Antibioﬁlm Activity of Antarctic Sponge-Associated Bacteria against Pseudomonas aeruginosa and Staphylococcus aureus

: Bioprospecting in unusual marine environments provides an innovative approach to search novel biomolecules with antibioﬁlm activity. Antarctic sponge-associated bacteria belonging to Colwellia , Pseudoalteromonas , Shewanella and Winogradskyella genera were evaluated for their ability to contrast the bioﬁlm formation by Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213, as model organisms. All strains were able to produce bioﬁlm at both 4 and 25 ◦ C, with the highest production being for Colwellia , Shewanella and Winogradskyella strains at 4 ◦ C after 24 h. Antibioﬁlm activity of cell-free supernatants (CFSs) differed among strains and on the basis of their incubation temperature (CFSs 4 ◦ C and CFSs 25 ◦ C ). The major activity was observed by CFSs 4 ◦ C against S. aureus and CFSs 25 ◦ C against P. aeruginosa , without demonstrating a bactericidal effect on their growth. Furthermore, the antibioﬁlm activity of crude extracts from Colwellia sp. GW185, Shewanella sp. CAL606, and Winogradskyella sp. CAL396 was also evaluated and visualized by confocal laser scanning microscopic images. Results based on the surface-coating assay and surface tension measurements suggest that CFSs and the crude extracts may act as biosurfactants inhibiting the ﬁrst adhesion of P. aeruginosa and S. aureus . The CFSs and the novel biopolymers may be useful in applicative perspectives for pharmaceutical and environmental purposes.


Introduction
Bacterial adhesion and biofilm formation processes have pervasive importance in environmental and human health. Compared with the free-living style, bacterial aggregations have considerable advantages in terms of self-protection, increasing microbial tolerance to exogenous stresses and the ability to escape to antibiotics or other biocides. Bacterial biofilms by pathogens represent a serious concern in public health, because they are involved in 65-80% of all human bacterial infections. Persistent infections (such as cystic fibrosis, urethritis, otitis, periodontitis, and endocarditis) have been associated with pathogenic bacteria able to form biofilm settings [1][2][3][4][5]. The biofilm lifestyle protects bacteria from the host immune response and confer them less susceptibility to antimicrobial agents, giving rise to chronic infections that are notoriously difficult to eradicate [6][7][8][9].
Consequently, the eradication of biofilm appears difficult with traditional pharmaceutical agents, and the exploration of novel anti-biofilm strategies, aimed at searching for new natural valuable compounds to prevent or eliminate biofilm is needed. After an initial attachment, the adhesion to abiotic and biotic surfaces becomes irreversible when the cells begin to secrete exopolymers with the subsequent aggregation of cells into microcolonies, and the formation and stabilization of biofilms [10]. Extracellular polymeric substances (EPSs), also known as exopolymers, mediate most of the cell-to-cell and cell-to-surface interactions that are necessary for the formation and maturation of biofilm [11]. So far more than 30 different biofilm matrix polysaccharides (for example, alginate and cellulose) have been characterized as major component of the biofilm scaffold [11][12][13]. On the other hand, bacterial exopolymers, including exopolysaccharides, involved in the biofilm formation, could also possess the ability to counteract the adhesion and the biofilm formation of a wide spectrum of bacteria and fungi [14]. Exopolysaccharides might act as signaling molecules that modulate gene expression of recipient bacteria [15], or in the competitive inhibition of multivalent carbohydrate-protein interactions [16].
Marine microorganisms, as free-living or associated with different hosts, represent until now untapped sources of molecules with a broad range of activity including the biofilm inhibition [17,18]. Increased attention is given to the discovery of new antibiofilm EPSs with potential applications in different fields, ranging from environmental, such as the inhibition of biofouling on immersed substrates [19] or water treatment and detoxification [20], to the prevention and eradication of biofilm-based infections [4]. Recent findings suggested that the exopolysaccharide produced by the marine thermophilic Bacillus licheniformis T14 possesses the ability to inhibit the biofilm formation of several pathogenic bacteria [18]. This exopolysaccharide did not possess antibacterial effects, suggesting that its antibiofilm activity is therefore mediated by different mechanisms other than growth inhibition. Most antibiofilm exopolymers showed surfactant activities, which influence the physical characteristics of bacterial cells and abiotic surfaces [18].
Among extremophiles, cold-adapted bacteria from polar habitats represent a potential source of structurally and functionally novel biologically active molecules with biotechnological potential [21,22]. Several cold-adapted bacteria of the genera Colwellia, Flavobacterium, Marinobacter, Polaribacter, Pseudoalteromonas, Pseudomonas, Shewanella and Winogradskyella have been reported as able to produce EPSs [23][24][25][26]. Pseudoalteromonas is the most frequently reported EPS-producer from Antarctica, mainly from sea-ice and sea-water [22]. Supernatants obtained from cold-adapted bacteria belonging to Pseudoalteromonas, Psychrobacter and Psychromonas were reported able to produce different antibiofilm molecules active against Pseudomonas aeruginosa PAO1, Staphylococcus aureus and S. epidermidis [27]. For instance, a mixture of small hydrophobic molecules, rather than polysaccharides, produced by the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 was reported as active against S. epidermidis biofilm formation [28]. More recently, novel isolates from Antarctic seawater [24,25] and sponge specimens [26] were reported as able to produce EPSs potentially useful in biotechnological applications as cryoprotectant agents.
In this context, the present study was aimed at exploring Antarctic sponge-associated cold-adapted bacteria (in the genera Colwellia, Pseudoalteromonas, Shewanella and Winogradskyella) as producers of antibiofilm agents. Antibiofilm activity was tested against Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213, as biofilm models of clinically significant bacteria.

Antarctic Sponge-Associated Bacteria
Cold-adapted strains used in this work are listed in Table 1. They were previously isolated from Antarctic sponge specimens collected at Terra Nova Bay (Ross Sea, Antarctica) [26,[29][30][31]. Sponge treatment and bacterial isolation procedures were described previously [32]. Strains tested for antibiofilm activity were selected among 1583 isolates for their highly mucous aspect on Marine Agar 2216 (MA; Difco Laboratories, Detroit, MI, USA) plates supplemented with different sugars (0.6%, w/v) [26]. Bacterial isolates belong to the Italian Collection of Antarctic Bacteria of the National Antarctic Museum (CIBAN-MNA), kept at the University of Messina (Italy). All strains grow in the temperature value range from 4 to 30 • C. Cultures are routinely grown at 4 • C.

Biofilm Formation Assay
Biofilm formation by P. aeruginosa ATCC 27853 and S. aureus ATCC 29213 was investigated in 96-well polystyrene microplates (Falcon ® , Fisher Scientific, Milan, Italy), as previously reported by O'Toole et al. [33]. Suitable aliquots (200 µL) of each overnight culture in LB or TSB (OD 600nm = 0.1 equivalent to 1.5 × 10 8 bacteria/mL) were poured in the microwells and the microplates were incubated at 37 • C for 48 h (for P. aeruginosa) or 24 h (for S. aureus), without shaking. Nonadherent bacteria were removed by washing 5 times with distilled water, by gentle aspiration. Biofilms were stained with 0.1% (w/v) crystal violet solution for 20 min. Excess stain was removed by aspiration, and the plates were washed (5 times) and air dried (for 45 min). The stained biofilms were solubilized with absolute ethanol. Biofilm mass was spectrophotometrically determined (OD 585nm ) by the level of the crystal violet present in the de-staining solution, using a microtiter plate reader (Multiskan GO, Thermo Scientific, Waltham, MA, USA). Each data point was averaged from eight replicated microwells, and the standard deviation (SD) was calculated.
As reported above, suitable aliquots (200 µL) of each overnight culture in VNSS+PYS (OD 600nm = 0.1, equivalent to 1.5 × 10 8 bacteria/mL) were poured in the microwells and the microplates were incubated at 4 • C for 96 h or 25 • C for 48 h, without shaking. To evaluate the biofilm masses the microplates were treated as described above.

Cell-Free-Supernatants from Antarctic Sponge-Associated Bacteria
Aliquots (10%, v/v) of each bacterial culture in the exponential growth phase were inoculated into 25 mL medium VNSS+PYS, and cultures were incubated at 4 • C for 4 days or at 25 • C for 48 h. In order to remove all bacterial cells, cultures were centrifuged at 10,000× g for 30 min, and each supernatant was filtered through a 0.2-µm-pore-size membrane (Sartorius, Göttingen, Germany). Aliquots of 100 µL of each cell-free supernatant (CFS 4 • C and CFS 25 • C from bacterial cultures incubated at 4 and 25 • C, respectively) were spreadplated onto MA plates at 4 and 25 • C for 4 and 2 days, respectively to ensure that no cells remained in each supernatant.

Cell Free Supernatants
CFSs obtained from bacterial isolates were evaluated for their capacity to inhibit biofilm formation by P. aeruginosa and S. aureus in 96-well polystyrene microplates, as previously reported [18,33]. Aliquots (20 µL) of each CFS were added (final concentration 10%, v/v) to 180 µL of overnight cultures of the two pathogens in TSB (OD 600nm = 0.1, equivalent to 1.5 × 10 8 bacteria/mL), and microplates were incubated at 37 • C for 48 h (P. aeruginosa) or 24 h (S. aureus) without shaking and treated as described above. The reduction of biofilm formation by each pathogenic strain was expressed as antibiofilm activity (%) by applying the following formula: (OD control − OD sample /OD control ) × 100. Each data point was averaged from eight replicated wells, and the SD was calculated.

Crude Extracts
The antibiofilm activity against the two pathogens was evaluated for the crude extracts obtained from the most active CFSs. Crude extracts from CFSs were obtained as reported previously [26]. Briefly, after centrifugation (8000× g for 10 min at 4 • C), the supernatant was treated with 1 volume of cold absolute ethanol added under stirring. Crude extracts were obtained by centrifugation and the pellets were dissolved in hot water. The final water solution was dialyzed against tap water and distilled water, and then freeze-dried.
In 96-well microtiter plates, 20 µL of each crude extract diluted in Phosphate Buffer Saline (PBS) (Sigma Aldrich) at final concentration of 100, 200 or 400 µg/mL, or 20 µL of PBS as control, were added to overnight cultures (180 µL) of P. aeruginosa or S. aureus (OD 600nm = 0.1) and plates were incubated at 37 • C for 48 h (P. aeruginosa) or 24 h (S. aureus). OD6 00nm values were recorded after 24 h or 48 h, and antibiofilm capacity of the crude extracts was evaluated as described above.
The reduction of biofilm formation by each pathogenic strain was expressed as antibiofilm activity (%), calculated as described above. Each data point was averaged from four replicated wells and the SD was calculated.

Surface Coating Assay
A volume of 50 µL of each CFS or 20 µL of each crude extract diluted in PBS (400 µg/mL final concentration) were transferred to the center of a 24-well polystyrene microtiter plate (Falcon no. 353047) [35]. The plates were incubated at 37 • C for 30 min to allow complete water evaporation. The wells were filled with 1 mL of diluted overnight cultures containing 10 5 CFU/mL of P. aeruginosa or S. aureus in LB and TSB, respectively, and the plates were incubated at 37 • C for 18 h, in static conditions. Wells were washed (two times) with distilled water and stained with 1 mL of 0.1% crystal violet solution. To remove the excess of crystal violet, stained biofilms were rinsed with distilled water and air dried, and finally the wells were photographed.

Surface Tension
Surface tension (ST) of each CFS was measured by using a Digital Tensiometer Gibertini Elettronica™ TSD (Gibertini Elettronica, Milan, Italy) by the Wilhelmy Plate method, as previously described [36]. A ST lower than 40 mN/m was considered as an expression of biosurfactant production [37,38].

Confocal Microscopic Observation
Aliquots (800 µL) of P. aeruginosa (in LB) or S. aureus (in TSB) overnight cultures (adjusted to OD 600nm = 0.1) were poured in a 24 well plates (Falcon) with inside sterilized round microscope glass cover slides coverslips (18 mm in diameter). After the addition of 200 µL of each crude extract diluted in PBS (400 µg/mL final concentration), plates were incubated at 37 • C for 24 h (for S. aureus) or 48 h (for P. aeruginosa). Not-attached bacteria were removed by washing with PBS, and the adherent cells on the coverslip were heat-fixed and stained with 20 µg/mL of propidium iodide (PI, Sigma Aldrich).
Coverslips were incubated in the dark at 30 • C for 5 min. Biofilm formation of P. aeruginosa and S. aureus were observed using the Confocal Laser Scanning Microscopy (CLSM) to TCS SP2 microscope (Leica Microsystems Heidelberg, Mannheim, Germany), equipped with Ar/Kr laser, and coupled to a microscope (Leica DMIRB).

Biofilm Formation Assay
All tested Antarctic sponge-associated bacterial showed the biofilm-forming ability ( Table 2). Overall, biofilm masses produced at 25 • C were lower than at 4 • C. The best biofilm production was observed for Colwellia sp. GW185, followed by Shewanella sp. CAL606 and Winogradskyella sp. CAL396. Pseudoalteromonas isolates resulted less efficient in biofilm formation than the other tested strains.

Antibiofilm Activity of CFSs
The antibiofilm activity of CFSs 4 • C and CFSs 25 • C from Antarctic strains against P. aeruginosa and S. aureus is shown in Figure 1. Overall, the presence of CFSs did not significantly influenced the growth values of P. aeruginosa and S. aureus, thus indicating the absence of any antibacterial activity ( Figure S1). . The data were analyzed by One-way ANOVA. Statistically significant differences (p ≤ 0.05) between the control (C) and treated samples are indicated by an asterisk (*). Refer to Table 1 for bacterial strain affiliation.

Surface Coating Assay with CFSs
Most of CFSs were able to prevent the adhesion of P. aeruginosa and S. aureus t polystyrene surfaces after 18 h treatment, with the only exception of Pseudoalteromona CAL260 (Table 3 and Figure S2). Overall, CFSs4°C were numerically less active (4/11) than CFSs25°C against both P. aeruginosa and S. aureus; however, each CFS showed a differen pattern of action. For instance, CFSs from Pseudoalteromonas sp. CAL416 contrasted th adhesion of P. aeruginosa but not of S. aureus. CFSs25°C from Pseudoalteromonas sp. strain CAL433 and CAL451 were active only against S. aureus, whereas CFSs25°C from Pseudoalteromonas sp. TB42 and Shewanella CAL62 were active only against P. aeruginosa. . The data were analyzed by One-way ANOVA. Statistically significant differences (p ≤ 0.05) between the control (C) and treated samples are indicated by an asterisk (*). Refer to Table 1 for bacterial strain affiliation.
The biofilm reduction against S. aureus was observed for all CFSs 4 • C (Figure 1b). Most of CFSs 4 • C exhibited a biofilm reduction ≥20%, with the only exception of Pseudoalteromonas CAL433 (10.7%). The highest inhibition activity was shown by the CFS 4 • C obtained from Shewanella sp. CAL606 (42.4%). With the exception of CFSs 25 • C from Pseudoalteromonas sp. strains TB42, CAL260 and CAL433, all the other CFSs were able to reduce the S. aureus biofilm formation. A moderate activity was observed for CFSs 25 • C from Winogradskyella CAL396, Shewanella CAL606 and Colwellia GW185 (range 20.0-26.3%).

Surface Coating Assay with CFSs
Most of CFSs were able to prevent the adhesion of P. aeruginosa and S. aureus to polystyrene surfaces after 18 h treatment, with the only exception of Pseudoalteromonas CAL260 (Table 3 and Figure S2). Overall, CFSs 4 • C were numerically less active (4/11) than CFSs 25 • C against both P. aeruginosa and S. aureus; however, each CFS showed a different pattern of action. For instance, CFSs from Pseudoalteromonas sp. CAL416 contrasted the adhesion of P. aeruginosa but not of S. aureus. CFSs 25 • C from Pseudoalteromonas sp. strains CAL433 and CAL451 were active only against S. aureus, whereas CFSs 25 • C from Pseudoalteromonas sp. TB42 and Shewanella CAL62 were active only against P. aeruginosa. Table 3. Inhibition of Pseudomonas aeruginosa and Staphylococcus aureus adhesion to polystyrene surfaces in absence (Control) or pre-coated with CFSs from Antarctic sponges-associated bacteria obtained after incubating at 4 • C (CFS 4 • C ) or 25 • C (CFS 25 • C ), after a 18 h treatment.

P. aeruginosa ATCC 27853
S. aureus ATCC 29213 CFSs from Colwellia sp. GW185 and Shewanella sp. strains CAL242 showed a similar pattern of action against the adhesion of the two pathogens. Finally, CFSs obtained from Shewanella CAL606 and Winogradskyella sp. CAL396 after incubation at both the temperature tested (4 and 25 • C) were able to contrast the adhesion of P. aeruginosa and S. aureus on polystyrene surfaces.

Antibiofilm of Crude Extracts
A slight reduction (≤10%) of biofilm formation of P. aeruginosa ATCC 27,853 and S. aureus ATCC 2913 was observed by all the crude extracts. The highest reduction was observed in the presence of the crude extracts (400 µg/mL) from CFS 25 • C of Colwellia sp. GW185 and CFSs 4 • C Shewanella sp. CAL606 and Winogradskyella sp. CAL396 (Figure 3).

Surface Coating Assay with Crude Extracts
Only the crude extract from Shewanella sp. CAL606 was able to contrast the adhesion of P. aeruginosa and S. aureus to polystyrene surfaces, while those from Colwellia sp. GW185 and Winogradskyella CAL396 were active against P. aeruginosa (Table 4 and Figure S3).

Confocal Microscopic Observation
The antibiofilm activity of the crude extracts (400 µg/mL) from Colwellia sp. GW185, Shewanella sp. CAL606 and Winogradskyella sp. CAL396 against P. aeruginosa and S. aureus was also observed onto glass surfaces after 24 h treatment, by confocal laser scanning microscopic images (Figure 4). The crude extracts from Shewanella sp. CAL606 and Winogradskyella sp. CAL396 showed a visible reduction of the biofilm formation of P. aeruginosa, while Colwellia sp. GW185 did not. The crude extracts from Colwellia sp. GW185, Shewanella sp. CAL606 and Winogradskyella sp. CAL396 were able to inhibit the biofilm formation of S. aureus.

Surface Coating Assay with Crude Extracts
Only the crude extract from Shewanella sp. CAL606 was able to contrast the adhesion of P. aeruginosa and S. aureus to polystyrene surfaces, while those from Colwellia sp. GW185 and Winogradskyella CAL396 were active against P. aeruginosa (Table 4 and Figure  S3).

Confocal Microscopic Observation
The antibiofilm activity of the crude extracts (400 µ g/mL) from Colwellia sp. GW185, Shewanella sp. CAL606 and Winogradskyella sp. CAL396 against P. aeruginosa and S. aureus was also observed onto glass surfaces after 24 h treatment, by confocal laser scanning microscopic images (Figure 4). The crude extracts from Shewanella sp. CAL606 and Winogradskyella sp. CAL396 showed a visible reduction of the biofilm formation of P. aeruginosa, while Colwellia sp. GW185 did not. The crude extracts from Colwellia sp. GW185, Shewanella sp. CAL606 and Winogradskyella sp. CAL396 were able to inhibit the biofilm formation of S. aureus.

Discussion
The biofilm formation is relevant in a wide range of domains, from medicine and food industry to marine environmental aspects. Bacterial communities involved in symbiotic relationships with marine invertebrates have recently captured the attention of researchers because they have been proven to be promising for bioprospecting purposes [39]. Even if for a long time many marine invertebrates, and particularly sponges (phylum Porifera), have been considered the direct source of bioactive compounds of therapeutic importance [40,41], more recently it has been demonstrated that the real responsible of metabolite production are their bacterial symbionts [42].

Discussion
The biofilm formation is relevant in a wide range of domains, from medicine and food industry to marine environmental aspects. Bacterial communities involved in symbiotic relationships with marine invertebrates have recently captured the attention of researchers because they have been proven to be promising for bioprospecting purposes [39]. Even if for a long time many marine invertebrates, and particularly sponges (phylum Porifera), have been considered the direct source of bioactive compounds of therapeutic importance [40,41], more recently it has been demonstrated that the real responsible of metabolite production are their bacterial symbionts [42]. Sponge-associated bacteria may either be transient food sources, symbiotic microbes, or pathogens [43]. It is expected that bacterial symbionts producing bioactive compounds, such as antimicrobial agents or inhibitors of bacterial communication systems (quorum sensing), are able to regulate or prevent the colonization by other microorganisms; and therefore, symbiotic relationships provide protection to the microbial symbionts as well as to the host organisms [44]. Classical symbiosis may involve sponge specialists (present in only one species), sponge associated (not found in the surrounding seawaters) or generalists (found in sponges and seawaters) [45]. These aspects were mainly investigated in temperate and tropical areas, while for cold environments, such as Antarctica, it is quite underexplored [21].
The aim of this work was to contribute to search for bacteria associated with Antarctic sponges with applicative perspectives for pharmaceutical and environmental purposes. Eleven cold-adapted strains isolated from Antarctic sponges, belonging to Colwellia, Pseudoalteromonas, Shewanella and Winogradskyella genera, were assayed for their ability to form biofilm, as indicative for the production of extracellular polymeric substances (EPSs) and then for the evaluation of their antibiofilm activity against P. aeruginosa ATCC 27853 and S. aureus ATCC 29213, as model organisms.
All Antarctic strains here investigated were able to form biofilm at both 4 • C and 25 • C incubation temperatures, with the best production by Colwellia, Shewanella and Winogradskyella strains at 4 • C rather than at 25 • C, suggesting that low incubation temperature greatly influence the production of exopolymers in psychrotrophic strains.
The antibiofilm activity of the cell-free supernatants against the two pathogens differed both among strains and on the base of the incubation temperatures, and the major activity was observed by CFSs at 4 • C against S. aureus and at 25 • C against P. aeruginosa, indicating a different action against Gram-positive and Gram-negative bacteria, that are known to produce biofilms with different features [46,47].
CFSs from Colwellia GW 185, Shewanella CAL606 and Winogradskyella CAL 396 were the most active against the biofilm formation by the two pathogens, without demonstrating bactericidal effects on growth, confirming that supernatants acted specifically against the biofilm formation. Similar activity toward staphylococci and P. aeruginosa biofilms have been displayed by cell-free supernatants from different marine bacteria [18,48] and also from cold-adapted bacteria isolated from Arctic and Antarctic seawater samples [27,28,[49][50][51][52].
To evaluate whether the reduction of the biofilm formation by CFSs produced at different incubation temperatures was due to the inhibition of the initial attachment, surface coating assays were performed. Our results suggested that the CFSs perform antibiofilm activity against P. aeruginosa and S. aureus at the initial phase of biofilm formation interfering with the cellular adhesion on surfaces ( Figure 2). Many molecules with antibiofilm properties act as surfactants, by modifying the bacterial cell physical features and their interaction with abiotic surfaces [17,53,54]. Biosurfactants generally alter the surface properties such as wettability and charge and therefore wane bacteria-surface and bacteria-bacteria interactions, reducing the ability to form biofilms [17,48,[55][56][57][58][59] The surface tension measurements confirmed the biosurfactant activity of CFSs, although with different effects due to the temperature incubation.
Differences in the antibiofilm activity of CFSs suggest that active molecules produced at different temperatures may differ quantitatively or alternatively may be not identical. A few studies provide evidences that changes in cultivation conditions lead to production of biofilm-specific metabolites and polymers [60][61][62][63]. Chemical characterization of crude extracts from the most active CFSs indicated that the strains produced different molecules (Table 5). These bacterial exopolymers have been recently reported to possess attractive properties useful in different biotechnological applications as thickeners and cryoprotectants, and also as bioflocculant, adhesives or antiadhesive, and heavy metals adsorbers for bioremediation purposes [22,24].  The different physical-chemical nature of exopolymers produced by Colwellia sp. GW185, Shewanella sp. CAL606 and Winogradskyella CAL396 can be responsible for their anti-biofilm activity. Results previously reported on the emulsifying activity of these extracts (Table 3) [26] are in agreement with our results in confirming a possible inhibition of the first phase of the biofilm formation. The antiadhesive activity by the crude extracts assessed by microplates assay was also validated by CLSM observations (Figure 4).
Other few bacterial purified EPSs from marine environments, mainly exopolysaccharides, have been reported to possess antibiofilm activities against pathogenic bacteria ( Table 6). Table 6. Bacterial exopolysaccharides with antibiofilm activity against pathogenic bacteria ( a Caruso et al. [26]). For instance, the exopolysaccharides EPS-T14 produced by Bacillus licheniformis T14 [18] and A101 isolated from the marine Vibrio sp. QY101 [48] were reported to inhibit the initial adhesion of both Gram-negative and Gram-positive bacteria. In addition, the A101 polysaccharide also affected P. aeruginosa cell-to-cell interactions and induced biofilm dispersion of P. aeruginosa but not of S. aureus. Because after the first adhesion bacteria establish strict surface bonds and connections, the initial biofilm formation step can be impacted by several nonbiocidal bacterial activities.

Conclusions
Cold-adapted bacteria belonging to Colwellia, Pseudoalteromonas, Shewanella and Winogradskyella genera, isolated from Antarctic sponges, are able to produce exopolymers allowing them to form biofilm as well as to contrast the biofilm formation of Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213, as biofilm models and clinically relevant bacteria. Antibiofilm activity of cell-free supernatants (CFSs) differed among strains and on the basis of their incubation temperature (CFSs 4 • C and CFSs 25 • C ). The major activity was observed by CFSs 4 • C against S. aureus and CFSs 25 • C against P. aeruginosa, without demonstrating a bactericidal effect on their growth.
Our results suggest that both CFSs and crude extracts from Colwellia sp. GW185, Shewanella sp. CAL606, and Winogradskyella sp. CAL396 may act as biosurfactants inhibiting the first adhesion of the two pathogens.
The sponge bacterial symbionts here studied confirm their previously suggested value as novel sources for bioprospecting. Further analyses are envisaged to deeply improve the optimization of production conditions of exopolymers and to investigate the genetic mechanisms at the base of the biofilm inhibition. These exopolymers, as nonbiocidal agents able to prevent the formation of bacterial biofilms, could lead to novel antibiofilm strategies useful in applicative perspectives, spanning from pharmaceutical and medical interests to environmental purposes.