If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
Corresponding author. Address: Department of Microbiology, Hospital Clínic, IDIBAPS, School of Medicine, University of Barcelona, Villarroel 170, 08036 Barcelona, Spain. Tel.: +34 932 275522; fax: +34 932 279372.
Acinetobacter baumannii is emerging as an important hospital pathogen, which can persist in the environment for extended periods of time. It is known to produce biofilms, a community of bacteria enclosed within a protective polymeric matrix.
Aim
To establish whether the effect of biofilm formation by Acinetobacter baumannii may be associated with persistence in the hospital environment.
Methods
The effect of biofilm formation on the survival of A. baumannii on dry surfaces was investigated in biofilm-forming compared to non-biofilm-forming strains. Survival assays were determined by viable counts of the cells inoculated on to glass cover slips and stored under controlled conditions of temperature and relative humidity.
Findings
The survival times for the biofilm-forming strains were longer than for the non-biofilm-forming ones (36 vs 15 days, respectively, P<0.001). Scanning and transmission electron microscopy studies showed a polysaccharide layer and appendages in the biofilm-forming strains, not in the non-biofilm forming ones.
Conclusion
Biofilm formation increases the survival rate of A. baumannii on dry surfaces and may contribute to its persistence in the hospital environment, increasing the probability of causing nosocomial infections and outbreaks.
Acinetobacter baumannii is an important pathogen capable of causing nosocomial infections, including pneumonia, wound and urinary tract infections, bacteraemia, and meningitis, especially in patients in the intensive care unit.
A. baumannii can survive on fingertips and inanimate objects such as glass, plastic and other environmental surfaces, even after exposure to dry conditions, during extended periods of time, and the environment has been implicated as a transmission route in some outbreaks.
Since A. baumannii can produce biofilm, the resistance phenotype could be attributed to the ability of A. baumannii clinical strains to form biofilms on abiotic surfaces.
Bacteria in a biofilm, as a structural community, are enclosed in a polymeric matrix constituting a protective mechanism to survive in harsh environments and during host infection. These bacteria become more resistant to antimicrobial stressors, antibiotics or cleaning and therefore this biofilm structure represents an important virulence factor.
To understand the effect of biofilm formation on the persistence of Acinetobacter on dry surfaces, we performed survival assays with biofilm-forming and non-biofilm-forming strains on glass cover slips in a desiccated environment and analysed the structure of the resulting biofilms.
Methods
Bacterial strains
We selected four isolates, two biofilm-forming and two non-biofilm-forming ones, from a set of 92 clonally unrelated isolates from a collection of 221 A. baumannii clinical isolates collected during the GEIH-Ab 2000 project.
In this study it was found that all clonally related isolates shared either an ability or an inability to form biofilm. Accordingly, the isolates for the present study were clonally unrelated and randomly selected. Susceptibility of the isolates to antimicrobial agents was tested by microdilution method according to the Clinical and Laboratory Standards Institute.
Biofilm formation was determined as follows. Overnight cultures were diluted to an OD600 of 0.01 in Mueller–Hinton broth (Oxoid, Madrid, Spain), deposited in 96-well plates and incubated at 37 °C for 48 h without shaking. Biofilm was stained with 0.5% Crystal Violet (w/v) and quantified at 550 nm after solubilization with 95% ethanol. The experiment was performed in duplicate. OD550 values for each well were subtracted from those of the blank, which only contained MH broth without inoculum.
Survival assay
A 1 mL aliquot of overnight nutrient broth culture at 30 °C was placed in a 1.5 mL Eppendorf tube and centrifuged for 5 min at 11, 600 g. Cells recovered were washed once and resuspended in distilled water. Approximately 20 μL of these suspensions were deposited on to sterile rounded glass cover slips, placed in uncovered Petri dishes and kept in airtight transparent plastic boxes. Relative humidity inside the plastic boxes was maintained at 31%±3 by the presence of a saturated salt solution of CaCl2·6H2O in an open 50 mL beaker.
Viable counts were determined at zero time, 24 h and every 72 h thereafter until the colony count was ≤20. For viable counts, each glass cover slip was vortexed vigorously for 15 s in 2 mL of sterile distilled water and 100 μL aliquots were inoculated on to nutrient agar plates, after appropriate dilutions. Three glass cover slips were washed separately for each count, and the whole survival assay described above was repeated on three occasions.
Electron microscopy assays
Strains were grown on to glass cover slips as previously described in the survival assays and were also grown in liquid medium to compare the biofilm morphology with that formed on dry surfaces.
Scanning electron microscopy (SEM)
Biofilms developed on glass cover slips in liquid medium were fixed in 1.2% glutaraldehyde in 0.1 M sodium cacodylate (pH=7.4) containing 0.05% Ruthenium Red.
Samples were postfixed in osmium tetroxide in cacodylate buffer and dehydrated in acetone, then treated with the critical point drying method (Polaron CPD 7501) and coated with gold (Bio-RAD SC510). Glass cover slips treated under dry conditions were fixed with glutaraldehyde and osmium vapours during ≥24 h in a closed chamber and coated with gold (Bio-Rad SC510). Images were performed in a Zeiss DSM 940 A at 15 kV and a Hitachi H-4100FE.
Transmission electron microscopy (TEM)
Samples were prepared on glass cover slips and thermanox. Thermanox was dehydrated in alcohol and the glass coverslips were processed for embedding in Spurr resin. Semithin and thin sections were cut on an Ultracut E (Reichert–Jung) maintaining the thermanox. Semithin sections were stained with Methylene Blue and observed under light microscopy (Olympus). Thin sections were stained in 2% uranyl acetate and lead citrate for observation on TEM. Images were performed in a JEOL 1010 at 80 kV using a Bioscan charge-couple device camera (Gatan).
Statistical analysis
The data were analysed using Statistical Package for the Social Sciences version 16 (SPSS Inc., Chicago, IL, USA). Comparisons between viable counts, time and biofilm/non-biofilm-forming strains were performed using the Bonferroni test. P<0.001 was considered statistically significant.
Results
The ability of strains Ab033 and Ab053 to form biofilm, and the inability of strains Ab001 and Ab143 to form biofilm was confirmed by Crystal Violet staining. The means of the duplicate OD600 values were: strain Ab033 (1.814), Ab053 (2.174), Ab001 (0.24), Ab143 (0.109). The interpretation for these OD values is: biofilm negative (<0.4), low biofilm-forming (0.4–1), and strong biofilm-forming (>1.5).
Biofilm-forming strains were less resistant to almost all the antimicrobials than their non-biofilm-forming counterparts. Non-biofilm-forming strains were resistant to piperacillin, ceftazidime, cefepime, ciprofloxacin, gentamicin, tobramycin; one of them, strain Ab001, was resistant also to imipenem and meropenem. The antimicrobial susceptibility of the strains is summarized in Table I.
Table IMinimum inhibitory concentration (MIC) of biofilm-forming and non-biofilm-forming Acinetobacter baumannii strains
A statistically significant difference (P<0.001) in the survival curves was observed; the maximum survival time for the biofilm-forming strains was 36 days, being 15 days for the non-biofilm-forming strains (Figure 1). The pairwise comparisons of the viable counts between the two groups of strains showed no statistically significant difference between the strains included in each group: Ab033 vs Ab053, P=0.019; Ab001 vs Ab143, P=0.022, contrary to the comparison between strains of the two groups: Ab001 vs Ab033 and Ab053, P<0.001; Ab143 vs Ab033 and Ab053, P<0.001. The results of the repetition of the assays were very similar. A mean of 13.8 cfu/mL difference between experiments with a confidence interval of 95% (8.4–19.4cfu/mL) was considered not relevant for the experiment.
Figure 1Survival curve. Comparison between biofilm-forming (Ab033, ▪; Ab053, ▴) and non-biofilm-forming (Ab001, ♦; Ab143, ●) strains.
SEM analysis of samples in liquid medium showed few cells clustered together in the non-biofilm-forming strains and large groups of conglomerate cells in the biofilm-forming strains (Figure 2A). In both cases, the cells’ morphology remained unaltered. On dry surfaces (Figure 2B), tight and dense conglomerates of cells were constituted by biofilm-forming strains, seemingly forming a multilayer structure, with the cells covered by a film most likely representing the exopolysaccharide produced by A. baumannii. By contrast, this layer was absent in the non-biofilm-forming strains and the cells seemed dehydrated (Figure 2B). SEM also showed that the biofilm-forming strains were linked to each other through extracellular appendages, possibly pili structures, both in liquid medium and on dry surfaces (Figure 2A and B).
Figure 2Scanning electron microscopy (SEM) of A. baumannii: (A) in liquid medium, (B) on dry surfaces. Black arrows specify cell extensions or channels and white arrows the exopolysaccharide layer covering the cells. Transmission electron microscopy (TEM) of A. baumannii (C) in liquid medium, (D) on dry surfaces. Black arrows specify appendage structures (pili or fimbriae) and white arrow the thick exopolysaccharide layer. (a, b) A. baumannii biofilm-forming strains; (c, d) A. baumannii non-biofilm-forming strains. In all photographs images (b) and (d) correspond to higher magnification of (a) and (c), respectively, to provide more detail.
An important observation was the statistically significant (P<0.001) decrease in the cfu/mL in the non-biofilm-forming strains by the third day (Figure 1). This result correlated with SEM and TEM analysis after 48 h of incubation, with dehydration and altered cell morphology in the non-biofilm-forming strains.
TEM analysis of biofilm-forming strains in liquid medium and dry surfaces clearly showed the presence of appendages projecting from the surface of the cells, that were absent in non-biofilm-forming strains. Additionally, a thick light grey layer was observed on the cell surface in biofilm-forming strains (Figure 2C and D) which may correspond to the exopolysaccharide matrix secreted by A. baumannii as a mechanism of protection against desiccation. Changes in cell morphology (such as compressed cells) were observed only when strains were grown on dry surfaces (Figure 2D).
Discussion
A. baumannii is an important opportunistic pathogen, with the ability to colonize and persist in the hospital setting and on medical devices, and also constitutes a significant problem in intensive care units.
In our study, survival assays clearly indicated that A. baumannii strains can attach to glass cover slips and also form biofilm, allowing their survival under dry conditions for much longer than non-biofilm-forming strains.
In addition to biofilm formation, some authors have described resistance of acinetobacter to many antibiotics in bacteria embedded in the biofilm.
In the present study, non-biofilm-forming strains were particularly more resistant than biofilm-forming strains (Table I). Rodriguez-Baño et al. found that biofilm-forming isolates were more susceptible to imipenem and ciprofloxacin than their non-biofilm-forming counterparts.
Jawad et al. found no statistically significant differences between the survival times of sporadic and acinetobacter outbreak strains but did find outbreak strains to be significantly more resistant than sporadic strains.
Vidal et al. found that an acinetobacter biotype 9 isolate from a respiratory tract infection formed biofilm on glass cover slips and that this comprised an amorphous material similar to exopolysaccharide.
In our study, we found A. baumannii clinical isolates attached to glass cover slips forming biofilm under dry conditions, with an exopolysaccharide matrix covering the cells only in biofilm-forming strains, as identified by SEM and TEM analysis. Because the exopolysaccharide is highly hydrated, it may prevent lethal desiccation and may thus protect against variations in humidity. It may also contribute to mechanical stability, longer survival and antimicrobial resistance.
In our SEM and more specifically in TEM analysis, cells linked to each other with extracellular appendages that resemble fimbriae or pili were observed only in the biofilm-forming strains. Tomaras et al. demonstrated that A. baumannii ATCC 19606 adhered to and formed biofilm on abiotic surfaces and that pili production was essential for biofilm formation by this clinical strain.
Inactivation of csuE results in the abolition of pili production as well as cell attachment and biofilm formation. Therefore, these appendages could be an important factor for bacterial adherence to solid surfaces, and medical devices.
In summary, our findings show a relationship between biofilm formation and survival of A. baumannii clinical isolates, demonstrating that isolates producing biofilm survive longer than their non-biofilm forming counterparts on dry surfaces. Thus, biofilm production and resistance to desiccation of acinetobacter may enhance colonization and persistence in the hospital environment and also increase the probability of acquiring antimicrobial resistance and ability to cause nosocomial infections and outbreaks.
Acknowledgements
This study has been supported by the Spanish Ministry of Health (FIS 08/0195 to J.V.), by 2009 SGR 1256 from the Departament de Universitats, Recerca i Societat de la Informació de la Generalitat de Catalunya, the Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, Spanish Network for the Research in Infectious Disease (REIPI 06/0008), and by funding from the European Community (TROCAR contract HEALTH-F3-2008-223031). We want to thank the AlBan programme E07D401559CO for supporting P.A.E., as well as Dr N. Cortadellas and A. García of the Electronic Microscopy Unit, Medicine Faculty SCT, University of Barcelona.
Conflict of interest statement
None declared.
Funding sources
None.
References
Bergogne-Bérézin E.
Towner K.
Acinetobacter spp as nosocomial pathogens: microbiological, clinical and epidemiological features.
00345-8&id=gr1.jpg){kind=link}
00345-8&id=gr2.jpg){kind=link}