Effects of commercial enzymes on the adhesion of a marine biofilm-forming bacterium

The antifouling potential of commercial hydrolases, four proteases, seven glycosidases and one lipase was evaluated on the adhesion of marine Pseudoalteromonas sp. D41. The experimental method, adapted to screen antifouling agents, was based on bacterial adhesion in natural sterile sea water in a microtiter plate and on total biomass quantification by the fluorescent dye DAPI (4[prime]6-diamidino-2-phenylindole). Savinase (subtilisin) was the most effective hydrolase in both the prevention of bacterial adhesion and the removal of adhered bacteria. However, some enzymatic preparations tested such as Amano protease were not only ineffective but also increased the number of adhered bacterial cells. Enumeration using epifluorescence microscopy of CTC (5-cyano-2,3-ditolyl tetrazolium chloride) and DAPI stained adhered D41 cells confirmed these observations. Overall, these results demonstrated that hydrolases could either prevent adhesion and remove adhered bacterial cells effectively, or conversely increase bacterial adhesion, depending on enzymatic concentrations and the type of enzymes tested.     

A marine bacterial adhesion microplate test using the DAPI fluorescent dye: a new method to screen antifouling agents

AIMS: To develop a method to screen antifouling agents against marine bacterial adhesion as a sensitive, rapid and quantitative microplate fluorescent test. METHODS AND RESULTS: Our experimental method is based on a natural biofilm formed by mono-incubation of the marine bacterium Pseudoalteromonas sp. D41 in sterile natural sea water in a 96-well polystyrene microplate. The 4'6-diamidino-2-phenylindole dye was used to quantify adhered bacteria in each well. The total measured fluorescence in the wells was correlated with the amount of bacteria showing a detection limit of one bacterium per 5 microm(2) and quantifying 2 x 10(7) to 2 x 10(8) bacteria adhered per cm(2). The antifouling properties of three commercial surface-active agents and chlorine were tested by this method in the prevention of adhesion and also in the detachment of already adhered bacteria. The marine bacterial adhesion inhibition rate depending on the agent concentration showed a sigmoid shaped dose-response curve. CONCLUSIONS: This test is well adapted for a rapid and quantitative first screening of antifouling agents directly in seawater in the early steps of marine biofilm formation. Significance AND IMPACT OF THE STUDY: In contrast to the usual screenings of antifouling products which detect a bactericidal activity, this test is more appropriate to screen antifouling agents for bacterial adhesion removal or bacterial adhesion inhibition activities. This screening test focuses on the antifouling properties of the products, especially the initial steps of marine biofilm formation.     

Marine bacteria from Danish coastal waters show antifouling activity against the marine fouling bacterium Pseudoalteromonas sp. strain S91 and zoospores of the green alga Ulva australis independent of bacteriocidal activity

The aims of this study were to determine if marine bacteria from Danish coastal waters produce antifouling compounds and if antifouling bacteria could be ascribed to specific niches or seasons. We further assess if antibacterial effect is a good proxy for antifouling activity. We isolated 110 bacteria with anti-Vibrio activity from different sample types and locations during a 1-year sampling from Danish coastal waters. The strains were identified as Pseudoalteromonas, Phaeobacter, and Vibrionaceae based on phenotypic tests and partial 16S rRNA gene sequence similarity. The numbers of bioactive bacteria were significantly higher in warmer than in colder months. While some species were isolated at all sampling locations, others were niche specific. We repeatedly isolated Phaeobacter gallaeciensis at surfaces from one site and Pseudoalteromonas tunicata at two others. Twenty-two strains, representing the major taxonomic groups, different seasons, and isolation strategies, were tested for antiadhesive effect against the marine biofilm-forming bacterium Pseudoalteromonas sp. strain S91 and zoospores of the green alga Ulva australis. The antiadhesive effects were assessed by quantifying the number of strain S91 or Ulva spores attaching to a preformed biofilm of each of the 22 strains. The strongest antifouling activity was found in Pseudoalteromonas strains. Biofilms of Pseudoalteromonas piscicida, Pseudoalteromonas tunicata, and Pseudoalteromonas ulvae prevented Pseudoalteromonas S91 from attaching to steel surfaces. P. piscicida killed S91 bacteria in the suspension cultures, whereas P. tunicata and P. ulvae did not; however, they did prevent adhesion by nonbactericidal mechanism(s). Seven Pseudoalteromonas species, including P. piscicida and P. tunicata, reduced the number of settling Ulva zoospores to less than 10% of the number settling on control surfaces. The antifouling alpP gene was detected only in P. tunicata strains (with purple and yellow pigmentation), so other compounds/mechanisms must be present in the other Pseudoalteromonas strains with antifouling activity.     

Surfactin reduces the adhesion of food-borne pathogenic bacteria to solid surfaces

Aims:  To investigate the effect of the biosurfactants surfactin and rhamnolipids on the adhesion of the food pathogens Listeria monocytogenes , Enterobacter sakazakii and Salmonella Enteritidis to stainless steel and polypropylene surfaces.
Methods and Results:  Quantification of bacterial adhesion was performed using the crystal violet staining technique. Preconditioning of surfaces with surfactin caused a reduction on the number of adhered cells of Ent. sakazakii and L. monocytogenes on stainless steel. The most significant result was obtained with L. monocytogenes where number of adhered cells was reduced by 10² CFU cm⁻². On polypropylene, surfactin showed a significant decrease on the adhesion of all strains. The adsorption of surfactin on polystyrene also reduces the adhesion of L. monocytogenes and Salm. Enteritidis growing cells. For short contact periods using nongrowing cells or longer contact periods with growing cells, surfactin was able to delay bacterial adhesion.
Conclusions:  The prior adsorption of surfactin to solid surfaces contributes on reducing colonization of the pathogenic bacteria.
Significance and Impact of the Study:  This is the first work investigating the effect of surfactin on the adhesion of the food pathogens L. monocytogenes , Ent. sakazakii and Salm. Enteritidis to polypropylene and stainless steel surfaces.     

Molecular investigation of the distribution, abundance and diversity of the genus Pseudoalteromonas in marine samples

The genus Pseudoalteromonas has attracted interest because it has frequently been found in association with eukaryotic hosts, and because many Pseudoalteromonas species produce biologically active compounds. One distinct group of Pseudoalteromonas species is the antifouling subgroup containing Pseudoalteromonas tunicata and Ps. ulvae, which both produce extracellular compounds that inhibit growth and colonization by different marine organisms. PCR primers targeting the 16S rRNA gene of the genus Pseudoalteromonas and the antifouling subgroup were developed and applied in this study. Real-time quantitative PCR (qPCR) was applied to determine the relative bacterial abundance of the genus and the antifouling subgroup, and denaturing gradient gel electrophoresis (DGGE) was applied to study the diversity of the genus in 11 different types of marine samples from Danish coastal waters. The detection of Ps. tunicata that contain the antifouling subgroup was achieved through specific PCR amplification of the antibacterial protein gene (alpP). The Pseudoalteromonas species accounted for 1.6% of the total bacterial abundance across all samples. The Pseudoalteromonas diversity on the three unfouled marine organisms Ciona intestinalis, Ulva lactuca and Ulvaria fusca was found to be low, and Ps. tunicata was only detected on these three hosts, which all contain accessible cellulose polymers in their cell walls.     

Adhesion of food-borne bacteria to stainless steel is reduced by food conditioning films

Aims:  Preconditioning of stainless steel with aqueous cod muscle extract significantly impedes subsequent bacterial adhesion most likely due to repelling effects of fish tropomyosin. The purpose of this study was to determine if other food conditioning films decrease or enhance bacterial adhesion to stainless steel.
Methods and Results:  Attachment of Pseudomonas fluorescens AH2 to stainless steel coated with water-soluble coatings of animal origin was significantly reduced as compared with noncoated stainless steel or stainless steel coated with laboratory substrate or extracts of plant origin. Coating with animal extracts also decreases adhesion of other food-relevant bacteria. The manipulation of adhesion was not attributable to growth inhibitory effects. Chemical analysis revealed that the stainless steels were covered by homogenous layers of adsorbed proteins. The presence of tropomyocin was indicated by appearance of proteins with similar molecular weight based in sodium dodecyl sulfate–polyacrylamide gel electrophoresis, in several extracts that reduced adhesion but also extracts not containing this protein reduced bacterial adhesion, indicating that several molecular species may be involved in the phenomenon.
Conclusions:  It is a common perception that food materials facilitate bacterial adhesion to surfaces; however, this study demonstrates that aqueous coatings of food origin may actually reduce bacterial adhesion.
Significance and Impact of the Study:  Compounds from food extracts may potentially be used as nontoxic coatings to reduce bacterial attachment to inert surfaces.     

Cell surface Lactobacillus plantarum LA 318 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) adheres to human colonic mucin

Aims:  To characterize the adhesion molecule of Lactobacillus plantarum LA 318 that shows high adhesion to human colonic mucin (HCM).
Methods and Results:  The adhesion test used the BIACORE assay where PBS-washed bacterial cells showed a significant decrease in adherence to HCM than distilled water-washed cells. A component in the PBS wash fraction adhered to the HCM and a main protein was detected as a c . 40-kDa band using SDS-PAGE. Using homology comparisons of the N-terminal amino acid sequences compared with sequence databases, this protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The DNA sequence of LA 318 GAPDH was 100% identical to the GAPDH ( gapB ) of L. plantarum WCFS1. The purified GAPDH adhered to HCM.
Conclusions:  We found the adhesin of L. plantarum LA 318 to HCM in its culture PBS wash fraction. The molecule was identified as GAPDH. Because LA 318 possesses the same adhesin as many pathogens, the lactobacilli GAPDH may compete with pathogens infecting the intestine.
Significance and Impact of the Study:  This is the first report showing GAPDH expressed on the cell surface of lactobacilli adheres to mucin suggesting L. plantarum LA 318 adheres to HCM using GAPDH binding activity to colonize the human intestinal mucosa.     

Anti-adhesive properties of fish tropomyosins

Aims:  We have recently found that preconditioning of stainless steel surfaces with an aqueous fish muscle extract can significantly impede bacterial adhesion. The purpose of this study was to identify and characterize the primary components associated with this bacteria-repelling effect.
Methods and Results:  The anti-adhesive activity was assayed against Escherchia coli K-12, and bacterial adhesion was quantified by crystal violet staining and sonication methods. Proteolytic digestion, elution and fractionation experiments revealed that the anti-adhesive activity of the extract was linked to the formation of a proteinaceous conditioning film composed primarily of fish tropomyosins. These fibrous proteins formed a considerable anti-adhesive conditioning layer on and reduced bacterial adhesion to several different materials including polystyrene, vinyl plastic, stainless steel and glass. The protein adsorption profiles obtained from the various materials did not differ significantly, but elution was often incomplete making minor qualitative/quantitative differences indiscernible.
Conclusions:  The data highlights the significance of protein conditioning films on bacterial adhesion and emphasizes the importance of substratum's physiochemical properties and exposure time with regards to protein adsorption/elution efficiency and subsequent bacterial adhesion.
Significance and Impact of the Study:  Fish tropomyosin-coatings could potentially offer a nontoxic and relatively inexpensive measure of reducing bacterial colonization of inert surfaces.     

Effect of growth time on the surface and adhesion properties of Lactobacillus rhamnosus GG

Aims:  To investigate the changes in the surface properties of Lactobacillus rhamnosus GG during growth, and relate them with the ability of the Lactobacillus cells to adhere to Caco-2 cells.
Methods and Results:  Lactobacillus rhamnosus GG was grown in complex medium, and cell samples taken at four time points and freeze dried. Untreated and trypsin treated freeze dried samples were analysed for their composition using SDS-PAGE analysis and Fourier transform infrared spectroscopy (FTIR), hydrophobicity and zeta potential, and for their ability to adhere to Caco-2 cells. The results suggested that in the case of early exponential phase samples (4 and 8 h), the net surface properties, i.e. hydrophobicity and charge, were determined to a large extent by anionic hydrophilic components, whereas in the case of stationary phase samples (13 and 26 h), hydrophobic proteins seemed to play the biggest role. Considerable differences were also observed between the ability of the different samples to adhere to Caco-2 cells; maximum adhesion was observed for the early stationary phase sample (13 h). The results suggested that the adhesion to Caco-2 cells was influenced by both proteins and non-proteinaceous compounds present on the surface of the Lactobacillus cells.
Conclusion:  The surface properties of Lact. rhamnosus GG changed during growth, which in return affected the ability of the Lactobacillus cells to adhere to Caco-2 cells.
Significance and Impact of the Study:  The levels of adhesion of Lactobacillus cells to Caco-2 cells were influenced by the growth time and reflected changes on the bacterial surface. This study provides critical information on the physicochemical factors that influence bacterial adhesion to intestinal cells.     

Comparison of in vitro models to study bacterial adhesion to the intestinal epithelium

Aims:  To evaluate the adhesion ability of intestinal bacteria to different in vitro models of intestinal epithelia, and to estimate the suitability of these models and the type of interactions involved.
Methods and results:  The adhesion of probiotic ( Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp . lactis Bb12), commensal ( B. animalis IATA-A2 and B. bifidum IATA-ES2) and potentially pathogenic bacteria ( E. coli and L. monocytogenes ) was determined. The adhesion models used were polycarbonate-well plates, with or without mucin, and different configurations of Caco-2 and/or HT29-MTX cell cultures. All bacteria adhered to wells without mucin (2·6–27·3%), the values being highly variable depending on the bacterial strain. Adhesion percentages of potentially probiotic bacteria to Caco-2 cultures were remarkably lower ( P  <   0·05) than those to mucin, and more similar to those of pathogenic strains. The lowest adhesion of different bacterial strains was detected on HT29-MTX (0·5–2·3%) cultures and Caco-2/HT29-MTX (0·6–3·2%) cocultures, while these values were increased in Caco-2 cultures plus mucin.
Conclusions:  The results suggested that bacterial strains exhibit different capacities to adhere to cellular components and several types of mucin present in different models, showing preferences for intestinal MUC2.
Significance and impact of the study:  The use of Caco-2 cells monolayer plus mucin (type II) better approaches the physiological characteristics of in vivo situation, providing a reliable and suitable in vitro model to evaluate bacterial adhesion.     

Effect of Bacteria Associated with the Green Alga Ulva reticulata on Marine Micro- and Macrofouling

The green alga Ulva reticulata (Forsskal) is often free from biofouling in Hong Kong waters. An early study indicated that bioactive substances from this alga inhibit settlement of the polychaete Hydroides elegans (Haswell). It is also predicted that epibiotic bacteria protect this alga from micro- and macrofouling. In this study, bacterial strains from the surface of U. reticulata were isolated and their inhibitive activities on micro- and macrofouling assayed. The strains were identified by 16S rRNA analysis as belonging to the genera Alteromonas , Pseudoalteromonas and Vibrio . There was no significant effect of these strains or their extracts (aqueous and ethanol) on the growth of five Vibrio strains isolated from natural biofilm. Two bacterial strains ( Alteromonas sp. and Vibrio sp. 3) were non-toxic to the benthic diatom Nitzschia paleacea (Grunow) while the other five strains caused a low level of mortality. No one bacterial strain was toxic to the larvae of H. elegans . Aqueous extract of one of the isolated bacterial species, i.e. Vibrio sp. 2, significantly ( p <0.00001) inhibited the settlement and metamorphosis of H. elegans larvae. The putative antifouling compounds have a molecular weight of >100 kD. On the other hand, biofilm of Pseudoalteromonas sp. 2 and aqueous extract of Vibrio sp. 2 suppressed the settlement of larvae induced by 3-isobutyl-1-methylxanthine (IBMX). Other epibiotic bacteria and their extracts had neither inhibitive nor inductive effects on larval settlement of H. elegans . The results indicate that the antifouling mechanism of U. reticulata may be dependent not only on materials from the macroalga itself but also on the epibiotic bacteria on the algal surface.     

Two species of culturable bacteria associated with degradation of brown algae Fucus evanescens

The heterotrophic microbial enrichment community established during degradation of brown algae Fucus evanescens was characterized. A two-species bacterial community of marine culturable gamma-proteobacteria consisted of Pseudoalteromonas and Halomonas. The first member of the community, Pseudoalteromonas sp., was highly metabolically active, had bacteriolytic and hemolytic activities, produced proteinases (gelatinase and caseinase), lipases, DNases, and fucoidanhydrolases, laminaranases, alginases, pustulanases, beta-glucosidases, beta-galactosidases, beta-N-acetylglucosaminidases, and beta-xylosidases. The second member of the community, Halomonas marina, produced only caseinase and DNase, and it did not hydrolyze algal polysaccharides. Both members of the studied bacterial community utilized a range of easily assimilable monosaccharides and other low molecular weight organic substances. The results provide an evidence of the complex metabolic interrelations between two members of this culturable community. One of them Pseudoalteromonas sp., most likely plays the major role in the initial stages of algal degradation; the other one, H. marina, resistant to the bacteriolytic activity of the former, is able to utilize the products of degradation of polysaccharides.     

The examination of Seliberia stellata exopolymers using lectin assays

Holdfast exopolymers of the dimorphic oligotrophic bacterium Seliberia stellata were examined using fluorescent lectins under light microscopy and colloidal gold lectins using transmission electron microscopy. Examination using fluorescent-labeled lectins revealed that lectins specific for polysaccharides and monosaccharides such as glucose and/or mannose, galactose, N-acetylgalactosamine, and N-acetylglucosamine (and its dimer) adhered to holdfast structure. Colloidal gold-labeled lectin assays also suggested the presence of these sugars. Both the holdfast that mediates swarmer cell adhesion and the holdfast that facilitates rosette formation gave similar results, suggesting the structures may be the same. Another exopolymer produced later in the growth cycle was observed using transmission electron microscopy. It appeared as an amorphous glycocalyx-like material very different from holdfast exopolymers. Retention of the gold lectin Wheat Germ Agglutinin (WGA), suggested the presence of N-acetylglucosamine, but fluorescent analyses were unsuccessful. The data suggest that S. stellata produces at least two different exopolymers: (a) the exopolymer of the swarmer cell and rosette holdfast whose function is adhesion and whose composition is (but may not be limited to) polysaccharides and (b) a slime-like exopolymer whose composition and function remain unknown. Correspondence: M.A. Hood     

In vitro activity of potential probiotic Lactobacillus murinus isolated from the dog

Aims:  The aim of this study was to isolate and identify Lactobacillus spp. isolates from faeces of a healthy dog, and to characterize their potential as probiotics in order to evaluate their possible use as probiotics for dogs.
Methods and Results:  An in vitro approach was used to characterize the isolates as potential probiotics including the evaluation of pH and bile salts tolerance, production of antimicrobial substances, biofilm formation on glass and polystyrene surfaces, aggregation ability and adhesion to canine intestinal mucus. The isolates survived to different pH and bile salts conditions, inhibited the in vitro growth of Escherichia coli and Clostridium perfringens , and adhered to glass and intestinal mucus.
Conclusions:  The properties shown by these isolates may indicate that they could colonize and persist in the gastrointestinal tract and induce beneficial effects to the host.
Significance and Impact of Study:  The evaluation of native canine isolates and future exprimental feeding assays may be useful tools to develop probiotics to improve animal health and reduce the risk of gastrointestinal disorders.     

Optimization of 2D-PAGE protocols for proteomic analysis of two nonaxenic toxin-producing freshwater cyanobacteria: Cylindrospermopsis raciborskii and Raphidiopsis sp.

Aims:  To optimize a protocol for the extraction and an in-depth analysis of the soluble protein fraction of two nonaxenic toxin-producing cyanobacteria Cylindrospermopsis raciborskii (hepatotoxin-producing), and Raphidiopsis sp. (neurotoxin-producing), using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).
Methods and Results:  The soluble protein fractions from strains of C. raciborskii and Raphidiosis sp. with different toxicity phenotypes were analysed by 2D-PAGE. Specific protocols were optimized specifically for each strain. Between 500 and 700 sharp protein spots were distinguished in a single 4–7 pH range 2D-PAGE for each cyanobacterium. Comparison of the protein maps of C. raciborskii CS-505 (a cylindrospermopsin-producing strain) and Raphidiopsis sp. D9 (saxitoxin-producing strain) against the nontoxic C. raciborskii strain CS-509 revealed many unique proteins in each protein map. We confirmed that the resolved proteins were cyanobacterial by identifying three randomly chosen protein spots from a Raphidiopsis sp. strain D9 2D-PAGE, using high-performance liquid chromatography (HPLC) tandem mass spectrometry (MS).
Conclusions:  The 2D-PAGE conditions presented here provide a robust protocol for proteomic studies in two CYN- and STX-producing model organisms, C. raciborskii and Raphidiopsis sp.
Significance and Impact of the Study:  We present the first protocols for proteomic analyses of Cylindrospermopsis raciborskii and Raphidiopsis sp.     

Sensory, chemical and bacteriological changes during storage of iced squid (Todaropsis eblanae)

AIMS: To relate sensory shelf-life of iced whole and gutted squid to bacterial growth and chemical changes. METHODS AND RESULTS: Cooked mantles from whole and gutted individuals were rejected after 10 and 12 days of storage, respectively, due to ammoniacal off-odours. Rate of production of both ammonia and trimethylamine was highest in the whole lot. Agmatine, which was only present in trace amounts in freshly-caught squid, increased rapidly in both lots. The main microflora at the time of sensory rejection of iced whole squid included Gram-negative, motile and non-fermentative rods, which were psychrophilic and had a requirement for NaCl. 16S rDNA sequence analyses identified the strains as belonging to the genus Pseudoalteromonas. Shewanella putrefaciens, Pseudoalteromonas sp. and Pseudomonas sp. dominated in spoiled gutted squid. Identification of strains from the stomach and digestive gland of recently-captured squid showed that the main flora consisted of Photobacterium phosphoreum. CONCLUSIONS: Spoilage of iced squid is likely to result from a combination of autolytic and bacterial changes. Agmatine seems to be an excellent freshness indicator. Photobacterium phosphoreum may contribute to spoilage through activity in the digestive gland, followed by diffusion of volatile compounds and amines to the mantle. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the psychrophilic nature of P. phosphoreum and Pseudoalteromonas sp., spread-plating and low temperature incubation are recommended for bacteriological evaluation of iced squid.     

Protein-mediated adhesion of the dissimilatory Fe(III)-reducing bacterium Shewanella alga BrY to hydrous ferric oxide.

The rate and extent of bacterial Fe(III) mineral reduction are governed by molecular-scale interactions between the bacterial cell surface and the mineral surface. These interactions are poorly understood. This study examined the role of surface proteins in the adhesion of Shewanella alga BrY to hydrous ferric oxide (HFO). Enzymatic degradation of cell surface polysaccharides had no effect on cell adhesion to HFO. The proteolytic enzymes Streptomyces griseus protease and chymotrypsin inhibited the adhesion of S. alga BrY cells to HFO through catalytic degradation of surface proteins. Trypsin inhibited S. alga BrY adhesion solely through surface-coating effects. Protease and chymotrypsin also mediated desorption of adhered S. alga BrY cells from HFO while trypsin did not mediate cell desorption. Protease removed a single peptide band that represented a protein with an apparent molecular mass of 50 kDa. Chymotrypsin removed two peptide bands that represented proteins with apparent molecular masses of 60 and 31 kDa. These proteins represent putative HFO adhesion molecules. S. alga BrY adhesion was inhibited by up to 46% when cells were cultured at sub-MICs of chloramphenicol, suggesting that protein synthesis is necessary for adhesion. Proteins extracted from the surface of S. alga BrY cells inhibited adhesion to HFO by up to 41%. A number of these proteins bound specifically to HFO, suggesting that a complex system of surface proteins mediates S. alga BrY adhesion to HFO.     

The anti-biofilm activity secreted by a marine Pseudoalteromonas strain

Bacterial biofilms occur on all submerged structures in marine environments. The authors previously reported that the marine bacterium Pseudoalteromonas sp. 3J6 secretes antibiofilm activity. Here, it was discovered that another Pseudoalteromonas sp. strain, D41, inhibited the development of strain 3J6 in mixed biofilms. Confocal laser scanning microscope observations revealed that the culture supernatant of strain D41 impaired biofilm formation of strain 3J6 and another marine bacterium. A microtiter plate assay of the antibiofilm activity was set up and validated with culture supernatants of Pseudoalteromonas sp. 3J6. This assay was used to determine the spectra of action of strains D41 and 3J6. Each culture supernatant impaired the biofilm development of 13 marine bacteria out of 18. However, differences in the spectra of action and the physical behaviours of the antibiofilm molecules suggest that the latter are not identical. They nevertheless share the originality of being devoid of antibacterial activity against planktonic bacteria.     

Characterization and anti-biofilm activity of extracellular polymeric substances produced by the marine biofilm-forming bacterium Pseudoalteromonas ulvae strain TC14

This study investigated soluble (Sol-EPS), loosely bound (LB-EPS), and tightly bound extracellular polymeric substances (TB-EPS) harvested from biofilm and planktonic cultures of the marine bacterium Pseudoalteromonas ulvae TC14. The aim of the characterization (colorimetric methods, FTIR, GC-MS, NMR, HPGPC, and AFM analyses) was to identify new anti-biofilm compounds; activity was assessed using the BioFilm Ring Test®. A step-wise separation of EPS was designed, based on differences in water-solubility and acidity. An acidic fraction was isolated from TB-EPS, which strongly inhibited biofilm formation by marine bacterial strains in a concentration-dependent manner. The main constituents of this fraction were characterized as two glucan-like polysaccharides. An active poly(glutamyl-glutamate) fraction was also recovered from TB-EPS. The distribution of these key EPS components in Sol-EPS, LB-EPS, and TB-EPS was distinct and differed quantitatively in biofilm vs planktonic cultures. The anti-biofilm potential of the fractions emphasizes the putative antifouling role of EPS in the environment.