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Acinetobacter on dry surfaces, we performed survival assayswith biofilm-forming and non-biofilm-forming strains on glasscover slips in a desiccated environment and analysed thestructure 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 unrelatedisolates from a collection of 221 A. baumannii clinical isolatescollected during the GEIH-Ab 2000 project.8 In this study it wasfound that all clonally related isolates shared either an abilityor an inability to form biofilm. Accordingly, the isolates for thepresent study were clonally unrelated and randomly selected.Susceptibility of the isolates to antimicrobial agents was testedby microdilution method according to the Clinical and Labo-ratory Standards Institute.14

Biofilm formation

Biofilm formation was determined as follows. Overnightcultures were diluted to an OD600 of 0.01 in Mueller eHintonbroth (Oxoid, Madrid, Spain), deposited in 96-well plates andincubated at 37 C for 48 h without shaking. Biofilm was stainedwith 0.5% Crystal Violet (w/v) and quantified at 550 nm after solubilization with 95% ethanol. The experiment was per-formed in duplicate. OD550 values for each well were sub-tracted from those of the blank, which only contained MH brothwithout inoculum.

Survival assay

A 1 mL aliquot of overnight nutrient broth culture at 30

Cwas placed in a 1.5 mL Eppendorf tube and centrifuged for 5 min at 11, 600 g . Cells recovered were washed once andresuspended in distilled water. Approximately 20 mL of thesesuspensions were deposited on to sterile rounded glass cover slips, placed in uncovered Petri dishes and kept in airtighttransparent plastic boxes. Relative humidity inside the plasticboxes was maintained at 31% 3 by the presenceof a saturatedsalt solution of CaCl2$6H2O in an open 50 mL beaker.

7

Viable counts were determined at zero time, 24 h and every72 h thereafter until the colony count was 20. For viablecounts, each glass cover slip was vortexed vigorously for 15 s in2 mL of sterile distilled water and 100 mL aliquots were inocu-lated 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 onthree occasions.

Electron microscopy assays

Strains were grown on to glass cover slips as previouslydescribed in the survival assays and were also grown in liquidmedium to compare the biofilm morphology with that formedon 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

(pH¼ 7.4) containing 0.05% Ruthenium Red.15 Samples werepostfixed in osmium tetroxide in cacodylate buffer and dehy-drated in acetone, then treated with the critical point dryingmethod (Polaron CPD 7501) and coated with gold (Bio-RADSC510). Glass cover slips treated under dry conditions werefixed with glutaraldehyde and osmium vapours during 24 h ina closed chamber and coated with gold (Bio-Rad SC510). Images

were performed in a Zeiss DSM 940 A at 15 kV and a HitachiH-4100FE.

Transmission electron microscopy (TEM)Samples were prepared on glass cover slips and thermanox.

Thermanox was dehydrated in alcohol and the glass coverslipswere processed for embedding in Spurr resin. Semithin and thinsections were cut on an Ultracut E (ReicherteJung) maintainingthe thermanox. Semithin sections were stained with MethyleneBlue and observed under light microscopy (Olympus). Thinsections were stained in 2% uranyl acetate and lead citrate for observation on TEM. Images were performed in a JEOL 1010 at80 kV using a Bioscan charge-couple device camera (Gatan).

Statistical analysis

The data were analysed using Statistical Package for theSocial Sciences version 16 (SPSS Inc., Chicago, IL, USA).Comparisons between viable counts, time and biofilm/non-biofilm-forming strains were performed using the Bonferronitest. P < 0.001 was considered statistically significant.

Results

The ability of strains Ab033 and Ab053 to form biofilm, andthe inability of strains Ab001 and Ab143 to form biofilm wasconfirmed by Crystal Violet staining. The means of the dupli-

cate OD600 values were: strain Ab033 (1.814), Ab053 (2.174),Ab001 (0.24), Ab143 (0.109). The interpretation for these ODvalues is: biofilm negative (1.5).

Biofilm-forming strains were less resistant to almost all theantimicrobials than their non-biofilm-forming counterparts.Non-biofilm-forming strains were resistant to piperacillin,ceftazidime, cefepime, ciprofloxacin, gentamicin, tobramy-cin; one of them, strain Ab001, was resistant also to imipenemand meropenem. The antimicrobial susceptibility of the strainsis summarized in Table I.

Table IMinimum inhibitory concentration (MIC) of biofilm-forming and

non-biofilm-forming Acinetobacter baumannii strains

Isolates MIC (mg/L)

AMP PIP CFT CFP SUL IMP MER CIP GEN TOB AMK

Ab001 256 512 32 256 16 8 8 64 128 256 256

Ab033a 32 32 2 2 1 0.12 0.5 32 1 0.5 0.5

Ab053a 16 16 4 2 2 0.25 0.5 4 1 0.5 1

Ab143 256 512 16 16 4 0.25 1 64 128 64 1

AMP, ampicillin; PIP, piperacillin; CFT, ceftazidime; CFP, cefepime;

SUL, sulbactam; IMP, imipenem; MER, meropenem; CIP, cipro-

floxacin; GEN, gentamicin; TOB, tobramycin; AMK, amikacin.a

Biofilm-forming strains.

P. Espinal et al. / Journal of Hospital Infection 80 (2012) 56e60 57

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A statistically significant difference (P < 0.001)in thesurvivalcurves wasobserved; the maximum survivaltime for thebiofilm-forming strains was 36 days, being 15 days for the non-biofilm-forming strains (Figure 1). The pairwise comparisons of theviable counts between the two groups of strains showed nostatistically significant difference between the strains includedin each group: Ab033 vs Ab053, P ¼ 0.019; Ab001 vs Ab143,

P ¼ 0.022, contraryto the comparison betweenstrainsof the twogroups: Ab001 vs Ab033 and Ab053, P < 0.001; Ab143 vs Ab033and Ab053, P < 0.001. The results of the repetition of the assayswere very similar. A mean of 13.8 cfu/mL difference betweenexperiments with a confidence intervalof 95% (8.4e19.4cfu/mL)was considered not relevant for the experiment.

SEM analysis of samples in liquid medium showed few cellsclustered together in the non-biofilm-forming strains and largegroups of conglomerate cells in the biofilm-forming strains(Figure 2A). In both cases, the cells’ morphology remainedunaltered. On dry surfaces (Figure 2B), tight and denseconglomerates of cells were constituted by biofilm-formingstrains, seemingly forming a multilayer structure, with thecells covered by a film most likely representing the exopoly-

saccharide produced by A. baumannii. By contrast, this layer was absent in the non-biofilm-forming strains and the cellsseemed dehydrated (Figure 2B). SEM also showed that thebiofilm-forming strains were linked to each other throughextracellular appendages, possibly pili structures, both inliquid medium and on dry surfaces (Figure 2A and B).

An important observation was the statistically significant(P < 0.001) decrease in the cfu/mL in the non-biofilm-formingstrains by the third day (Figure 1). This result correlated withSEM and TEM analysis after 48 h of incubation, with dehydrationand altered cell morphology in the non-biofilm-forming strains.

TEM analysis of biofilm-forming strains in liquid medium anddry surfaces clearly showed the presence of appendages pro-

jecting 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 exopoly-saccharide matrix secreted by A. baumannii as a mechanism ofprotection against desiccation. Changes in cell morphology(such as compressed cells) were observed only when strainswere grown on dry surfaces (Figure 2D).

Discussion

A. baumannii is an important opportunistic pathogen, withthe ability to colonize and persist in the hospital setting and onmedical devices, and also constitutes a significant problem inintensive care units.3,16 This micro-organism survives onnutrient-limited surfaces for several days and is also capable of

resisting desiccation and disinfection.4,6,17

It is hypothesizedthat its ability to persist in these environments, as w ell as itsvirulence, is a result of its capacity to form biofilms.10 Theseproperties, combined with the role of A. baumannii as a noso-comial pathogen, make it responsible for hospital-acquiredinfections.7

Previous studies have already reported that A. baumanniisurvives desiccation better than other Acinetobacter species.18

In our study, survival assays clearly indicated that A. baumanniistrains 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 havedescribed resistance of acinetobacter to many antibiotics inbacteria embedded in the biofilm.3,6,9 In the present study,non-biofilm-forming strains were particularly more resistantthan biofilm-forming strains (Table I). Rodriguez-Baño et al.found that biofilm-forming isolates were more susceptible toimipenem and ciprofloxacin than their non-biofilm-formingcounterparts.8 However , these results differ from those re-ported in other studies.3,6 Jawad et al. found no statisticallysignificant differences between the survival times of sporadicand acinetobacter outbreak strains but did find outbreakstrains to be significantly more resistant than sporadic strains.4

Nonetheless, their studies did not evaluate the effect of biofilmformation observed in our study.

A few previous reports have described the ability of clinical

isolates of A. baumannii to attach to, and form, biofilms onglass surfaces.5,18 Vidal et al. found that an acinetobacter biotype 9 isolate from a respiratory tract infection formedbiofilm on glass cover slips and that this comprised an amor-phous material similar to exopolysaccharide.18

In our study, we found A. baumannii clinical isolatesattached to glass cover slips forming biofilm under dry condi-tions, with an exopolysaccharide matrix covering the cells onlyin biofilm-forming strains, as identified by SEM and TEM anal-ysis. Because the exopolysaccharide is highly hydrated, it mayprevent lethal desiccation and may thus protect against vari-ations in humidity. It may also contribute to mechanicalstability, longer survival and antimicrobial resistance.13,19

In our SEM and more specifically in TEM analysis, cells linked

to each other with extracellular appendages that resemblefimbriae or pili were observed only in the biofilm-formingstrains. Tomaras et al. demonstrated that A. baumannii ATCC19606 adhered to and formed biofilm on abiotic surfaces andthat pili production was essential for biofilm formation by thisclinical strain.5 Inactivation of csuE results in theabolition of piliproduction 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.20

In summary, our findings show a relationship between bio-film formation and survival of A. baumannii clinical isolates,demonstrating that isolates producing biofilm survive longer than their non-biofilm forming counterparts on dry surfaces.

1400

1200

1000

800

600

400

200

00 5 10 15 20 25

Time (days)

c f u / m L

30 35 40

Figure 1. Survival curve. Comparison between biofilm-forming

(Ab033, -; Ab053,:) and non-biofilm-forming (Ab001,A; Ab143,

C) strains.


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Thus, biofilm production and resistance to desiccation of aci-netobacter may enhance colonization and persistence in thehospital environment and also increase the probability of

acquiring antimicrobial resistance and ability to cause noso-comial infections and outbreaks.

Acknowledgements

This study has been supported by the Spanish Ministry ofHealth (FIS 08/0195 to J.V.), by 2009 SGR 1256 from theDepartament de Universitats, Recerca i Societat de la Infor-mació de la Generalitat de Catalunya, the Ministerio de Sani-dad y Consumo, Instituto de Salud Carlos III, Spanish Networkfor the Research in Infectious Disease (REIPI 06/0008), and byfunding from the European Community (TROCAR contractHEALTH-F3-2008-223031). We want to thank the AlBan pro-

gramme E07D401559CO for supporting P.A.E., as well as

Dr N. Cortadellas and A. Garcı́a of the Electronic MicroscopyUnit, Medicine Faculty SCT, University of Barcelona.

Conflict of interest statementNone declared.

Funding sources

None.

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Figure 2. Scanning 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

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3. Lee H, Koh Y, Kim J, et al. Capacity of multidrug-resistant clinicalisolates of Acinetobacter baumannii to form biofilm and adhere toepithelial cell surfaces. Clin Microbiol Infect 2008;14:49e54.

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