+ MODEL

Research in Microbiology xx (2013) 1e7www.elsevier.com/locate/resmic

Anti-biofilm activity of the Antarctic marine bacterium Pseudoalteromonashaloplanktis TAC125

Rosanna Papa a, Ermenegilda Parrilli b, Filomena Sannino b,c, Gaetano Barbato d,Maria Luisa Tutino b, Marco Artini a, Laura Selan a,*

aDepartment of Public Health and Infectious Diseases, Sapienza University, Piazzale Aldo Moro 5, 00185 Rome, ItalybDepartment of Chemical Sciences, Federico II University, Complesso Universitario Monte Sant’Angelo, Via Cinthia 4, 80126 Naples, Italy

c Institute of Protein Biochemistry, CNR, Via Pietro Castellino 111, 80131 Naples, ItalydDepartment of Science and Chemical Technologies, University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133 Rome, Italy

Received 24 September 2012; accepted 24 January 2013

Abstract

Considering the increasing impact of bacterial biofilms on human health, industrial and food-processing activities, the interest in thedevelopment of new approaches for the prevention and treatment of adhesion and biofilm formation capabilities has increased. A viable approachshould target adhesive properties without affecting bacterial vitality in order to avoid the rapid appearance of escape mutants.

It is known that marine bacteria belonging to the genus Pseudoalteromonas produce compounds of biotechnological interest, including anti-biofilm molecules. Pseudoalteromonas haloplanktis TAC125 is the first Antarctic Gram-negative strain whose genome was sequenced. In thiswork the anti-biofilm activity of P. haloplanktis supernatant was examined on different staphylococci. Results obtained demonstrated thatsupernatant of P. haloplanktis, grown in static condition, inhibits biofilm of Staphylococcus epidermidis. In order to define the chemical nature ofthe biofilm-inhibiting compound, the supernatant was subject to various treatments. Data reported demonstrated that the biologically activecomponent is sensible to treatment with sodium periodate suggesting its saccharidic nature.� 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: Anti-biofilm; Staphylococcus; Pseudoalteromonas; Antarctic

1. Introduction

Biofilm is the predominant mode of growth for bacteria inmost environments (Bendaoud et al., 2011) and particularlywith respect to chronic infections (Dohar et al., 2009). Theyare relevant in a wide range of clinical domains includingmedicine and surgery (Francolini and Donelli, 2010) and foodindustry (Lequette et al., 2010). Biofilms also contaminatea wide variety of infrastructure elements such as water and airpurification systems (Sublette et al., 2006), optical sensors

* Corresponding author. Tel.: þ39 0649694261; fax: þ39 0649694298.

E-mail addresses: rosanna.papa@uniroma1.it (R. Papa), erparril@unina.it

(E. Parrilli), mena.10@libero.it (F. Sannino), barbato.g@fastwebnet.it

(G. Barbato), tutino@unina.it (M.L. Tutino), marco.artini@uniroma1.it

(M. Artini), laura.selan@uniroma1.it (L. Selan).

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Microbiology (2013), http://dx.doi.org/10.1016/j.resmic.2013.01.010

0923-2508/$ - see front matter � 2013 Institut Pasteur. Published by Elsevier Ma

http://dx.doi.org/10.1016/j.resmic.2013.01.010

(Kerr et al., 1998), marine and industrial equipments(Holmstrom et al., 2002).

In medical settings, biofilms are the cause of persistentinfections-implicated in 80% or more of all microbial cases-releasing harmful toxins and even obstructing indwellingcatheters (Epstein et al., 2012) or causing orthopaedic implantinfections (Drago et al., 2013). Staphylococci are recognizedas the most frequent causes of biofilm-associated infections(Otto, 2008; Arciola et al., 2012). Taking into account theincreasing impact of bacterial biofilms, the interest in thedevelopment of new approaches for the prevention and treat-ment of adhesion and biofilm formation capabilities hasamplified. A viable approach should target adhesive propertieswithout affecting bacterial vitality in order to avoid the rapidappearance of escape mutants. Many bacterial biofilms secretemolecules such as quorum sensing signals (Ni et al., 2009),

rctic marine bacterium Pseudoalteromonas haloplanktis TAC125, Research in

sson SAS. All rights reserved.

2 R. Papa et al. / Research in Microbiology xx (2013) 1e7

surfactants (Kiran et al., 2010), enzymes (Kaplan, 2010), andpolysaccharides (Qin et al., 2009; Valle et al., 2006) thatfunction regulating biofilm architecture or mediating therelease of cells from biofilms during the dispersal stage of thebiofilm life cycle (Kaplan, 2010).

These compounds often exhibit broad-spectrum biofilm-inhibiting or biofilm-detaching activity when tested againstbiofilms cultured in vitro. Such compounds may representa novel source of anti-biofilm compounds for technologicaldevelopment.

Marine bacteria belonging to the genus Pseudoalteromonasproduce compounds of biotechnological interest, includinganti-biofilm molecules (Klein et al., 2011). Marine bacteriafrom Antarctica represent an untapped reservoir of bio-diversity, indeed, Antarctic microorganisms can synthesizea broad range of potentially valuable bioactive compounds(Jayatilake et al., 1996).

Pseudoalteromonas haloplanktis TAC125 is the first Ant-arctic Gram-negative strain whose genome was sequenced(Medigue et al., 2005). Genomic and metabolic features of thisstrain, accounting for its remarkable versatility, were discoveredby combining genome sequencing and further in silico andin vivo analyses. P. haloplanktis TAC125 was also the firstAntarctic bacterium in which an efficient gene-expressiontechnology was set up allowing the production of homologousand heterologous recombinant proteins (Rippa et al., 2012;Corchero et al., 2012). The host versatilitywas recentlywidenedby the development of an efficient genetic scheme for the con-struction of genome targeted insertion/deletion mutants, whichallows a deeper understanding of P. haloplanktis TAC125physiology (Giuliani et al., 2012; Parrilli et al., 2010).

In this work we examined the anti-biofilm activity of P.haloplanktis culture supernatant, grown in static or in dynamiccondition, on different Staphylococcus epidermidis andStaphylococcus aureus strains. P. haloplanktis TAC125 secre-ted an anti-biofilm activity which impaired biofilm develop-ment of S. epidermidis. Results obtained demonstrated thatthe cell-free supernatants of P. haloplanktis grown in staticcondition strongly inhibited bacterial adhesion. In particular, S.epidermidis showed the highest susceptibility to the treatment.This strong inhibitory effect was also observed on the maturebiofilm of S. epidermidis. Interestingly, the P. haloplanktissupernatant was devoid of antibacterial activity against free-

Table 1

Strains used in this study.

Strain Origin

P. haloplanktis TAC125 Antarctic sea watera

S. aureus 6538P Clinical isolate

S. aureus 25923 Clinical isolate

S. aureus 20372 Clinical isolate from septi

S. aureus 1511 Clinical isolate from infec

S. epidermidis RP62A Reference strain isolated f

S. epidermidis O-47 Clinical isolate from septi

S. epidermidis XX-17 Clinical isolate from infec

S. epidermidis 1521 Clinical isolate from infec

a Isolated from Antarctic coastal sea water sample collected in the vicinity of the F

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living bacteria, showing that its activity was specificallydirected against biofilm. The chemical nature of the biofilm-inhibiting compound was preliminarily determined using var-ious treatments. Treatment with sodium periodate impairedP. haloplanktis supernatant capacity to inhibit biofilm forma-tion suggesting that the biologically active component could bea polysaccharide.

2. Materials and methods

2.1. Bacterial strains and culture conditions

Bacterial strains used in this work are listed in Table 1.Bacteria were grown in Brain Heart Infusion broth (BHI,Oxoid, UK). Biofilm formation was assessed in static assayand planktonic cultures were performed under vigorous agi-tation (180 rpm). P. haloplanktis TAC125 was grown at 4 �Cwhile staphylococci were grown at 37 �C.

2.2. Biofilm formation of P. haloplanktis TAC125

Biofilm formation of P. haloplanktis TAC125 was obtainedat 4 �C in Brain Heart Infusion broth (BHI, Oxoid, UK). Thewells of a sterile 24-well flat-bottomed polystyrene plate werefilled with 1 ml of BHI, and an opportune dilution of Antarcticbacterial culture in exponential growth phase (about 0.1 OD600 nm) was added into each well. The plates were aerobicallyincubated for 96 h at 4 �C. After rinsing with PBS, adheredcells were stained with 0.1% crystal violet, rinsed twice withdouble-distilled water, and thoroughly dried as previouslydescribed (Christensen et al., 1985). The dye bound toadherent cells was resolubilized with 20% (v/v) glacial aceticacid and 80% (v/v) ethanol per well. The OD of each well wasmeasured at 590 nm. Each data point is composed of threeindependent samples.

2.3. Preparation of P. haloplanktis TAC125 supernatants

SN4B is the supernatant of a liquid culture of P. hal-oplanktis TAC125 grown without shaking (biofilm) andSN4A is the supernatant of a liquid culture of P. haloplanktisTAC125 grown with shaking (planktonic growth),respectively.

Reference and/or source

Medigue et al., 2005

ATCC collection

ATCC collection

c arthritis ATCC collection

ted catheter Our collection

rom infected catheter ATCC collection

c arthritis Heilmann et al., 1996

ted catheter Our collection

ted catheter Our collection

rench Antarctic station Dumont d’Urville, Terre Adelie (66�400 S; 140� 010 E).

rctic marine bacterium Pseudoalteromonas haloplanktis TAC125, Research in

3R. Papa et al. / Research in Microbiology xx (2013) 1e7

2.3.1. Preparation of SN4BThe wells of a sterile 24-well flat-bottomed polystyrene

plate were filled with 900 ml of BHI and 100 ml of overnight P.haloplanktis TAC125 bacterial culture was added into eachwell. The plates were incubated at 4 �C monitoring biofilmformation each 24 h. After 96 h, supernatant was recoveredand centrifuged at 13,000 rpm. Supernatant was then trans-ferred into a Centricon tube (Millipore, Billerica, MA, USA)with 10 kDa MWCO. It was 10-fold concentrated by cen-trifugation at 4000 rpm, 4 �C. Supernatant was sterilized byfiltration through membranes with a pore diameter of 0.22 mm,and stored at 4 �C until use.

2.3.2. Preparation of SN4AP. haloplanktis TAC125 bacterial culture was grown in

planktonic form at 4 �C under vigorous agitation (180 rpm).Supernatant was recovered by centrifugation at 13,000 rpm at4 �C and processed as above described.

2.4. Biofilm formation of staphylococci

Biofilm formation of staphylococcal species was evaluatedin the presence of SN4A and SN4B supernatants. Quantifi-cation of in vitro biofilm production was based on methodpreviously reported (Artini et al., 2011). Briefly, the wells ofa sterile 48-well flat-bottomed polystyrene plate were filledwith 400 ml of the appropriate medium. 1/100 dilution ofovernight bacterial cultures was added into each well. The firstrow contained the untreated bacteria, while each of theremaining rows contained serial dilutions of SN4A and SN4Bstarting from 1:2. The plates were incubated aerobically for24 h at 37 �C. After rinsing with PBS, adhered cells werestained with 0.1% crystal violet, rinsed twice with double-distilled water, and thoroughly dried as previously described(Christensen et al., 1985). The dye bound to adherent cells wasresolubilized with 20% (v/v) glacial acetic acid and 80% (v/v)ethanol per well. The OD of each well was measured at590 nm. Each data point is composed of three independentsamples.

Assay on preformed biofilm was also performed. Thewells of a sterile 48-well flat-bottomed polystyrene plate werefilled with 400 ml of 1/100 diluted overnight bacterial culturesgrown in BHI. The plates were aerobically incubated for 24 hat 37 �C. After 24 h the content of the plates was poured offand the wells washed with sterile distilled water to removethe unattached bacteria. The remaining attached bacteriawere treated with 400 ml of the appropriate medium con-taining serial dilution of SN4A and SN4B (starting from100%) aerobically incubated for 2, 4, 8 and 24 h at 37 �C.After each time-point the plates were treated as previouslydescribed. Each data point is composed of four independentsamples.

2.5. Initial attachment assay

The attachment assay was carried out as previously reportedwith slight modifications (Nithya and Pandian, 2010).

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Overnight cultures were diluted 1/100 in BHI and grownat 37 �C up to reaching 0.5 OD ml�1. The wells of a sterile48-well flat-bottomed polystyrene plate were filled with 100 mlof bacterial cultures and 300 ml of the appropriate mediumcontaining serial dilution of SN4B. The assay was carried outfor 1 h at 37 �C to allow the cells to adhere to the surface. Afterincubation, the plates were processed as previously described.Each data point is composed of four independent samples.

2.6. Physico-chemical characterization of SN4B anti-biofilm compound

To examine the heat sensitivity, the culture supernatantswere incubated for 1 h in a water bath at 50 �C and cooled onice. For the protease treatment, proteinase K (Sigma Aldrich,St Louis, MO) was added to SN4B at a final concentration of1 mg ml�1 and the reactions were incubated for 1 h at 37 �C.As controls, SN4B was incubated for 1 h at 37 �C withoutproteinase K, which did not impair the anti-biofilm activities.For the polysaccharide treatment, NaIO4 at a final concentra-tion of 20 mM was added to supernatant for 12 h at 37 �C. Ascontrols the same treatment was performed on BHI broth toexclude an anti-biofilm effect due to the NaIO4. For each ofthe above tests, the anti-biofilm activities of treated and un-treated culture supernatants were compared using the micro-titer plate assay against S. epidermidis O-47. Each data point iscomposed of four independent samples.

3. Results

3.1. Effect of P. haloplanktis TAC125 exoproducts onbiofilm formation of staphylococci

Biofilm formation of P. haloplanktis TAC125 was evaluatedat 4 �C in BHI with over a 96 h period. P. haloplanktis TAC125was grown in the same medium used for staphylococci culturesto avoid interference due to the medium composition. The bestproduction (more than about 2.5 OD 590 nm) of P. haloplanktisTAC125 biofilm was obtained incubating the cells in static for96 h at 4 �C (data not shown).

The effects of filter sterilized supernatants of P. hal-oplanktis TAC125 cultures grown in planktonic and sessileform at 4 �C on different S. epidermidis and S. aureus strains(Table 1) were examined. SN4B was defined the supernatantof P. haloplanktis deriving from static growth and SN4A thesupernatant of P. haloplanktis liquid culture grown in dynamiccondition, respectively. Preliminary experiments were carriedout to assess the effect of supernatants on the growth rate of S.epidermidis and S. aureus. Bacterial cultures were separatelytreated with SN4B and SN4A supernatants at a concentrationof 50% and growths were monitored over 24 h. In Fig. 1A thegrowth curves of S. epidermidis O-47 in the presence and inthe absence of above described supernatants were reported. Asshown, SN4B and SN4A did not affect S. epidermidis dupli-cation rate. Bacterial growth curves were found to be nearlysuperimposible both in the presence and in the absence ofsupernatants. Same results were obtained evaluating the effect

rctic marine bacterium Pseudoalteromonas haloplanktis TAC125, Research in

Fig. 1. Inhibition of S. aureus and S. epidermidis biofilm formation by P. haloplanktis TAC125 SN4B supernatant. (A) Growth curves of S. epidermidis O-47 in the

presence and in the absence of 50% SN4B or SN4A supernatant. Growth was monitored by measuring the absorbance of the cultures at 600 nm. (B) Biofilm

formation by S. epidermidis O-47 in 48-well microtiter plate wells in the presence and in the absence of 50% SN4B supernatant. (C) Effect of SN4B treatment on

biofilm formation for each staphylococcal strain. SN4B supernatant was tested using serial dilutions starting from 1:2 (50%). Each data point represents the

mean � SD of three independent experiments. S. a., S. aureus species; S.e., S. epidermidis species.

4 R. Papa et al. / Research in Microbiology xx (2013) 1e7

of SN4B and SN4A supernatants on the S. aureus growth rate(data not shown).

SN4A and SN4B supernatants effect on biofilm productionof staphylococci was evaluated and the SN4B supernatantshowed to have a good anti-biofilm effect on S. epidermidisspecies (Fig. 1B) while it did not have effect on S. aureusspecies (Fig. 1C). By contrast SN4A did not show anyinhibitory effect on tested strains (data not shown). SN4B hada similar effect on all S. epidermidis strains tested showinga species-specific effect. The inhibitory effect is clearly dose-dependent with an efficacy higher than 50% already at 1:8dilution (17% residual biofilm for S. epidermidis O-47).

Attachment behaviour of S. epidermidis biofilm formation,in the presence and in the absence of SN4B culture

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supernatant was investigated. Initial attachment of the bacteriato a solid surface is the first step in biofilm formation. Asreported in Fig. 2 SN4B culture supernatant at a concentrationof 50% decreased the attachment of S. epidermidis and thiseffect was more pronounced on strongest biofilm former strainO-47 (biofilm reduction of about 40%).

3.2. P. haloplanktis TAC125 exoproducts impair maturebiofilm of S. epidermidis

The effect of SN4B on S. epidermidis mature biofilm wasalso tested. Single-species mature biofilm of S. epidermidisRP62A and S. epidermidis O-47 was separately treated withserial dilutions of SN4B starting from a concentration of 100%.

rctic marine bacterium Pseudoalteromonas haloplanktis TAC125, Research in

Fig. 2. Effect of SN4B supernatant on initial attachment of S. epidermidis

species. Bacteria were treated with serial dilution of SN4B starting from 50%.

Each data point is composed of four independent samples.

Fig. 3. Effect of SN4B supernatant on 24 h biofilm of S. epidermidis species.

Data are reported as percentage of residual biofilm. The mature biofilm was

treated with serial dilution of SN4B starting from 100%. Each data point is

composed of four independent samples.

5R. Papa et al. / Research in Microbiology xx (2013) 1e7

The effect on preformed biofilm was evaluated at differenttreatment time, SN4B supernatant didn’t disaggregate the S.epidermidis mature biofilm after 2, 4, and 8 h (data not shown)but it was able to impair mature biofilm of S. epidermidis after24 h of treatment (Fig. 3). Indeed, the results showed a signifi-cant reduction in the absorbance of the treated samples, dem-onstrating that SN4B was also extremely effective in thedispersal of S. epidermidis preformed biofilm (S. epidermidisO-47 untreated bacteria OD 590 nm ¼ 1.863 � 0.219; SN4B-treated bacteria OD 590 nm ¼ 0.457 � 0.122) (Fig. 3). Thisresult suggested that the supernatant action was not restricted toinitial bacterial attachment on abiotic surface but was alsoeffective on mature biofilm.

Table 2

Effect of various treatments on the anti-biofilm activity of SN4B.

a

3.3. Physico-chemical characterization of P.haloplanktis TAC125 anti-biofilm compounds

Treatment Percentage of residual biofilm

None 0 � 5.2

50 �C, 1 h 8.5 � 0.7

Proteinase K, 1 h at 37 �C 24.3 � 2.2

NaIO4, 12 h at 37 �C 94.7 � 9.6

a Inhibition of SN4B was determined against S. epidermidis O-47 using the

microtiter plate assay and was reported as percentage of residual biofilm after

the treatment. The values were calculated from four replicates.

To have information about the chemical nature of thebiofilm-inhibiting compound, SN4B supernatant was dis-pensed in several aliquots, each submitted to different chem-ical and physical treatments. Percentage of inhibition of eachtreated aliquot of SN4B was determined against S. epidermidisO-47 (Table 2). Neither proteinase K treatment nor heat

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treatment affected the biofilm-inhibiting activity of SN4B(Table 2). The biofilm-inhibiting material was heat stable, infact it retained about 91.5% of its inhibitory activity after 1 hat 50 �C. Treatment of SN4B supernatant sodium metaper-iodate significantly reduced its biofilm-inhibiting activity(about 5% of residual activity). It has been shown that NaIO4

is able to hydrolyse compounds by oxidizing the carbonsbearing vicinal hydroxyl groups and cleaving the CeC bonds,this hydrolytic activity was demonstrated also against poly-saccharides (Bendaoud et al., 2011). These data suggested thatthe biologically active component could be a polysaccharide.

4. Discussion

Disrupting the multicellular structure of bacterial biofilmwas proposed as the most promising strategy for increasing thesensitivity of pathogens in biofilm to antibiotics and host im-mune systems (Costerton and Stewart, 2001). The rationale ofthis work is to look for new compounds inhibiting virulencerather than bacterial growth; in fact this choice may imposea weaker selective pressure for the development of antibioticresistance relative to current antibiotics. Moreover, even in thisera of combinatorial chemistry, compounds from natural ori-gin still provide a high number of interesting structures.

Marine bacteria produce several compounds whichmay be ofpotential biotechnological interest, and in particular culture su-pernatants derived frommost of themhave been shown to exhibit

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anti-biofilm activity against both Gram positive and Gram neg-ative bacteria, including Acinetobacter, S. aureus, Salmonellatyphimurium, Shigella sonnei, Listeria monocytogenes andseveral Bacillus species (Rendueles et al., 2013).

Antarctic marine bacterium P. haloplanktis TAC125 hasseveral genes and operons that may play an important role incolonization of both biotic and abiotic surfaces (Medigue et al.,2005) and in the experimental conditions here used a strongbiofilm formation on polystyrene plates was observed. In thiswork we examined the anti-biofilm activity of cell-free super-natant of P. haloplanktis grown in sessile and in planktoniccondition on different staphylococci. Results obtained demon-strated that only supernatant of P. haloplanktis grown in staticcondition (SN4B) inhibits biofilm of S. epidermidis. This latterresult could be explained in consideration that specific envi-ronmental conditions prevailing within biofilms may induceprofound genetic andmetabolic rewiring of the biofilm-dwellingbacteria (Beloin andGhigo, 2005). This could lead to productionof biofilm-specific metabolites or polymers, some of which mayalso exhibit an antagonist effect over competing microorgan-isms. Consistent with this hypothesis, several anti-biofilm mol-ecules have been identifilig;ed in cell-free extracts isolateddirectly from mature in vitro cultured biofilms (Rendueles et al.,2013). Although it has been considered that SN4B supernatantderived from a static growthwhere could coexist both planktonicand biofilm phenotypes. Thus the active compounds could beproduced by the planktonic cells stimulated by the oxygenlimitation since bacteria are grown without shaking.

It is interesting to note that previously characterized anti-biofilm compounds have often a broad-spectrum biofilm inhi-bition activity (Bendaoud et al., 2011; Klein et al., 2011;Rendueles et al., 2013) while the P. haloplanktis TAC125 anti-biofilm molecule seemed to be species-specific. P. haloplanktisTAC125 supernatant is, in fact, inactive against biofilm of S.aureus and Pseudomonas aeruginosa (data not shown).

Most of the known anti-biofilm molecules are also anti-bacterial (bactericidal or bacteriostatic). By contrast, P. hal-oplanktis TAC125 supernatant lacks of antibacterial activityagainst free-living bacteria, making its activity specificallydirected against biofilms. Very few natural molecules werereported to display anti-biofilm activity without being also anti-bacterial. Such molecules could be polysaccharides (Valle et al.,2006), biosurfactants (Das et al., 2009), or quorum sensing in-hibitors (Rasmussen and Givskov, 2006; Estrela et al., 2009).

Since the P. haloplanktis TAC125 anti-biofilm molecule hasbeen shown to exhibit no bacteriostatic nor bactericidal ac-tivity, its anti-biofilm activity is likely to be mediated bymechanisms other than growth inhibition. There are threehypothetical non-antibacterial modes of action.

The first hypothesis is that the anti-biofilm molecule couldact as a surfactant molecule that modifies the physical charac-teristics of bacterial cells and abiotic surfaces. Another possiblemode of action is competitive inhibition of multivalent carbo-hydrateeprotein interactions (Wittschier et al., 2007). Thus, theanti-biofilm compound might block lectins or sugar bindingproteins present on the surface of bacteria, or block tip adhesinsof fimbriae and pili (Zinger-Yosovich and Gilboa-Garber,

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2009). The third hypothesis is that the P. haloplanktis TAC125anti-biofilm compound might act as a signalling molecule thatmodulates the gene expression of recipient bacteria (Kim et al.,2009). Indeed, bacterial communication is one of the regulatorymechanisms suggested to be involved in biofilm formation(Parsek and Greenberg, 2005).

The results of the initial attachment assay indicate that theculture supernatant SN4B inhibited the biofilm formation bycontrasting the initial attachment of bacterial cells to the surface.

Moreover the results here obtained demonstrated that the P.haloplanktis TAC125 anti-biofilm action is effective on maturebiofilm, it is interesting to note that the action on maturebiofilm is not immediate but requires a prolonged time. Thislatter result suggests that the anti-biofilm compound could actas a signal that downregulated the adhesive properties of thecell surface or the biofilm matrix rather than a surfactant thatrapidly carries its action.

There are different bacterial communication systems, bothstrict intraspecific and interspecific systems. One systemsuggested to serve as a bacterial intra- and interspecies com-munication system uses the autoinducer-2 (AI-2) produced byluxS gene, as a signalling molecule (Federle and Bassler, 2003;Yoshida et al., 2005). LuxS has also been identified in S.epidermidis (Xu et al., 2006; Li et al., 2008), but P. hal-oplanktis TAC125 genome analysis revealed that the Antarcticbacterium is devoid of luxS gene (Medigue et al., 2005) sug-gesting the anti-biofilm activity could be due to a not identifiedsignalling molecule.

The anti-biofilm effects of P. haloplanktis exoproductscould be due to a novel molecule or the synergistic actions ofdifferent molecules. A preliminary physico-chemical charac-terization of SN4B supports the hypothesis of a compound ofpolysaccharide nature because treatment with periodateimpaired its capacity to inhibit biofilm formation.

It is well known that bacterial exopolysaccharides exhibithighly variable structures and it is likely that they also performadditional functions besides their implied function in matrixstabilization and energy storage (Rendueles et al., 2013). Infact, several studies showed that certain bacterial mutantsdeficient in capsular polysaccharide production exhibitincreased biofilm formation (Valle et al., 2006; Flahaut et al.,2008). These observations suggest that some bacterial exo-polysaccharides can perform functions that inhibit or desta-bilize the biofilm. Understanding the mode of action of SN4Bwill require identification of the active molecules.

Since S. epidermidis is the main microorganism responsiblefor most of the chronic graft infections, medical applicationscould be proposed. The present study was intended atobtaining new molecules with anti-adhesive properties whoseuse could be proposed during persistent infection sustained bystaphylococci in combination therapy with antibiotics.

Acknowledgements

This work was supported by P.N.R.A. (Programma Nazio-nale di Ricerche in Antartide 2009e2011) and by PharmaseaPF7 2012e16.

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References

Arciola, C.R., Hansch, G.M., Visai, L., Testoni, F., Maurer, S., Campoccia, D.,

Selan, L., Montanaro, L., 2012. Interactions of staphylococci with osteo-

blasts and phagocytes in the pathogenesis of implant-associated osteo-

myelitis. Int. J. Artif. Organs 35, 713e726.

Artini, M., Scoarughi, G.L., Papa, R., Cellini, A., Carpentieri, A., Pucci, P.,

Amoresano, A., Gazzola, S., Cocconcelli, P.S., Selan, L., 2011. A new

anti-infective strategy to reduce adhesion-mediated virulence in Staphy-

lococcus aureus affecting surface proteins. Int. J. Immunopathol. Phar-

macol. 24, 661e672.

Beloin, C., Ghigo, J.M., 2005. Finding gene-expression patterns in bacterial

biofilms. Trends Microbiol. 13, 16e19.

Bendaoud, M., Vinogradov, E., Balashova, N.V., Kadouri, D.E.,

Kachlany, S.C., Kaplan, J.B., 2011. Broad-spectrum biofilm inhibition by

Kingella kingae exopolysaccharide. J. Bacteriol. 193, 3879e3886.Christensen, G.D., Simpson, W.A., Younger, J.J., Baddour, L.M., Barrett, F.F.,

Melton, D.M., Beachey, E.H., 1985. Adherence of coagulase-negative staphy-

lococci to plastic tissue culture plates: a quantitative model for the adherence

of staphylococci to medical devices. J. Clin. Microbiol. 22, 996e1006.Corchero, J.L., Gasser, B., Resina, D., Smith, W., Parrilli, E., Vazquez, F.,

Abasolo, I., Giuliani, M., Jantti, J., Ferrer, P., Saloheimo, M.,

Mattanovich, D., Schwartz Jr., S., Tutino, L., Villaverde, A., 2012. Un-

conventional microbial systems for the cost-efficient production of high-

quality protein therapeutics. Biotechnol. Adv. http://dx.doi.org/10.1016/j.

biotechadv.2012.09.001

Costerton, J.W., Stewart, P.S., 2001. Battling biofilmsdthe war is against

bacterial colonies that cause some of the most tenacious infections known.

The weapon is knowledge of the enemy’s communication system. Sci. Am.

285, 74e81.

Das, P., Mukherjee, S., Sen, R., 2009. Antiadhesive action of a marine

microbial surfactant. Colloids Surf. B. Biointerfaces 71, 183e186.

Dohar, J.E., Hebda, P.A., Veeh, R., Awad, M., Costerton, J.W., Hayes, J.,

Ehrlich, G.D., 2009. Mucosal biofilm formation on middle-ear mucosa in

a nonhuman primate model of chronic suppurative otitis media. The

Laryngoscope 115, 1469e1472.

Drago, L., Vecchi, E.D., Mattina, R., Romano, C.L., 2013. Activity of N-

acetyl-L-cysteine against biofilm of Staphylococcus aureus and Pseudo-

monas aeruginosa on orthopedic prosthetic materials. Int. J. Artif. Organs

(Epub ahead of print).

Epstein, A.K., Wong, T.S., Belisle, R.A., Boggs, E.M., Aizenberg, J., 2012.

Liquid-infused structured surfaces with exceptional anti-biofouling per-

formance. Proc. Natl. Acad. Sci. U.S.A. 109, 13182e13187.

Estrela, A.B., Heck, M.G., Abraham, W.R., 2009. Novel approaches to control

biofilm infections. Curr. Med. Chem. 16, 1512e1530.

Federle, M.J., Bassler, B.L., 2003. Interspecies communication in bacteria. J.

Clin. Invest. 112, 1291e1299.

Flahaut, S.., Vinogradov, E., Kelley, K.A., Brennan, S., Hiramatsu, K., Lee, J.C.,

2008. Structural and biological characterization of a capsular polysaccharide

produced by Staphylococcus haemolyticus. J. Bacteriol. 190, 1649e1657.Francolini, I., Donelli, G., 2010. Prevention of biofilm-based medical-device-

related infections. FEMS Immunol. Med. Microbiol. 59, 227e238.

Giuliani, M., Parrilli, E., Pezzella, C., Rippa, V., Duilio, A., Marino, G.,

Tutino, M.L., 2012. A novel strategy for the construction of genomic

mutants of the Antarctic bacterium Pseudoalteromonas haloplanktis

TAC125. Methods Mol. Biol. 824, 219e233.

Heilmann, C., Gerke, C., Perdreau-Remington, F., Gotz, F., 1996. Character-

ization of Tn917 insertion mutants of Staphylococcus epidermidis affected

in biofilm formation. Infect. Immun. 64, 277e282.

Holmstrom, C., Egan, S., Franks, A., McCloy, S., Kjelleberg, S., 2002.

Antifouling activities by marine surface associated Pseudoalteromonas

species. FEMS Microbiol. Ecol. 41, 47e58.Jayatilake, G.S., Thornton, M.P., Leonard, A.C., Grimwade, J.E., Baker, B.J.,

1996 Mar. Metabolites from an Antarctic sponge-associated bacterium,

Pseudomonas aeruginosa. J. Nat. Prod. 59 (3), 293e296. PubMed PMID:

8882433.

Kaplan, J.B., 2010. Biofilm dispersal: mechanisms, clinical implications and

potential therapeutic uses. J. Dent. Res. 89, 205e218.

Please cite this article in press as: Papa, R., et al., Anti-biofilm activity of the Anta

Microbiology (2013), http://dx.doi.org/10.1016/j.resmic.2013.01.010

Kerr, A., Cowling, M.J., Beveridge, C.M., Smith, M.J., Parr, A.C.S., 1998. The

early stages of marine biofouling and its effect on two types of optical

sensors. Environ. Int. 24, 331e343.

Kim, H.S., Kim, S.M., Lee, H.J., Park, S.J., Lee, K.H., 2009. Expression of the

cpdA gene, encoding a 3’,5’-cyclic AMP (cAMP) phosphodiesterase, is

positively regulated by the cAMP-cAMP receptor protein complex. J.

Bacteriol. 191, 922e930.

Kiran, G.S., Sabarathnam, B., Selvin, J., 2010. Biofilm disruption potential of

a glycolipid biosurfactant from marine Brevibacterium casei. FEMS

Immunol. Med. Microbiol. 59, 432e438.

Klein, G.L., Soum-Soutera, E., Guede, Z., Bazire, A., Compere, C.,

Dufour, A., 2011. The anti-biofilm activity secreted by a marine Pseu-

doalteromonas strain. Biofouling 27, 931e940.

Lequette, Y., Boels, G., Clarisse, M., Faille, C., 2010. Using enzymes to

remove biofilms of bacterial isolates sampled in the food-industry. Bio-

fouling 26, 421e431.Li, M., Villaruz, A.E., Vadyvaloo, V., Sturdevant, D.E., Otto, M., 2008. AI-2-

dependentgene regulation inStaphylococcusepidermidis.BMCMicrobiol. 8, 4.

Medigue, C., Krin, E., Pascal, G., Barbe,V., Bernsel, A., Bertin, P.N., Cheung, F.,

Cruveiller, S., D’Amico, S., Duilio, A., Fang, G., Feller, G., Ho, C.,

Mangenot, S., Marino, G., Nilsson, J., Parrilli, E., Rocha, E.P., Rouy, Z.,

Sekowska, A., Tutino,M.L., Vallenet, D., vonHeijne, G., Danchin, A., 2005.

Coping with cold: the genome of the versatile marine Antarctica bacterium

Pseudoalteromonas haloplanktis TAC125. Genome Res. 15, 1325e1335.

Ni, N., Li, M., Wang, J., Wang, B., 2009. Inhibitors and antagonists of bac-

terial quorum sensing. Med. Res. Rev. 29, 65e124.

Nithya, C., Pandian, S.K., 2010. The in vitro activity of selectedmarine bacterial

culture supernatants against Vibrio spp. Arch. Microbiol. 192, 843e854.

Otto, M., 2008. Staphylococcal biofilm. Curr. Top. Microbiol. Immunol. 322,

207e228.Parrilli, E., Giuliani, M., Giordano, D., Russo, R., Marino, G., Verde, C.,

Tutino, M.L., 2010. The role of a 2-on-2 haemoglobin in oxidative and

nitrosative stress resistance of Antarctic Pseudoalteromonas haloplanktis

TAC125. Biochimie 92, 1003e1009.Parsek, M.R., Greenberg, E.P., 2005. Sociomicrobiology: the connections

between quorum sensing and biofilms. Trends Microbiol. 13, 27e33.

Qin, Z., Yang, L., Qu, D., Molin, S., Tolker-Nielsen, T., 2009. Pseudomonas

aeruginosa extracellular products inhibit staphylococcal growth, and dis-

rupt established biofilms produced by Staphylococcus epidermidis.

Microbiology 155, 2148e2156.

Rasmussen, T.B., Givskov, M., 2006. Quorum-sensing inhibitors as anti-

pathogenic drugs. Int. J. Med. Microbiol. 296, 149e161.

Rendueles, O., Kaplan, J.B., Ghigo, J.M., 2013. Antibiofilm polysaccharides.

Environ. Microbiol. 15, 334e346.Rippa, V., Papa, R., Giuliani, M., Pezzella, C., Parrilli, E., Tutino, M.L.,

Marino, G., Duilio, A., 2012. Regulated recombinant protein production in

the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.

Methods Mol. Biol. 824, 203e218.

Sublette, K., Peacock, A., White, D., Davis, G., Ogles, D., Cook, D.,

Kolhatkar, R., Beckmann, D., Yang, X., 2006. Monitoring subsurface

microbial ecology in a sulfate-amended, gasolin-contaminated aquifer.

Ground Water Monit. R 26, 70e78.

Valle, J., Da Re, S., Henry, N., Fontaine, T., Balestrino, D., Latour-Lambert, P.,

Ghigo, J.M., 2006. Broad-spectrum biofilm inhibition by a secreted bac-

terial polysaccharide. Proc. Natl. Acad. Sci. U.S.A. 103, 12558e12563.

Wittschier, N., Lengsfeld, C., Vorthems, S., Stratmann, U., Ernst, J.F.,

Verspohl, E.J., Hensel, A., 2007. Large molecules as anti-adhesive com-

pounds against pathogens. J. Pharm. Pharmacol. 59, 777e786.

Xu, L., Li, H., Vuong, C., Vadyvaloo, V., Wang, J., Yao, Y., Otto, M., Gao, Q.,

2006. Role of the luxS quorum-sensing system in biofilm formation and

virulence of Staphylococcus epidermidis. Infect. Immun. 74, 488e496.

Yoshida, A., Ansai, T., Takehara, T., Kuramitsu, H.K., 2005. LuxS-based

signaling affects Streptococcus mutans biofilm formation. Appl. Environ.

Microbiol. 71, 2372e2380.

Zinger-Yosovich, K.D., Gilboa-Garber, N., 2009. Blocking of Pseudomonas

aeruginosa and Ralstonia solanacearum lectins by plant and microbial

branched polysaccharides used as food additives. J. Agric. Food Chem. 57,

6908e6913.

rctic marine bacterium Pseudoalteromonas haloplanktis TAC125, Research in