# Virstatin inhibits biofilm formation and motility of Acinetobacter baumannii

Yassine Nait Chabane, Mohamed Ben Mlouka, Stephane Alexandre, Marion

Nicol, Sara Marti, Martine Pestel-Caron, Jordi Vila, Thierry Jouenne,
Emmanuelle Dé

► To cite this version:

Yassine Nait Chabane, Mohamed Ben Mlouka, Stephane Alexandre, Marion Nicol, Sara Marti, et al. Virstatin inhibits biofilm formation and motility of Acinetobacter baumannii. BMC Microbiology, 2014, 14 (1), pp.62. 10.1186/1471-2180-14-62. hal-02326869

HAL Id: hal-02326869

https://hal.science/hal-02326869

Submitted on 25 May 2021

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Virstatin inhibits biofilm formation and motility of *Acinetobacter baumannii*

Yassine Nait Chabane<fnref n="1" />,<fnref n="2" />, Mohamed Ben Mlouka<fnref n="1" />,<fnref n="2" />, Stéphane Alexandre<fnref n="1" />,<fnref n="2" />, Marion Nicol<fnref n="1" />,<fnref n="2" />, Sara Marti<fnref n="1" />,<fnref n="2" />, Martine Pestel-Caron<fnref n="1" />,³, Jordi Vila⁴, Thierry Jouenne<fnref n="1" />,<fnref n="2" /> and Emmanuelle Dé<fnref n="1" />,<fnref n="2" />*

**Abstract**

**Background:** *Acinetobacter baumannii* has emerged as an opportunistic nosocomial pathogen causing infections worldwide. One reason for this emergence is due to its natural ability to survive in the hospital environment, which may be explained by its capacity to form biofilms. Cell surface appendages are important determinants of the *A. baumannii* biofilm formation and as such constitute interesting targets to prevent the development of biofilm-related infections. A chemical agent called virstatin was recently described to impair the virulence of *Vibrio cholerae* by preventing the expression of its virulence factor, the toxin coregulated pilus (type IV pilus). The objective of this work was to investigate the potential effect of virstatin on *A. baumannii* biofilms.

**Results:** After a dose-response experiment, we determined that 100 µM virstatin led to an important decrease (38%) of biofilms formed by *A. baumannii* ATCC17978 grown under static mode. We demonstrated that the production of biofilms grown under dynamic mode was also delayed and reduced. The biofilm susceptibility to virstatin was then tested for 40 clinical and reference *A. baumannii* strains. 70% of the strains were susceptible to virstatin (with a decrease of 10 to 65%) when biofilms grew in static mode, whereas 60% of strains respond to the treatment when their biofilms grew in dynamic mode. As expected, motility and atomic force microscopy experiments showed that virstatin acts on the *A. baumannii* pili biogenesis.

**Conclusions:** By its action on pili biogenesis, virstatin demonstrated a very promising antibiofilm activity affecting more than 70% of the *A. baumannii* clinical isolates.

**Keywords:** Pellicle, Protein-protein interactions, Type IV pili, Virstatin

**Background**

*Acinetobacter baumannii*, a microorganism with a worldwide epidemic spread, causes a wide range of infections, including pneumonia and blood-stream infections. This increasing threat in hospitals is mainly due to the occurrence of multidrug-resistant strains, associated with the real problem of eradication in the hospital wards [1]. Biofilm formation may facilitate the environmental survival of *A. baumannii* by conferring resistance to antibiotics, desiccation or nutritional stress and explain the success of particular strains in hospitals [2,3]. Several factors have been proved to play a role in this biofilm formation or maturation, like the poly-β-(1–6)-N-acetyl glucosamine

extracellular polysaccharide, the biofilm-associated protein, the autotransporter Ata or the systems of protein glycosylation [4-7]. In addition, extracellular appendages are often involved in different stages of bacterial biofilm [8] as exemplified by the *csuA/BABCDE* chaperon-usher system, coding for fimbriae, and required for initial steps of *A. baumannii* biofilm development [6,9,10]. Recent findings have also demonstrated that more than one cell surface appendages system may be involved in the maintenance of the biofilm structure, especially when the biofilm is formed at the air-liquid interface [11]. Finally, the presence of a type IV pili system and its involvement in motility was recently described in *A. baumannii* [12]. It could also play a role in biofilm development (initial adhesion, microcolonies formation and in maturation, last step of biofilm formation) as demonstrated for other bacterial species [8,13]. Therefore, the contribution of extracellular

<footnote>* Correspondence: emmanuelle.de@univ-rouen.fr</footnote>

<footnote n="1">¹Normandie University, Caen, France</footnote>

<footnote n="2">²Laboratory "Polymères, Biopolymères, Surfaces", UMR 6270 & FR 3038 CNRS, IRIB, University of Rouen, Mont-Saint-Aignan, Cedex 76821, France</footnote>

<footnote>Full list of author information is available at the end of the article</footnote>
appendages to the biofilm structuration makes them very
attractive as therapeutic targets [14].

In this study, we experienced this new approach by tes-
ting the use of the chemical agent virstatin. This small or-
ganic molecule has been demonstrated to inhibit Vibrio
cholerae virulence and its orogastric administration would
protect infant mice from V. cholerae intestinal coloni-
zation [15,16]. Virstatin acted in preventing expression of
the two major Vibrio cholerae virulence factors, cholera
toxin and the toxin coregulated pilus (a type IV pilus,
T4P). It would disrupt protein-protein interactions, i.e. the
dimerization of the transcriptional regulator ToxT, stop-
ping thus the activation of ctx and tcp genes [15,16].

We present here the efficacy of the virstatin as an inhibi-
tor of the pili system synthesis to prevent A. baumannii
biofilm formation.

**Methods**

**Bacterial strains and MICs determination**

We used *A. baumannii* ATCC 17978 and ATCC 19606 as
reference strains, as well as 38 clonally unrelated *A.*

*baumannii* clinical isolates, among which multidrug-
resistant (MDR) and extensively drug-resistant (XDR)
strains [2,17]. Nineteen clinical isolates formed biofilm on
solid support and the remaining 19 isolates had the ability
to form a pellicle [18]. Determination of MICs of virstatin
(4-[N-(1,8-naphthalimide)]-n-butyric acid; Bachem, Weil
am Rhein, Germany) solubilized in dimethyl sulfoxide
(DMSO, Sigma, Saint Louis, USA) was performed by the
microdilution method as described by Wiegand *et al.* [19].

**Virstatin effect on biofilms formed in static mode**

A. baumannii biofilms were grown on 24-wells plates in Mueller Hinton (MH) broth with or without virstatin added at 20, 50 or 100 µM using DMSO as control. Plates were incubated for 24 h at 37°C without shaking. Attached cells were quantified by the protocol described by O'Toole and Kolter [20] or counted after their detachment by sonication. 25 µL were plated on MH agar and Colony Forming Units (CFU) were counted after 24 h of additional growth at 37°C. For testing the dispersing effect of virstatin, biofilms were grown for 24 h then

<figure><img src="image_2.png" /></figure>
challenged with increasing concentrations of virstatin
(from 25 to 400 µM). Attached cells were similarly quan-
tified after 24 h of additional growth.

All experiments were performed at least in triplicate.
One way ANOVA was used to assess significant diffe-
rences between a biofilm growth with and without virsta-
tin. All data were statistically analyzed using Prism Graph
Pad 5.

Virstatin effect on biofilms formed in dynamic mode

The effect of virstatin on biofilms formed in dynamic
mode was estimated with the BioFlux device (Fluxion Bio-
sciences, South San Francisco, CA) as described by Benoit
et al. [21] with some modifications. This system consists
of a network of microfluidic laminar flow channels in
which the growth of biofilms is controlled by shear force.
The microfluidic channels (depth, 70 µm; width, 370 µm
in PDMS : PolyDiMéthylSiloxane) were wetted with MH
medium and inoculated with 10⁷ CFU.mL⁻¹ of A. bau-
mannii ATCC 17978 or of the other 39 strains. Following
1 h of incubation for cell attachment at 37°C, 1 mL fresh
MH broth containing or not 100 µM virstatin was
pumped at a flow rate of 0.3 dyn/cm² from inlet wells
through the channels to outlet wells. To obtain images,
A. baumannii strains were grown for 24 h with a flow rate
of 1 dyn/cm². During biofilm formation, images were
obtained using an inverted video-microscope (Leica DM
IRBE), a digital camera (CoolSNAP Fx), and treated by
Metamorph 7.0 software. Biofilm quantification was per-
formed by image analysis using the Bioflux 200 software
and the 'area coverage' module in 'greyscale' mode. The
amount of sessile bacterial covering the flow cells was
estimated in a defined and representative window, by an
average value of coverage (µm²) depending on time (from
0-24 h). These values were then compared in absence or
presence of 100 µM virstatin.

Motility experiments

Motility of all *A. baumannii* strains was tested on poly-
styrene Petri dishes containing 10 g/L tryptone, 5 g/L
NaCl with 0.3% agar (TSA; Difco) supplemented with
100 µM of virstatin or with the same volume of DMSO
and incubated at 37°C overnight [22].

Atomic force microscopy (AFM)

*A. baumannii* ATCC 17978 pellicles were transferred to
collodion-coated glass slides after 8 h growth in MH broth
supplemented or not with 100 µM of virstatin [11]. AFM
imaging was performed as described in Marti *et al*. [11].

Results

Virstatin effect on A. baumannii growth in planktonic
mode

We first examined the toxicity of virstatin on planktoni-
cally growing *A. baumannii* ATCC 17978 by generating
growth curves with and without this compound and by
counting CFU after addition of different concentrations
of virstatin (Additional file 1: Figure S1-A&B). Neither
virstatin nor its solvent (DMSO) inhibited *A. baumannii*
growth at concentrations used in next steps of this
study. The MIC of virstatin was finally measured at
1.6 mM.

Virstatin effect on *A. baumannii* sessile bacteria

To investigate the preventing effect of virstatin on bio-
film growth in static mode, we measured *A. baumannii*
ATCC 17978 biofilm production at varying virstatin con-
centrations. This quantitative determination was moni-
tored by crystal violet colorimetric assay. Figure 1A shows
clearly that virstatin exhibited an anti-biofilm activity,
which was optimal for 100 µM with 38% biofilm forma-
tion inhibition. These data were confirmed by bacterial
counting within the biofilms depending on virstatin

<figure><img src="image_3.png" /><figcaption>**Figure 2** Virstatin activity on biofilm formation by *A. baumannii* clinical isolates: Percentages measured the 24 h-biofilm formation in presence of 100 µM virstatin compared to biofilm formation without virstatin (but DMSO as control). 100% denoted no activity of virstatin. *A. baumannii* strains forming biofilm on solid supports in black bars (■), strains forming pellicle in dark grey bars (■).</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Figure 3** (See legend on next page.)</figcaption></figure>
(See figure on previous page.)

**Figure 3** *Virstatin affects bacterial motility and pili production.* A&B- Plates of motility of *A. baumannii* ATCC 17978 strain in presence (B) or absence (A) of virstatin in semi-solid 0.3% TSA after 24 h growth at 37°C. Typical plates are shown. The diameter of the motility zone decreased when medium is supplemented with 100 µM virstatin. The experiments were performed in triplicate. C-F - AFM images of *A. baumannii* ATCC 17978 water-facing side pellicles after 8 h growth: C&E without virstatin; D&F with 100 µM virstatin. Topographic representation using an uncommon scale made to emphasize the bacterial pili and the 'EPS' (extropolymeric substances) surrounding layer.

concentrations (Additional file 1: Figure S1-B). We then
examined the dispersing effect of virstatin on preformed
biofilms. After growth for 24 h, biofilms were challenged
by different concentrations of virstatin. Additional file 2:
Figure S2 shows slight biofilm dispersion (12% for 400 µM)
whatever the virstatin concentration. Therefore, virstatin
exerted a marked inhibitory effect on *A. baumannii* biofilm
formation but a weak dispersing effect on preformed
biofilms.

We also investigated the effect of virstatin on biofilms
formed in dynamic mode under flow shear, using the
Bioflux microfluidic device. Figure 1B shows light micro-
graphs taken from 0 to 24 h after the flow was initiated in
the presence or absence of 100 µM virstatin. At 6 h, the
micrographs showed a reduction of biofilm formation
within the microfluidic channel irrigated by broth en-
riched in virstatin. After 20 h, in absence of virstatin, the
channel was nearly filled by biofilm, whereas broth loaded
with virstatin could still freely flow through the channel.
The Figure 1C showed the kinetic of biofilm formation
within the channels for both conditions. It clearly demon-
strated that virstatin induced a time lag of nearly 10 hours
for biofilm formation and that the biofilm production is
reduced after 24 h. These data confirmed an antibio-
film action of virstatin within the first steps of biofilm
formation.

Virstatin effect on biofilms formed by clinical isolates

To assess more broadly the anti-biofilm activity of vir-
statin, we tested this molecule on different *A. baumannii*
clinical isolates as well as on the two reference strains.
We examined a panel of 20 isolates forming pellicle and
20 other strains forming only biofilm on solid support
[18]. As shown by Figure 2, 70% of clinical isolates pre-
sented at least a 10% decrease in their ability to form a
biofilm. This decrease reached more than 65% for
100 µM virstatin. It should be noticed that the activity of
virstatin was more marked on strains forming pellicle
(75% of strains with a decrease from 10 to 65%) than on
strains forming only biofilm on solid support (65% of
strains with a decrease from 10 to 47%). The activity of
virstatin was also examined on biofilms grown in dy-
namic mode. If all the tested strains were able to develop
a biofilm in dynamic growth mode, virstatin could in-
duce a time lag only for 60% of strains (Additional file 3:
Table S1) and its activity was also more marked on

strains forming pellicle (75%) than on strains forming
biofilm on solid support (45%).

Virstatin affects bacterial motility and pili production

As virstatin may affect pili production, we performed mo-
tility assays. 0.3% TSA plates containing or not 100 µM
virstatin were inoculated by *A. baumannii* ATCC 17978
[12]. Figure 3A & B shows that virstatin significantly
inhibited bacterial migration. This motility assay was per-
formed for all the strains (Additional file 3: Table S1) and
the data showed that over the 30 strains that were mobile,
60% underwent a decrease of their motility.

In order to check the presence of pili after virstatin
action, the pellicles of *A. baumannii* ATCC 17978 were
examined by AFM. In absence of virstatin, observation of
the water-facing side of the pellicles revealed the signifi-
cant presence of pili (0.5 to 2 µm in length) around bac-
teria, which were located on the border of the colonies
(Figure 3C). In contrast when bacteria grew with virstatin,
the amount of pili drastically decreased (Figure 3D).
Moreover, we observed a layer surrounding bacteria, the
average height and width of which were greater in pre-
sence (height: 100 nm, width: 1–1.5 µm) than in absence
of virstatin (height: 50 nm, width: 0.1 µm, Figure 3C & D).
This was most likely extropolymeric substances (EPS)
overproduced in presence of virstatin. At a lower reso-
lution, images showed strong differences between both
pellicles (Figure 3E & F). The pellicle formed in absence of
virstatin was quite dense and was formed of a single bac-
terial layer, giving a colony mean height of (0.20 ± 0.05)
µm. In presence of virstatin, the pellicle appeared less
dense and formed with more than one layer of bacteria. In
this case, the colony was about (0.50 ± 0.05) µm in height.

Discussion

Targeting essential protein-protein interactions in bacterial
function is a very promising but challenging therapeutic
approach which is increasingly developed [14,15]. In this
context, small organic molecules (ring fused 2-pyridones)
were tested as pilicide or curlicide on uropathogenic
*Escherichia coli* (UPEC) with success. In targeting either
type 1 pili or amyloid fibers biogenesis, these com-
pounds can prevent biofilm formation and attenuate
UPEC virulence [14]. Virstatin is similarly used to de-
crease the expression of T4P which are virulence factors
in *V. cholerae* [15,16].
In this study, we demonstrated that virstatin presents also an interesting antibiofilm activity as it could reduce by 65% the biofilm production in some A. baumannii clinical isolates and is active on more than 70% of the tested strains. Virstatin remained also active against biofilms formed under flow shear inducing clearly a delay of the development of this type of biofilm. These results are in favor of its action on pili biogenesis. Indeed, these cell surface appendages are known to be involved in the first stages of biofilm development promoting, initial adhesion and surface colonization but also microcolonies formation [8]. Thus the decrease of virstatin activity at a later phase of biofilm development (48 h) is not surprising, as pili expression may not be required. Moreover, the AFM observations confirmed the hypothesis suggesting an inhibitory effect of virstatin on the biogenesis of pili: they pointed out a drastic under-production of these appendages in the presence of the molecule (Figure 3C & D). Finally, motility experiments demonstrated that virstatin clearly affected the migration of A. baumannii on semisolid surface. This motility has been examined by Clemmer and colleagues [12] who showed that the loss of *pilT*, a gene involved in twitching mediated by T4P, resulted in a 54% reduction in motility of non-flagellar A. baumannii. In addition, AFM micrographs showed the overexpression of EPS in biofilms grown with virstatin. Recently Wang et al. [23] demonstrated that if deletion of T4P in *Pseudomonas aeruginosa* resulted in a reduction of biofilm biomass in flow cell and in pelicles, it concomitantly resulted in an overproduction of the Psl polysaccharide in the microcolonies. Overall, these results are in favor of an action of virstatin on T4P pili biogenesis.

## Conclusions

The emergence of A. baumannii as one of the most problematic nosocomial pathogen has made necessary the development of new strategies to impair its ability to persist in hospital environment. Here, we have tested a strategy based on the prevention of the pili biosynthesis to reduce the A. baumannii biofilm production. This was successful with the use of a small organic compound, the virstatin, which demonstrated an activity on 70% of the clinical isolates with a decrease in biofilm production that could reach 65%.

## Additional files

**Additional file 1: Figure S1.** Virstatin effect on A. baumannii ATCC 17978 growth in planktonic and biofilm modes. A- Growth curves of A. baumannii in MH broth (□), MH broth with 0.5% DMSO (volume used for the addition of 100 µM virstatin) (◇), or MH broth with 100 µM virstatin (Δ). B- A. baumannii colony forming units after a 24 h planktonic (♦) and biofilm (●) growth depending on virstatin concentrations. Curves are given as an average of 3 replicates.

**Additional file 2: Figure S2.** Dispersing effect of virstatin. Virstatin added at 100 µM on 24 h preformed biofilms. Quantification of biofilm biomass was made by crystal violet staining after additional 24 h growth. "*" for *P* < 0.05.

**Additional file 3: Table S1.** Virstatin effect on A. baumannii clinical isolates.

## Competing interests

The authors declare that they have no competing interests.

## Authors' contributions

YN-C, TJ, ED conceived and designed the experiments. YN-C, MB-M, MP-C, SM, SA, MN performed the experiments. YN-C, SA, TJ, JV, ED analyzed the data. MP-C, SM, SA, JV contributed to materials/technical support. ED wrote the paper. All authors read and approved the final manuscript.

## Acknowledgements

We thank the Spanish Group for the Study of Nosocomial Infections (GEIH) to provide us A. baumannii clinical isolates, the Primacen microscopy Platform for their technical help and Dr. G.A. Junter for helpful discussions. YN-C. has a doctoral fellowship from the Haute-Normandie region and MB-M. from "Vaincre La Mucoviscidose".

## Author details

¹Normandie University, Caen, France. ²Laboratory "Polymères, Biopolymères, Surfaces", UMR 6270 & FR 3038 CNRS, IRIB, University of Rouen, Mont-Saint-Aignan, Cedex 76821, France. ³University of Rouen, Rouen University Hospital, GRAM, Rouen, EA 2656, France. ⁴Department of Microbiology, Hospital Clinic, Barcelona, Spain.

Received: 25 September 2013 Accepted: 25 February 2014
Published: 12 March 2014

## References

1. Dijkshoorn L, Nemec A, Seifert H: An increasing threat in hospitals: multidrug-resistant *Acinetobacter baumannii*. *Nat Rev Microbiol* 2007, 5(12):939-951.

2. Rodriguez-Bano J, Marti S, Soto S, Fernandez-Cuenca F, Cisneros JM, Pachon J, Pascual A, Martinez-Martinez L, McQueary C, Actis LA, Vila J, Spanish Group for the Study of Nosocomial Infections (GEIH): Biofilm formation in *Acinetobacter baumannii*: associated features and clinical implications. *Clin Microbiol Infect* 2008, 14(3):276-278.

3. Giannouli M, Antunes LC, Marchetti V, Triassi M, Visca P, Zarrilli R: Virulence-related traits of epidemic *Acinetobacter baumannii* strains belonging to the international clonal lineages I-III and to the emerging genotypes ST25 and ST78. *BMC Infect Dis* 2013, 13:282.

4. Bentancor LV, O'Malley JM, Bozkurt-Guzel C, Pier GB, Maira-Litran T: Poly-N-acetyl-beta-(1-6)-glucosamine is a target for protective immunity against *Acinetobacter baumannii* infections. *Infect Immun* 2012, 80(2):651-656.

5. Choi AH, Salami L, Avci FY, Pier GB, Maira-Litran T: The pgaABCD locus of *Acinetobacter baumannii* encodes the production of poly-beta-1-6-N-acetylglucosamine, which is critical for biofilm formation. *J Bacteriol* 2009, 191(19):5953-5963.

6. Gaddy JA, Actis LA: Regulation of *Acinetobacter baumannii* biofilm formation. *Future Microbiol* 2009, 4(3):273-278.

7. Iwashkiw JA, Seper A, Weber BS, Scott NE, Vinogradov E, Stratilo C, Reiz B, Cordwell SJ, Whittal R, Schild S, Feldman MF: Identification of a general O-linked protein glycosylation system in *Acinetobacter baumannii* and its role in virulence and biofilm formation. *PLoS Pathog* 2012, 8(6):e1002758.

8. Mikkelsen H, Sivaneson M, Filloux A: Key two-component regulatory systems that control biofilm formation in *Pseudomonas aeruginosa*. *Environ Microbiol* 2011, 13(7):1666-1681.

9. Tomaras AP, Dorsey CW, Edelmann RE, Actis LA: Attachment to and biofilm formation on abiotic surfaces by *Acinetobacter baumannii*: involvement of a novel chaperone-usher pili assembly system. *Microbiology* 2003, 149(Pt 12):3473-3484.

10. Di Nocera PP, Rocco F, Giannouli M, Triassi M, Zarrilli R: Genome organization of epidemic *Acinetobacter baumannii* strains. *BMC Microbiol* 2011, 11:224.
11. Marti S, Nait Chabane Y, Alexandre S, Coquet L, Vila J, Jouenne T, De E: Growth of *Acinetobacter baumannii* in pellicle enhanced the expression of potential virulence factors. *PloS one* 2011, 6(10):e26030.

12. Clemmer KM, Bonomo RA, Rather PN: Genetic analysis of surface motility in *Acinetobacter baumannii*. *Microbiology* 2011, **157**(Pt 9):2534–2544.

13. Klausen M, Heydorn A, Ragas P, Lambertsen L, Aaes-Jorgensen A, Molin S, Tolkер-Nielsen T: *Biofilm formation by Pseudomonas aeruginosa* wild type, flagella and type IV pili mutants. *Mol Microbiol* 2003, **48**(6):1511–1524.

14. Cegelski L, Pinkner JS, Hammer ND, Cusumano CK, Hung CS, Chorell E, Aberg V, Walker JN, Seed PC, Almqvist F, Chapman MR, Hultgren SJ: Small-molecule inhibitors target *Escherichia coli* amyloid biogenesis and biofilm formation. *Nat Chem Biol* 2009, **5**(12):913–919.

15. Hung DT, Shakhnovich EA, Pierson E, Mekalanos JJ: Small-molecule inhibitor of Vibrio cholerae virulence and intestinal colonization. *Science* 2005, **310**(5748):670–674.

16. Shakhnovich EA, Hung DT, Pierson E, Lee K, Mekalanos JJ: Virstatin inhibits dimerization of the transcriptional activator ToxT. *Proc Natl Acad Sci USA* 2007, **104**(7):2372–2377.

17. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JF, Kahlmeter G, Olsson-Liljequist B, Paterson DL, Rice LB, Stelling J, Struelens MJ, Vatopoulos A, Weber JT, Monnet DL: Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. *Clin Microbiol Infec* 2012, **18**(3):268–281.

18. Marti S, Rodriguez-Bano J, Catel-Ferreira M, Jouenne T, Vila J, Seifert H, De E: Biofilm formation at the solid-liquid and air-liquid interfaces by *Acinetobacter* species. *BMC Res Notes* 2011, **4**:5.

19. Wiegand I, Hilpert K, Hancock RE: Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. *Nat Protoc* 2008, **3**(2):163–175.

20. O'Toole GA, Kolter R: Initiation of biofilm formation in *Pseudomonas fluorescens* WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. *Mol Microbiol* 1998, **28**(3):449–461.

21. Benoit MR, Conant CG, Ionescu-Zanetti C, Schwartz M, Matin A: New device for high-throughput viability screening of flow biofilms. *Appl Environ Microbiol* 2010, **76**(13):4136–4142.

22. McQueary CN, Actis LA: *Acinetobacter baumannii* biofilms: variations among strains and correlations with other cell properties. *J Microbiol* 2011, **49**(2):243–250.

23. Wang S, Parsek MR, Wozniak DJ, Ma LZ: A spider web strategy of type IV pili-mediated migration to build a fibre-like Psl polysaccharide matrix in *Pseudomonas aeruginosa* biofilms. *Environ Microbiol* 2013, **15**(8):2238–2253.

doi:10.1186/1471-2180-14-62

Cite this article as: Nait Chabane et al.: Virstatin inhibits biofilm formation and motility of *Acinetobacter baumannii*. *BMC Microbiology* 2014 **14**:62.

Submit your next manuscript to BioMed Central
and take full advantage of:

* Convenient online submission

* Thorough peer review

* No space constraints or color figure charges

* Immediate publication on acceptance

* Inclusion in PubMed, CAS, Scopus and Google Scholar

* Research which is freely available for redistribution

Submit your manuscript at
www.biomedcentral.com/submit

BioMed Central