, and 130 bp), whereas with Hae III,
four strains showed the same pattern (five bands of
290, 251, 239, 90, and 70 bp), but the fifth differed
from them by the presence of an additional band of
~400 bp (Fig. 1A). The evolutionary processes which
216
Nawrot U. et al.
resulted in the formation of the heteroallelic genotype C were discussed by many authors. McCullough
et al. (1999) proposed two hypotheses of genotype C
development. The first assumes that genotype C arises
after losing the intron by genotype B and the second
is that genotype C is formed due to the acquisition
of the intron by genotype A, most probably as a result
of sexual recombination, which has not yet been detected in C. albicans. Recently, Miletti-Gonzalez and
Leibowitz (2008) studied the genetic arrangement of
the CaLSU intron in the rDNA of an isolate with
genotype C and showed that intron-possessing rDNA
and intron-less rDNA copies are arrayed in tandem
and adjacent to each other, forming rDNA clusters
present in two R chromosomes. The authors observed
high variability in the number of rDNA complex copies among clones of genotype C. The simple RFLP
experiment performed in this study showed that the
diversity of genotype C described above can be wider
due to variability in the rDNA sequence.
In summary, the presented results indicate a high
homogeneity of the analyzed fragment of LSU rDNA
in the clinical isolates of C. albicans, in contrast to published data obtained with a Turkish collection of yeasts.
The restriction analysis of the amplicons obtained after
A, B, and C genotyping is a simple and reproducible
method enabling broader strain characteristics and can
be useful in epidemiological and evolutionary studies.
Acknowledgments
This study was supported by the Polish Ministry of Science
and Higher Education (Nr 402 055 31/1808)
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