1 Departments of Microbiology, Miami University, 40 Pearson Hall, Oxford, OH 45056, USA
2 Departments of Botany, Miami University, 40 Pearson Hall, Oxford, OH 45056, USA
Correspondence
Luis A. Actis
actisla{at}muohio.edu
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ABSTRACT |
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The GenBank accession number for the sequence reported in this paper is AY241696.
Present address: Department of Medical Microbiology and Immunology, Texas A&M University Health Science Center, College Station, TX, USA.
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INTRODUCTION |
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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 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 flagellum-mediated 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 well-established 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.
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METHODS |
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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 (15x125 mm), polystyrene (12x75 mm) or polypropylene (12x75 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 °C. 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 ethanolacetone 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 (100x100 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 °C 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 (1x1 cm) were cut 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 <R6Kori/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 µg kanamycin ml-1 were toothpicked into separate wells of 96-well polystyrene microtitre plates, each containing 200 µl LB broth. Plates were incubated stagnant at 37 °C 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 <R6K
ori/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 µg kanamycin ml-1 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. baumannii 19606 csuE-like gene, whose disruption by the insertion of EZ : : TN <R6Kori/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 (5'-CGGGATCCCGTATTGCTGCTAAACGTGGCTCGGGTGTTGTG-3') and 1672 (3'-CGGGATCCCGGCTATAGAGCTTATGCAAAACTCATGCAGTGCC-5') 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 DH5
. Plasmid DNA was isolated from a colony that was resistant to 100 µg ampicillin ml-1 and sensitive to 20 µg tetracycline ml-1 and electroporated into A. baumannii 19606 #37 and #144 cells. Transformants that grew after overnight incubation at 37 °C on LB agar containing 100 µg ampicillin ml-1 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.
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RESULTS |
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Genetic analysis of cell attachment and biofilm formation
The EZ : : TN <R6Kori/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 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 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 biofilm-deficient 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 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 csuE-like 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 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 4043 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.
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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 (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 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 <R6Kori/KAN-2> 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 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 PA4648PA4653 gene cluster, which showed significant similarity to the A. baumannii 19606 csu locus, is different from the cupA1A5 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 PA4648PA4653 gene cluster remains to be tested.
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DISCUSSION |
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The light microscopy of crystal-violet-stained cells and fluorescence microscopy of GFP-expressing bacteria showed that the conglomerates located at the liquidair 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 liquidair interface (Fig. 4b). The cell stacks appear to be 45 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.
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 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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ACKNOWLEDGEMENTS |
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Received 4 June 2003;
revised 27 August 2003;
accepted 5 September 2003.