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Microbiology (2003), 149, 3473–3484
DOI 10.1099/mic.0.26541-0
Attachment to and biofilm formation on abiotic
surfaces by Acinetobacter baumannii: involvement
of a novel chaperone-usher pili assembly system
Andrew P. Tomaras,1 Caleb W. Dorsey,13 Richard E. Edelmann2
and Luis A. Actis1
Departments of Microbiology1 and Botany2, Miami University, 40 Pearson Hall, Oxford,
OH 45056, USA
Correspondence
Luis A. Actis
[email protected]
Received 4 June 2003
Revised 27 August 2003
Accepted 5 September 2003
Acinetobacter baumannii causes severe infections in compromised patients, survives on abiotic
surfaces in hospital environments and colonizes different medical devices. In this study the
analysis of the processes involved in surface attachment and biofilm formation by the prototype
strain 19606 was initiated. This strain attaches to and forms biofilm structures on plastic and
glass surfaces, particularly at the liquid–air interface of cultures incubated stagnantly. The cell
aggregates, which contain cell stacks separated by water channels, formed under different culture
conditions and were significantly enhanced under iron limitation. Electron and fluorescence
microscopy showed that pili and exopolysaccharides are part of the cell aggregates formed by
this strain. Electron microscopy of two insertion derivatives deficient in attachment and biofilm
formation revealed the disappearance of pili-like structures and DNA sequencing analysis
showed that the transposon insertions interrupted genes with the highest similarity to hypothetical
genes found in Pseudomonas aeruginosa, Pseudomonas putida and Vibrio parahaemolyticus.
Although the products of these genes, which have been named csuC and csuE, have no known
functions, they are located within a polycistronic operon that includes four other genes, two of
which encode proteins related to chaperones and ushers involved in pili assembly in other bacteria.
Introduction of a copy of the csuE parental gene restored the adherence phenotype and the
presence of pili on the cell surface of the csuE mutant, but not that of the csuC derivative. These
results demonstrate that the expression of a chaperone-usher secretion system, some of whose
components appear to be acquired from unrelated sources, is required for pili formation and
the concomitant attachment to plastic surfaces and the ensuing formation of biofilms by
A. baumannii cells.
INTRODUCTION
Members of the genus Acinetobacter are non-motile,
ubiquitous Gram-negative bacteria that can be recovered
from a wide range of sources such as soil, water, food
products and medical environments (Bergogne-Berenzin &
Towner, 1996). The latter source is of particular significance
because A. baumannii is the Acinetobacter genomic species
of greatest importance in human medicine. A large number
of reports describe outbreaks of nosocomial infections that
3Present address: Department of Medical Microbiology and Immunology, Texas A&M University Health Science Center, College Station,
TX, USA.
Abbreviations: DIP, 2,29-dipyridyl; EDDHA, ethylenediamine-di-(ohydroxyphenyl) acetic acid; GFP, green fluorescent protein; SEM,
scanning electron microscopy; TEM, transmission electron microscopy.
The GenBank accession number for the sequence reported in this
paper is AY241696.
0002-6541 G 2003 SGM
include urinary tract infections, secondary meningitis,
wound and burn infections, and particularly nosocomial
pneumonia (Bergogne-Berenzin et al., 1993, 1996). Some
of the challenges in the prevention and treatment of the
infections caused by this opportunistic pathogen are its
remarkable widespread resistance to different antibiotics
and its ability to persist in nosocomial environments and
medical devices (Bergogne-Berenzin & Towner, 1996). A.
baumannii survives for several days on inanimate objects
and surfaces found normally in medical environments,
even in dry conditions on dust particles. These survival
properties most likely play a significant role in the outbreaks
caused by this pathogen.
The potential ability of A. baumannii to form biofilms could
explain its outstanding antibiotic resistance and survival
properties. This possibility is supported by a very limited
number of publications which showed that a clinical isolate
of this bacterium is able to attach to and form biofilm
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A. P. Tomaras and others
structures on glass surfaces (Vidal et al., 1996, 1997).
Bacterial biofilms, arrangements in which the cells are
morphologically, metabolically and physiologically different from their planktonic counterparts (Stoodley et al.,
2002), have been found on the surface of medical devices
such as intubation tubes, catheters, artificial heart valves,
water lines and cleaning instruments (Donlan & Costerton,
2002). The surfaces of all of these medical and dental devices
are normal targets for colonization by complex microbial
communities. Previous research efforts have shown that
biofilm formation proceeds via a series of steps which,
upon completion, produce a mature, three-dimensional
structure on both biotic and abiotic surfaces. Some of the
current working models for biofilm formation implicate the
participation of either bacterial surface motility mediated by
pili and flagella (O’Toole & Kolter, 1998a), the flagellummediated recruitment of planktonic cells by the developing biofilm from the liquid medium (Tolker-Nielsen et al.,
2000) or the formation and growth of microcolonies
formed as a consequence of the multiplication of cells
attached to solid surfaces (Heydorn et al., 2000). While
forming and establishing these multicellular structures,
the cells composing them secrete exopolysaccharides
which serve to fortify and maintain the structure of the
biofilm. It is not well understood whether these steps
and cell components are involved in the apparent ability
of A. baumannii to form biofilms on abiotic surfaces.
Furthermore, the mechanism by which this bacterium
forms biofilms may pose a challenge because of its wellestablished non-motile phenotype (Bergogne-Berenzin &
Towner, 1996).
In this work, we report the initial characterization of the
biofilm structures formed by the A. baumannii 19606 prototype strain under different culture conditions. In addition,
we have initiated the genetic and molecular analysis of some
of the factors that are involved in this important cellular
process that could explain the resistance and survival of
this opportunistic pathogen under harsh conditions such as
those found in patients and nosocomial environments.
METHODS
Bacterial strains, plasmids and culture conditions. The bacter-
ial strains and plasmids used in this work are listed in Table 1.
Luria–Bertani (LB) broth and agar (Sambrook et al., 1989) were
used to maintain all bacterial strains. M9 minimal medium (Miller,
1972) was used for sliding agar motility assays and growth under
chemically defined conditions. The effect of iron on biofilm formation was tested by using M9 minimal medium supplemented with
200 mM FeCl3, 200 mM ethylenediamine-di-(o-hydroxyphenyl) acetic
acid (EDDHA) or 200 mM 2,29-dipyridyl (DIP) to simulate ironreplete and two iron-limited conditions, respectively. The effect of
different carbon sources on biofilm production was examined
through the addition of either 10 mM sodium citrate, 25 mM
sodium succinate, 50 mM ethanol, 0?5 % pyruvate, 0?5 % acetate or
0?5 % lactate to Simmons salts. The latter consisted of 0?2 g MgSO4
l21, 1?0 g NH4H2PO4 l21, 1?0 g K2HPO4 l21 and 5?0 g NaCl g l21,
adjusted to pH 7?0. Transmission electron microscopy (TEM)
experiments were done using cells lifted from agar plates incubated
at 37 uC. Scanning electron microscopy (SEM) and light microscopy
experiments were done using cells cultured in LB broth. Swimming,
swarming, sliding and twitching agar plates (Rashid & Kornberg,
2000) were used to test different types of cell motility. Plates were
stab-inoculated through to the bottom with a sterile toothpick and
incubated at 37 uC for 16 h.
Table 1. Bacterial strains and plasmids used in this work
Strain/plasmid
Strains
A. baumannii
19606
19606 #37
Derivative of 19606
19606 #144
Derivative of 19606
E. coli
DH5a
Top10
EC100D
Plasmids
pCR-Blunt II-TOPO
pUC4K
pWH1266
pMU125
pMU243
pMU348
pMU349
Relevant characteristics*
Source/reference
Clinical isolate, prototype strain
csuC : : EZ : : TN <R6Kcori/KAN-2>
Bouvet & Grimont (1986)
This work
csuE : : EZ : : TN <R6Kcori/KAN-2>
This work
Used for recombinant DNA methods
Used for recombinant DNA methods
pir+; used for plasmid rescue
Gibco-BRL
Invitrogen
Epicentre
Used to clone amplicons
Source of the Kmr gene
Acinetobacter lwoffii plasmid cloned into pBR322 PvuII site; Apr Tcr
pWH1266 harbouring gfp, Apr
pWH1266 harbouring csuE, Apr
Plasmid rescued by self-ligation of NdeI-digested DNA from mutant #144
Plasmid rescued by self ligation of EcoRI-digested DNA from mutant #144
Invitrogen
PharmaciaBiotech
Hunger et al. (1990)
Dorsey et al. (2002)
This work
This work
This work
*Apr, ampicillin resistance; Kmr, kanamycin resistance; Tcr, tetracycline resistance.
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Acinetobacter baumannii biofilms
General DNA procedures. Total DNA was isolated either by ultra-
centrifugation in CsCl density gradients (Meade et al., 1982) or using
a mini-scale method adapted from previously published research
(Barcak et al., 1991). Plasmid DNA was isolated using commercial kits
(Qiagen). DNA digestions were performed with restriction enzymes as
indicated by the supplier (New England Biolabs) and size-fractionated
by agarose gel electrophoresis (Sambrook et al., 1989). Southern blot
analyses were conducted using standard protocols (Sambrook et al.,
1989) using high-stringency conditions (Graber et al., 1998). The
pUC4K aph gene, which encodes kanamycin resistance and was used
as a probe to detect the insertion of the EZ : : TN <R6Kcori/KAN-2>
transposon, was PCR-amplified with Pfu DNA polymerase (Stratagene) and the primers 133 (59-GGCGCTGAGGTCTGCCTCCTG-39)
and 134 (59-GAGCCATATTCAACGGG-39). The amplicon was
purified using the GeneClean II kit (QBiogene) and labelled with
[a-32P]dCTP (Feinberg & Vogelstein, 1983). The radioactive bands
were detected with a Storm 860 scanner (Molecular Dynamics).
Transcriptional analysis of gene expression. Expression of
polycistronic genes was tested by RT-PCR analysis using total RNA
isolated from bacteria grown in LB broth as described by Wu &
Janssen (1996). The RNA samples were treated with RNase-free
DNase I (Roche) and used with an RT-PCR commercial kit
(Qiagen), under the conditions suggested by the manufacturer. The
amplicons were analysed by agarose gel electrophoresis (Sambrook
et al., 1989). PCR of total RNA without reverse transcription was
used to test for DNA contamination of RNA samples. The nature of
the amplicons was confirmed by automated DNA sequencing.
Biofilm assays. One millilitre of fresh medium in borosilicate
(156125 mm), polystyrene (12675 mm) or polypropylene (126
75 mm) sterile tubes was inoculated with 0?01 ml of an overnight
culture. Duplicate cultures for each sample were incubated for 8 h
either shaking (at 200 r.p.m. in an orbital shaker) or stagnant at
37 uC. One tube was sonicated immediately for 5 s with a thin probe
and the OD600 of the culture was determined to estimate total cell
biomass. The supernatant of the other tube was aspirated and rinsed
thoroughly with distilled water. The cells attached to the tube walls
were visualized and quantified by staining with crystal violet and
solubilization with ethanol–acetone as described by O’Toole et al.
(1999). The OD580/OD600 ratio was used to normalize the amount
of biofilm formed to the total cell content of each sample tested to
avoid variations due to differences in bacterial growth under different experimental conditions. All assays were done at least twice
using fresh samples each time. In addition, every test was done in
duplicate each time. Square (1006100 mm) polystyrene Petri dishes
were also used to detect and characterize the structures formed by
A. baumannii cells on plastic surfaces.
Microscopy experiments. Cells attached to the bottom and the
side of square plates were visualized with regular (brightfield) light
microscopy after staining with crystal violet as described by O’Toole
et al. (1999). A. baumannii 19606 cells harbouring pMU125 and
expressing the green fluorescent protein (GFP) were visualized by
epifluorescence microscopy using a Nikon Eclipse E400 microscope
equipped with a SPOT digital camera (Diagnostic Instruments). The
same equipment was used to visualize samples stained with calcofluor white as described by Neu et al. (2002). Colonies grown overnight on Tris-M9 agar were sampled with Formvar-coated gold
grids, negative-stained with 1?5 % ammonium molybdate and visualized using a Zeiss EM 10C transmission electron microscope. For
SEM, the cells were cultured in square Petri dishes without shaking
at 37 uC either overnight or for up to 10 days replacing the culture
medium daily. Upon removal of the culture medium, the plates
were immediately flooded with 2?5 % gluteraldehyde in 0?05 M
sodium cacodylate and incubated at room temperature for 2 h. The
fixative was removed and replaced immediately with distilled water
to prevent sample dehydration. Small samples (161 cm) were cut
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from the sides of each plate, rinsed with distilled water, dehydrated
with increasing concentrations of ethanol, ranging from 25 to
100 %, and CO2 critical point dried. Samples were gold-coated and
visualized with a JEOL T200 scanning electron microscope.
Transposition mutagenesis and rescue of interrupted genes.
A. baumannii 19606 was mutagenized using the EZ : : TN <R6Kcori/
KAN-2> Tnp Transposome transposition mutagenesis system and
electroporation as described by Dorsey et al. (2002). Transformants
that grew after plating on LB agar containing 40 mg kanamycin ml21
were toothpicked into separate wells of 96-well polystyrene microtitre plates, each containing 200 ml LB broth. Plates were incubated
stagnant at 37 uC for 24 h, the culture supernatant was aspirated and
the wells were rinsed with water and stained with crystal violet.
Potential biofilm mutants were validated using tube and Petri dish
assays as described above. The genomic regions harbouring the
insertion of the EZ : : TN <R6Kcori/KAN-2> transposon were rescued by self-ligation of EcoRI- or NdeI-digested DNA and electroporation into Escherichia coli EC100D pir+ cells. Plasmid DNA
isolated from colonies that grew on LB agar containing 40 mg kanamycin ml21 was used as a template to determine the nucleotide
sequence of the genomic DNA flanking the transposon element with
the primers complementary to this insertion element that were
supplied with the mutagenesis kit. Further extension of nucleotide
sequences was done using custom-designed primers. DNA sequencing was conducted using the DYEnamic ET Terminator Cycle
Sequencing Kit (Amersham Pharmacia Biotech) and an ABI 3100
automated DNA sequencer (Applied Biosystems). Sequences were
examined and assembled using Sequencher 4.1.2 (Gene Codes).
Nucleotide and amino acid sequences were analysed with DNASTAR,
BLAST and the analysis tools available through the ExPASy Molecular
Biology Server (http://www.expasy.ch), such as SIGNALP, PSORT,
HMMTOP and TMHMM to predict the presence of signal peptides and
the cellular locations of proteins. G+C content was determined
with Artemis (http://www.sanger.ac.uk), using a 120 nt window.
Genetic complementation of a biofilm mutant. The A. bau-
mannii 19606 csuE-like gene, whose disruption by the insertion of
EZ : : TN <R6Kcori/KAN-2> caused a biofilm-deficient phenotype
in the derivative #144, was PCR-amplified from the parental strain
genome with Pfu DNA polymerase and the primers 1671 (59-CGGGATCCCGTATTGCTGCTAAACGTGGCTCGGGTGTTGTG-39)
and 1672 (39-CGGGATCCCGGCTATAGAGCTTATGCAAAACTCATGCAGTGCC-59) that included BamHI restriction sites. The
blunt-ended amplicon was ligated into pCR-Blunt II-TOPO and
transformed into E. coli Top10. Plasmid DNA, which was isolated
from a kanamycin-resistant colony and proved to have an insert
with the same nucleotide sequence as the parental DNA, was
digested with BamHI, ligated into the cognate site of the shuttle
vector pWH1266 and transformed into E. coli DH5a. Plasmid DNA
was isolated from a colony that was resistant to 100 mg ampicillin
ml21 and sensitive to 20 mg tetracycline ml21 and electroporated
into A. baumannii 19606 #37 and #144 cells. Transformants that
grew after overnight incubation at 37 uC on LB agar containing
100 mg ampicillin ml21 were tested for their ability to form biofilms
on plastic surfaces as described above. The presence of the complementing plasmid was verified by Southern blot analysis using csuE
amplified from the parental strain as a probe.
RESULTS
Attachment to and formation of biofilms on
abiotic surfaces
The initial assays showed that A. baumannii 19606 cells
which were taken from an agar plate with a bacteriological
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A. P. Tomaras and others
(b)
2
1
2
OD580/OD600
(a)
1
(a)
3
(b)
6
2
4
1
2
(c)
20
16
12
8
4
0
Fig. 1. Detection of biofilms formed on plastic surfaces. E. coli
DH5a (sample 1) and A. baumannii 19606 (sample 2) were
incubated either in polystyrene tubes (a) or square Petri dishes
(b) overnight at 37 6C without shaking in LB broth. Attached
cells were detected by staining with crystal violet after washing
with tap water.
loop, deposited on the surface of a Petri dish or a Teflon
strip and left for 10–15 min at room temperature remained
tightly adhered after washing with tap water and staining
with crystal violet. Incubation of A. baumannii 19606 cells
overnight in LB broth without shaking at 37 uC either in
polystyrene tubes or square Petri dishes also resulted in
their adherence to these surfaces. In contrast, no adherence
was detected with E. coli DH5a cells cultured under similar
conditions (compare tubes and plates shown in Fig. 1a
and b). These observations were confirmed further when
cell attachment to plastic was quantified by relating the
total cell mass to the stain retained by the attached cells
as described in Methods. It is interesting to note that the
A. baumannii 19606 cells form denser aggregates at the
liquid–air interface than on the side and bottom surfaces
of the tubes and plates. Furthermore, the biofilm grows
upwards from the liquid–air interface onto the walls of
the plate (Fig. 1b), a phenomenon that is not caused by
the movement of the broth, since the inoculated plate was
incubated without any movement until stained with crystal
violet.
Quantitative analysis showed that while the amount of
A. baumannii 19606 cells attached to polystyrene and
polypropylene is similar, their attachment to borosilicate
is significantly reduced when tested under the same
experimental conditions (Fig. 2a). Culture shaking also
reduced the amount of cells attached to these three abiotic
surfaces when compared with cultures incubated stagnantly
(Fig. 2a). The values shown in Fig. 2(a), particularly those
obtained with glass tubes, do not represent background
values since crystal violet does not attach by itself to the
surface of borosilicate. A. baumannii 19606 attaches to
polystyrene tubes when cultured stagnantly at different
temperatures, although this process is more efficient at
30 uC than at 37 uC (Fig. 2b). The addition of inorganic
iron to M9 minimal medium reduced the amount of
attached cells, while the supplementation of this chemically
defined medium with the iron chelators EDDHA or DIP
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2
0
0
3
1 2
Experimental conditions
1
2
3
4
Fig. 2. Quantification of A. baumannii 19606 biofilms formed
under different culture conditions on plastic and glass surfaces.
(a) Cells cultured in LB broth in polystyrene (1), polypropylene
(2) or borosilicate (3) tubes with (open bars) or without (closed
bars) shaking overnight at 37 6C. (b) Cells cultured in LB broth
in polystyrene tubes without shaking either at 30 (1) or 37 6C
(2). (c) Cells cultured in Tris-M9 minimal medium in polystyrene
with no supplementation (1) or supplemented with 200 mM
FeCl3 (2), 200 mM EDDHA (3) or 200 mM DIP (4) at 37 6C
overnight. OD600 was determined after the cultures were sonicated briefly to resuspend most of the cells to determine total
cell mass. OD580 was determined after the stained tubes were
incubated with ethanol-acetone.
increased cell attachment significantly (Fig. 2c). In contrast,
no significant effects were observed when the cells were
cultured in a chemically defined medium supplemented
with different carbon sources such as citrate, succinate,
ethanol, pyruvate, acetate or lactate (data not shown).
Microscopy analysis of cells attached to plastic
surfaces
Light microscopy examination of A. baumannii 19606 cells
attached to the bottom surface of a Petri dish and stained
with crystal violet showed the presence of discrete cell
clumps (Fig. 3a), which did not coalesce to form at least
a monolayer even after several days of incubation with or
without renewing the culture medium. In contrast, compact
cell aggregates were seen on the sides of the plates at the
liquid–air interface. Similar results were obtained when
A. baumannii 19606 cells producing GFP encoded by the
recombinant plasmid pMU125 were examined with epifluorescence microscopy (Fig. 3b). This figure also shows
that the fluorescent cellular aggregates are interspersed with
areas devoid of cells, an arrangement that is very similar
to those described in other bacteria capable of forming
biofilms on abiotic surfaces, such as Pseudomonas aeruginosa (Lawrence et al., 1991). Fluorescence microscopy of
samples stained with calcofluor white showed that the cells
attached either to the walls or the bottom of the plates were
embedded within a blue fluorescent material (Fig. 3c). This
non-specific UV-excitable dye that binds to exopolysaccharides such as those produced by Rhizobium meliloti
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Acinetobacter baumannii biofilms
(a)
(b)
(c)
Fig. 3. Light microscopy of A. baumannii 19606 cells attached to square Petri dishes. (a) Cells attached to the bottom
(upper panel) or the side (lower panel) were stained with crystal violet. (b) Cells harbouring the plasmid pMU125 attached
either to the bottom (upper panel) or the side (lower panel) were examined with epifluorescence microscopy. (c) Cells
attached to the bottom (upper panel) or the side (lower panel) were stained with calcofluor white and examined with
epifluorescence microscopy. All cells were cultured in LB broth without shaking overnight at 37 6C.
(Leigh et al., 1985) was recently used to study biofilms
formed by Salmonella enteritidis (Solano et al., 2002). These
reports together with our data support the hypothesis that
A. baumannii 19606 produces exopolysaccharides that are
part of the cellular structures formed by this bacterium
when it grows attached to a plastic surface.
SEM analysis of samples similar to those used for light
microscopy showed that while few cells were clustered
together on the sides of the plate below the meniscus of the
liquid medium, compact cell conglomerates separated by
channel-like spaces were formed at the meniscus of the
broth (compare Fig. 4b and c). Much denser and tighter
conglomerates were formed by cells located just above the
meniscus, which appear to form multilayer structures, with
the top cell layer covered with a film that most likely
represents the exopolysaccharides produced by this bacterium (Fig. 4a). Alternatively, this film could also have been
the result of the dehydration of the cell aggregates and the
exopolysaccharides that surround them, which are located
immediately above the liquid–air interface, during incubation.
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SEM also showed that the cells were linked to each other
through extracellular appendages that resemble pili structures (see insets of Fig. 4a and b). The presence of such a
type of appendage was investigated by TEM of cells cultured
on agar plates and stained with ammonium molybdate. This
approach showed the presence of structures that resemble
pili projecting from the surface of the cells (Fig. 5a), which
were found to be fairly equally distributed on the cell
surfaces without any particular organization.
Cell motility and the presence of cellular appendages have
been implicated in the formation of biofilms by other
bacteria (O’Toole & Kolter, 1998a; Tolker-Nielsen et al.,
2000). However, members of the Acinetobacter genus,
including A. baumannii, are taxonomically defined to lack
flagella and thus are non-motile (Bergogne-Berenzin &
Towner, 1996). To confirm this inability to move, we tested
19606 cells by inoculating defined solid media used to detect
swimming, swarming, sliding and twitching motility as
described by Rashid & Kornberg (2000). These assays
proved that A. baumannii 19606 lacked the ability to move
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A. P. Tomaras and others
Fig. 4. SEM of A. baumannii 19606 cells
attached to square Petri dishes. Attachment
of the parental strain (left) and the insertion
derivative #144 harbouring plasmid pMU243
(right) above (a and d), at (b and e) and
below (c and f) the liquid–air interface. Bars:
(a, c, d–f) 5 mm; (b) 10 mm; all insets, 1 mm.
via these mechanisms under the experimental conditions
used in this study.
Genetic analysis of cell attachment and biofilm
formation
analysis of the side of the plates in which the mutant #144
derivative was incubated showed the presence of very few
cell clusters, with no more than two or three cells grouped
together that did not display pili on their surfaces (data not
shown).
The EZ : : TN <R6Kcori/KAN-2> Tnp Transposome
system from Epicentre was used to initiate the identification and characterization of some of the genetic determinants required by A. baumannii 19606 to attach to and
form biofilms on plastic surfaces. As we reported recently
(Dorsey et al., 2002), screening of insertion derivatives of
this strain resulted in the identification of mutants affected
in cell attachment and biofilm formation without affecting
their growth in LB broth. The rescue cloning and initial
nucleotide sequence analysis of the mutant #144 resulted
in the identification of a gene encoding a protein highly
similar to that encoded by the Vibrio parahaemolyticus csuE
gene. TEM analysis of mutant #144 proved that the disruption of the A. baumannii 19606 csuE-like gene resulted
in the disappearance of the pili detected in the parental
strain (compare Fig. 5a and b). Although not shown, SEM
The role of the A. baumannii 19606 csuE-like gene was
confirmed further by genetic complementation of the
insertion derivative with the parental gene PCR-amplified
and cloned in the shuttle vector pWH1266 (Hunger et al.,
1990). Electroporation of the recombinant plasmid
pMU243 into the A. baumannii 19606 #144 insertion
derivative restored its ability to attach and form biofilms
in a fashion similar to that displayed by the 19606 parental
strain (sample 2 in Fig. 1a). In contrast, the electroporation
of pWH1266 did not change the phenotype of the biofilmdeficient derivative, which produced an image identical to
that displayed by sample 1 in Fig. 1(a). SEM and TEM
analyses also showed that the introduction of the parental
csuE-like gene restored the ability of A. baumannii 19606
#144 to form structures similar to those formed by the
parental strain (Fig. 4d–f) and the concomitant presence of
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Acinetobacter baumannii biofilms
total DNA isolated from this biofilm mutant, showed that
the A. baumannii 19606 csuE-like gene is the last component
of a gene cluster that encompasses six ORFs (Fig. 6a). The
mean G+C content of this region is 37?5 mol%; however,
ORF 2 (28?5 mol%) and 3 (29?7 mol%) displayed a
significantly lower value than the mean (Fig. 6b and
Table 2). This observation suggests that these two ORFs
were acquired from a source different from that of the
remaining ORFs that compose this apparently polycistronic
locus, whose G+C content is closer to the 40–43 mol%
values assigned to different A. baumannii isolates (Bouvet
& Grimont, 1986). The polycistronic nature of this locus
was confirmed by RT-PCR analysis, which produced
amplicons with the predicted sizes (Fig. 7), and nucleotide
sequences when the appropriate pairs of primers were
used. None of these amplicons could be detected when total
RNA was used as a template for PCR.
Fig. 5. TEM of A. baumannii 19606 cells cultured on agar and
stained with ammonium molybdate. (a) 19606 parental strain.
(b) Insertion mutant #144. (c) Insertion mutant #144 harbouring the plasmid pMU243. Bars, 100 nm.
pili on the surface of the mutant cells (Fig. 5c). Southern
blot analysis of total DNA isolated from A. baumannii 19606
#144 and the complemented mutant probed with the csuElike gene validated the attachment and biofilm results by
confirming the presence of the complementing plasmid
pMU243 as an independent replicon without detectable
rearrangements (data not shown).
Sequence analysis of plasmid DNA rescued
from mutant #144
Sequence analysis of plasmids pMU348 and pMU349, which
were rescued by self-ligation of NdeI- and EcoRI-digested
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The first ORF encodes a potential protein with a predicted
25 aa signal peptide, which showed the highest similarity
to the P. aeruginosa hypothetical protein PA4648 (Stover
et al., 2000) and the Pseudomonas putida PP2358–PP2360
hypothetical proteins (Nelson et al., 2002) (Table 2). All
these proteins contain the conserved domain COG5430,
which has been identified in other hypothetical proteins
produced by bacteria such as P. aeruginosa, Ralstonia
solanacearum and Yersinia pestis. These proteins were
annotated as potential secreted proteins related to the
type 1 pili subunit CsuA and CsuB proteins. Significant
homology was also detected with the Myxococcus xanthus
spore protein U, which is produced as a secretory precursor
and then secreted across the membrane to be assembled
on the spore surface (Gollop et al., 1990). Interestingly, the
protein U and other proteins annotated as protein U-like
proteins also showed significant similarity to the conserved
domain COG5430. This similarity may reflect the fact that
most of the proteins that belong to this family have been
annotated as secreted proteins and therefore may use
similar secretion mechanisms. The product of ORF 2,
which is predicted to have a 31 aa signal peptide, displayed
significant similarity only to the hypothetical V. parahaemolyticus CsuA protein (Table 2). The predicted product
of ORF 3 is a protein with a potential 20 aa signal sequence
that is related to the proteins PP2360, PA4648 and CsuB
from P. putida, P. aeruginosa and V. parahaemolyticus,
respectively (Table 2). Interestingly, all these proteins contain a sequence highly related to the conserved domain
COG5430 that has been found in proteins annotated as
type 1 pili subunit CsuA/B or CsuB in different Gramnegative bacteria, as well as sequences related to the spore
protein U. Further analysis using GAP showed that the
identity of the A. baumannii 19606 CsuA/B, CsuA and CsuB
proteins ranged from 23?7 to 27?7 %, while their similarity
was between 34?1 and 36?6 %.
The predicted product of the fourth ORF is highly related
to the hypothetical PP2361, CsuC and PA4651 proteins of
P. putida, V. parahaemolyticus and P. aeruginosa, respectively
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A. P. Tomaras and others
Fig. 6. Analysis of the A. baumannii 19606
DNA region harbouring the csu locus. (a)
Genetic and physical map of the csu locus.
The horizontal arrows and numbers indicate
the location and direction of transcription of
the ORFs identified by the computer analysis
of nucleotide sequence of the region
rescued from mutant #144 as plasmids
pMU348 and pMU349. Vertical lines indicate the location of the NdeI and EcoRI
restriction sites. The vertical arrows indicate
the transposon insertion site in the isogenic
mutants #37 and #144. The name of each
ORF was assigned based on its similarity
to genes described in other bacteria. (b)
Percentage G+C of the csu locus determined with Artemis using a 120 nt window.
(c) Alignment of csu-like ORFs present in
the genomes of different bacteria. The numbers within the arrows represent the gene
numbers assigned in the genome of the
cognate bacterial strains.
(Table 2). Analysis with BLASTP showed that all four proteins
are highly related to fimbrial chaperones and contain the
COG 3121.1 and Pfam 00345.6 conserved domains, which
have been either described or annotated as pili assembly
factors, with the FimC and PapD chaperones being some of
the best characterized members of this protein family that
Table 2. Sequence analysis of the ORFs located within the csu locus of A. baumannii 19606
ORF
G+C (mol%)
Protein (aa/kDa)
Similarity*
E valueD
Identity/similarityd
Accession no.
1
39?0
180/18?7
2
3
28?5
29?7
192/20?8
172/19?3
4
37?6
277/30?6
5
39?7
832/92?7
6
42?5
339/36?6
P. aeruginosa PA4648
P. putida PP2358
P. putida PP2360
P. putida PP2359
V. parahaemolyticus CsuA
P. putida PP2360
P. aeruginosa PA4648
V. parahaemolyticus CsuB
P. putida PP2361
V. parahaemolyticus CsuC
P. aeruginosa PA4651
P. putida PP2362
P. aeruginosa PA4652
P. putida PP2363
V. parahaemolyticus CsuE
P. aeruginosa PA4653
5e-19
2e-12
1e-10
7e-10
3e-06
4e-07
1e-06
3e-06
1e-35
2e-35
2e-32
e-144
e-143
4e-26
2e-23
6e-18
42?4/49?4
28?4/39?2
32?9/39?0
33?7/40?7
26?0/40?5
31?9/41?1
30?7/36?1
25?3/36?7
35?4/45?4
35?3/54?1
31?0/41?1
38?0/46?0
41?7/50?4
34?2/38?9
33?2/41?6
29?8/38?1
Q9HVE4
AAN67971
AAN67973
AAN67972
AF339087
AAN67973
Q9HVE4
AF339087
AAN67974
AF339087
Q9HVE1
AAN67975
Q9HVE0
AAN67976
AF339087
Q9HVD9
*Identification of bacterial products is based on BLASTP analysis.
DThe E values of the highest and more significant matches obtained during the
dValues were obtained with the GCG GAP program.
3480
BLASTP
analyses are shown.
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Microbiology 149
Acinetobacter baumannii biofilms
1
2
3
4
5
6
kbp
2.0
0.6
Fig. 7. Agarose gel electrophoresis of the amplicons obtained
by RT-PCR of total RNA extracted from A. baumannii 19606
cells. The primers used were designed to detect the expression
of ORF 1–ORF 2 (lane 1), ORF 2–ORF 3 (lane 2), ORF 3–
ORF 4 (lane 3), ORF 4–ORF 5 (lane 4) and ORF 5–ORF 6
(lane 5). Lane 6, HindIII-digested l DNA.
are involved in the biogenesis of pili in E. coli (Hermanns
et al., 2000; Sauer et al., 2000). The expression and role
of this gene is supported by the phenotype of the derivative #37, which harbours a transposon insertion in csuC
(Fig. 6a) and displays the same biofilm-deficient phenotype
as mutant #144 (data not shown). TEM analysis showed
that CsuC-deficient cells do not have pili on the cell surface, an observation that confirms the role of this protein
in the assembly of these cell appendages (data not shown).
The product of ORF 5 is a large protein that is highly
similar to the hypothetical proteins PP2363 and PA4652 of
P. putida and P. aeruginosa, respectively (Table 2). These
three proteins contain sequences related to COG3188 and
Pfam 00577, conserved domains that have been described
in fimbrial usher proteins involved in the biogenesis of
Gram-negative bacterial pili. This protein family includes
the FimD and PapC usher proteins that are required for the
biogenesis of pili (Klemm et al., 1985; Thanassi et al., 1998).
The last ORF of this A. baumannii 19606 gene cluster, whose
disruption by the insertion of the EZ : : TN <R6Kcori/KAN2> transposon (Fig. 6a) resulted in the isolation of the
attachment and biofilm deficient derivative #144, encodes
a predicted protein that has a 27 aa signal peptide. BLASTP
analysis showed that the most related proteins are all
hypothetical proteins, with the highest similarity to the
PP2363, CsuE and PA4653 proteins of P. putida, V. parahaemolyticus and P. aeruginosa, respectively (Table 2). This
predicted A. baumannii protein has sequences highly
related to the COG5430 conserved domain that has been
found in uncharacterized secreted proteins in P. aeruginosa,
Y. pestis, Ralstonia solanacearum and Rickettsia conorii.
Furthermore, a search of the Pfam database with MOTIFSCAN
showed that the product of this ORF has a motif with some
similarity to fimbrial proteins found in different bacteria. As
described for the products of ORFs 1 and 3, the product
of ORF 6 also has similarity with proteins harbouring the
protein U domain found in the family of spore coat proteins. Fig. 6(c) shows that the A. baumannii 19606 gene
cluster has the same number of genes and genetic organization as in P. aeruginosa (Stover et al., 2000) and Y. pestis
(Parkhill et al., 2001), while the cognate loci in P. putida
(Nelson et al., 2002) and V. parahaemolyticus (GenBank
http://mic.sgmjournals.org
accession no. AF339087; Makino et al., 2003) are one ORF
longer and shorter, respectively.
It is worthy to note that all the genes and gene products
described in Table 2 are hypothetical bacterial elements
described during the annotation of the cognate genomes.
Therefore, neither their expression nor biological role(s)
were tested experimentally as we have done with the A.
baumannii 19606 csu locus. Furthermore, the P. aeruginosa
PA4648–PA4653 gene cluster, which showed significant
similarity to the A. baumannii 19606 csu locus, is different
from the cupA1–A5 fimbrial gene cluster that specifies a
chaperone-usher pathway that was found to be involved
in the formation of biofilm formation by this pathogen
(Vallet et al., 2001). Nevertheless, the A. baumannii 19606
csu locus showed significant similarity with the cupA cluster
when compared with BLASTX. However, the similarity was
only with CupA3 (E value 1e-20), the usher component of
this P. aeruginosa secretory system that was annotated as
the PA2130 protein. Therefore, the expression and biological role of the P. aeruginosa PA4648–PA4653 gene
cluster remains to be tested.
DISCUSSION
Several reports have shown that Acinetobacter environmental isolates form biofilm communities composed of
different bacterial species. In contrast, only two brief
reports describe the ability of A. baumannii clinical isolates
to attach to and form biofilms on glass surfaces (Vidal
et al., 1996, 1997), a property that is most likely to be
associated with the capacity of this pathogen to survive in
hospital environments and medical devices, and cause
severe infections in compromised patients (BergogneBerenzin & Towner, 1996). In this report we have extended
these initial observations by showing that the prototype
strain 19606 also attaches to and forms biofilms on the
surface of different plastics (polystyrene, polypropylene
and Teflon), some of which are used in the fabrication of
medical devices. It is also evident that this bacterium
forms biofilms under static as well as dynamic conditions,
although the latter results in less biofilm when compared
with samples incubated stagnantly. This property could
explain the ability of this pathogen to persist successfully
in medical environments where cells could be subjected to
the shearing forces of a liquid stream, such as those found
with catheters and respiratory tubes, or allowed to persist
on less disturbed surfaces such as those of hospital furniture and bed linen (Bergogne-Berenzin & Towner, 1996).
The formation of biofilms at different temperatures and
the presence of extracellular material surrounding the
attached cells that is stained with calcofluor white are also
in accordance with the ability of this pathogen to grow
within a wide range of temperatures and produce capsule
and exopolysaccharides (Bergogne-Berenzin & Towner,
1996; Towner et al., 1991). It is also clear that A. baumannii
19606 attaches to and forms biofilm structures on hydrophobic (plastic) as well as hydrophilic (glass) surfaces, with
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A. P. Tomaras and others
much denser cell conglomerates located at the liquid–air
interface. This observation is significant because A.
baumannii is a strict aerobic bacterium, a phenotype that
could explain this behaviour as well as the observation that
the bottom of the plates or tubes did not have a contiguous
cell layer even after extended incubation. No significant
effects on biofilm formation were observed when the cells
were cultured in a chemically defined medium supplemented
with different carbon sources. In contrast, the incubation
of A. baumannii 19606 cells under iron limitation resulted
in a significant increase of biofilm when compared to that
obtained with cells cultured under iron-rich conditions.
This expressional behaviour could be explained by the
presence of the nucleotide sequence 59-TACAAATCTAATCATTATA-39 10 nt upstream of the first ORF, which
matches 13 of the 19 nt of the binding site for the Fur iron
repressor protein that controls the expression of ironregulated protein in bacteria (Calderwood & Mekalanos,
1988). These observations are quite significant since these
bacterial cells would most likely be exposed to iron-limiting
conditions either in the host or the nosocomial environment
(Neilands, 1981).
The light microscopy of crystal-violet-stained cells and
fluorescence microscopy of GFP-expressing bacteria
showed that the conglomerates located at the liquid–air
interface indeed form biofilm structures similar to those
described for other bacteria, with channels that would
provide nutrients and remove waste products. The presence
of these channels is supported further by the data obtained
by SEM, which showed stacks of cells surrounded by open
areas on the sides of the plates at the liquid–air interface
(Fig. 4b). The cell stacks appear to be 4–5 cells tall with
the cells attached to each other by pili-like structures and
amorphous material, with the latter being more evident in
the structures formed just above the meniscus of the broth.
The pili-like structures, whose presence was confirmed
by TEM analysis, were also seen on the surface of A.
calcoaceticus RAG-1, a hydrocarbon-degrading environmental isolate that adheres to plastic surfaces and hexadecane droplets (Rosenberg et al., 1982). Interestingly, a
mutation that abolished the formation of these cell-surface
appendages impaired the ability of this bacterium to attach
to hydrophobic surfaces and grow in the presence of hexadecane as sole carbon source. This attachment phenotype
that depends on the formation of thin filaments is very
similar to what we have observed with A. baumannii 19606,
which is discussed in more detail below. The amorphous
material seen between cells, cell stacks and particularly
covering the top cell layer formed above the broth meniscus
may represent exopolysaccharides, compounds known to
be produced by environmental and clinical isolates of
Acinetobacter (Towner et al., 1991). Taken together, the
data indicate that environmental and clinical members of
the Acinetobacter genus use similar strategies to attach to
solid surfaces and to initiate the formation of biofilm
structures that allow them to persist and proliferate under
harsh conditions.
3482
Cell motility mediated either by appendages such as
flagella and pili or the action of glycopeptidolipids is
required for biofilm formation by bacteria such as
P. aeruginosa (O’Toole & Kolter, 1998a), P. fluorescens
(O’Toole & Kolter, 1998b) and Mycobacterium smegmatis
(Recht et al., 2000), respectively. The lack of this cell
behaviour was one of the criteria used by Baumann et al.
(1968) to name this genus Acinetobacter, although twitching and gliding motility have been observed when some
isolates were tested on semi-solid media (Towner et al.,
1991). All our attempts failed to detect any type of motility
with the A. baumannii 19606 prototype strain, indicating
that its ability to form biofilms does not depend on this
cell property, at least under the experimental conditions
used in this work. Based on this behaviour, it is possible
to speculate that the multiplication and growth of cells
and microcolonies already attached to solid surfaces is the
mechanism by which this strain forms biofilms on plastics
and glass, as described for some Pseudomonas strains
(Heydorn et al., 2000).
The genetic approach used in this work proved that
the presence of pili-like structures on the surface of A.
baumannii 19606 cells is essential in the early steps of the
process that leads to the formation of biofilm structures
on plastic surfaces. The disruption of the csuC and csuE
ORFs resulted in non-piliated cells and abolished cell
attachment and biofilm formation. These defects were
restored to levels similar to those of the parental strain
when the insertion derivative #144 was electroporated with
a shuttle vector harbouring the wild-type copy of the csuE
gene. In contrast, this recombinant construct could not
reverse the phenotype of the mutant #37, in which the
insertion interrupted the csuC chaperone-encoding gene, to
that of the parental strain. Taken together, the data show
that this is indeed a polycistronic operon that encodes
functions required for pili assembly and biofilm formation. Based on the nucleotide composition, it appears that
part of this gene cluster has been acquired by A. baumannii
19606 from an unrelated source by lateral gene transfer, a
possibility that is in accordance with the well known natural
competence and ability of members of this genus to acquire
DNA easily from different sources (Towner et al., 1991).
While the translational products of the fourth and fifth
ORFs are the most recognizable since they are highly related
to chaperone and usher bacterial proteins, respectively, the
four remaining ORFs encode hypothetical proteins potentially involved in pili assembly. Searches with PSI-BLAST,
SCANPROSITE and MOTIFSCAN failed to identify the ORF
encoding the major pilus protein among the four ORFs
whose predicted products are not related to chaperone and
usher proteins. On one hand, this observation may indicate
that the A. baumannii 19606 gene encoding this subunit is
one of the four ORFs located in this cluster, although its
translation product is not significantly related to known pili
subunit sequences. On the other hand, it is possible that
the gene encoding this subunit is located outside of this
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Microbiology 149
Acinetobacter baumannii biofilms
gene cluster. This analysis also showed that the systems
most related to that of A. baumannii 19606 are found in
the human pathogens P. aeruginosa (Stover et al., 2000)
and Y. pestis, and the environmental bacterium P. putida
(Nelson et al., 2002), all of which have been annotated as
hypothetical genes and proteins. In addition, a similar
locus was found in the strains BB22 (GenBank accession
no. AF339087) and RIMD 2210633 (KXV237) (Makino
et al., 2003) of the food-borne pathogen V. parahaemolyticus. This observation suggests that the csu operon is
widespread among unrelated bacteria, which could play a
central role in the ability of these micro-organisms to attach
to and form biofilms on abiotic surfaces, a property that
would allow them to persist in their natural environments.
In summary, the results presented in this study demonstrate
the ability of the prototype strain 19606 of the opportunistic
pathogen A. baumannii to attach to and form structures
typical of bacterial biofilms on abiotic surfaces under
different experimental conditions. In addition, the genetic
approach used in this work proved that the expression of
csuC and csuE, which belong to a gene cluster related to
bacterial loci encoding secretion and pili assembly functions
and the production of pili are required in the early steps of
the process that leads to biofilm formation. Prior to this
study, the loci most closely related to the A. baumannii
19606 csu locus were annotated as hypothetical genes and
proteins. This study demonstrates the expression of and
identifies a biological function for one member of this
uncharacterized group of operons of the chaperone-usher
superfamily.
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