Investigating the anti-bioadhesion properties of short, medium chain length, and amphiphilic polyhydroxyalkanoate films.

Silicone materials are widely used in fouling release coatings, but developing eco-friendly protection via biosourced coatings, such as polyhydroxyalcanoates (PHA) presents a major challenge. Anti-bioadhesion properties of medium chain length PHA and short chain length PHA films are studied and compared with a reference Polydimethylsiloxane coating. The results highlight the best capability of the soft and low-roughness PHA-mcl films to resist bacteria or diatoms adsorption as compared to neat PDMS and PHBHV coatings. These parameters are insufficient to explain all the results and other properties related to PHA crystallinity are discussed. Moreover, the addition of a low amount of PEG copolymers within the coatings, to create amphiphilic coatings, boosts their anti-adhesive properties. This work reveals the importance of the physical or chemical ambiguity of surfaces in their anti-adhesive effectiveness and highlights the potential of PHA-mcl film to resist the primary adhesion of microorganisms.

Anti-Biofilm Activity of a Hyaluronan-like Exopolysaccharide from the Marine Vibrio MO245 against Pathogenic Bacteria.

Biofilms, responsible for many serious drawbacks in the medical and marine environment, can grow on abiotic and biotic surfaces. Commercial anti-biofilm solutions, based on the use of biocides, are available but their use increases the risk of antibiotic resistance and environmental pollution in marine industries. There is an urgent need to work on the development of ecofriendly solutions, formulated without biocidal agents, that rely on the anti-adhesive physico-chemical properties of their materials. In this context, exopolysaccharides (EPSs) are natural biopolymers with complex properties than may be used as anti-adhesive agents. This study is focused on the effect of the EPS MO245, a hyaluronic acid-like polysaccharide, on the growth, adhesion, biofilm maturation, and dispersion of two pathogenic model strains, Pseudomonas aeruginosa sp. PaO1 and Vibrio harveyi DSM19623. Our results demonstrated that MO245 may limit biofilm formation, with a biofilm inhibition between 20 and 50%, without any biocidal activity. Since EPSs have no significant impact on the bacterial motility and quorum sensing factors, our results indicate that physico-chemical interactions between the bacteria and the surfaces are modified due to the presence of an adsorbed EPS layer acting as a non-adsorbing layer.

Temperate and tropical coastal waters share relatively similar microbial biofilm communities while free-living or particle-attached communities are distinct.

Free-living (FL) marine microbial communities differ from those attached to particles (PA). Likewise, biofilms (B) colonizing artificial surfaces, including plastics or ship hulls, hardly resemble their planktonic surroundings. However, few studies have examined the effect of the environment on these lifestyles and on the source of organisms colonizing marine surfaces. Using 16S rRNA gene metabarcoding, we identified specificities of marine prokaryotic community lifestyles (FL, PA or B) sampled in three coastal polluted locations with dissimilar environmental conditions: the North-Western Mediterranean Sea and the Atlantic and Indian Oceans. Biofilms developed over polyvinyl chloride (PVC) were found to be significantly different from FL or PA collected during the immersions. Alpha-diversity increased from FL to PA and to B, illustrating the integrative aspect of the latter, with little proportion of operational taxonomic units shared with the first two. Beta-diversity clustered first the lifestyles and then the sites. FL and PA were more affected by water quality, especially by trace metal contamination, whereas B were as sensitive to trace metals as to nutrients. Although biofilms should be supplied by the planktonic (ultra) rare biosphere, source tracking could only detect small contributions of FL or PA taxa to B communities.

Phidianidine A and Synthetic Analogues as Naturally Inspired Marine Antifoulants.

Stationary and slow-moving marine organisms regularly employ a natural product chemical defense to prevent being colonized by marine micro- and macroorganisms. While these natural antifoulants can be structurally diverse, they often display highly conserved chemistries and physicochemical properties, suggesting a natural marine antifouling pharmacophore. In our current report, we investigate the marine natural product phidianidine A, which displays several chemical properties found in highly potent marine antifoulants. Phidianidine A and synthetic analogues were screened against the settlement and metamorphosis of Amphibalanus improvisus cyprids, and several of the compounds displayed inhibitory activities at low micromolar concentrations with IC50 values down to 0.7 µg/mL observed. The settlement study highlights that phidianidine A is a potent natural antifoulant and that the scaffold can be tuned to generate simpler and improved synthetic analogues. The bioactivity is closely linked to the size of the compound and to its basicity. The study also illustrates that active analogues can be prepared in the absence of the natural constrained 1,2,4-oxadiazole ring. A synthetic lead analogue of phidianidine A was incorporated in a coating and included in antifouling field trials, where it was shown that the coating induced potent inhibition of marine bacteria and microalgae settlement.

Epinephrine affects motility, and increases adhesion, biofilm and virulence of Pseudomonas aeruginosa H103.

Microbial endocrinology has demonstrated for more than two decades, that eukaryotic substances (hormones, neurotransmitters, molecules of the immune system) can modulate the physiological behavior of bacteria. Among them, the hormones/neurotransmitters, epinephrine (Epi) and norepinephrine (NE), released in case of stress, physical effort or used in medical treatment, were shown to be able to modify biofilm formation in various bacterial species. In the present study, we have evaluated the effect of Epi on motility, adhesion, biofilm formation and virulence of Pseudomonas aeruginosa, a bacterium linked to many hospital-acquired infections, and responsible for chronic infection in immunocompromised patients including persons suffering from cystic fibrosis. The results showed that Epi increased adhesion and biofilm formation of P. aeruginosa, as well as its virulence towards the Galleria mellonella larvae in vivo model. Deciphering the sensor of this molecule in P. aeruginosa and the molecular mechanisms involved may help to find new strategies of treatment to fight against this bacterium.

Pseudomonas aeruginosa resistance of monosaccharide-functionalized glass surfaces.

Preventing microorganism colonization on a surface is a great challenge in the conception of medical, food and marine devices. Here, we describe the formation of carbohydrate functionalized glass surfaces with D-glucose, D-galactose and D-mannose and how they efficiently affected the bacterial attachment. The carbohydrate entities were covalently attached to the pre-functionalized surface by click chemistry thanks the copper catalysed alkyl-azide cycloaddition. Water contact angle and X-ray photoelectron spectroscopy characterisations showed a homogeneous and quantitative cycloaddition at the scale of microorganisms. The adhesion assays with Pseudomonas aeruginosa, used as model of opportunistic pathogen, indicated a significant diminution of almost 40% of the bacterial accumulation on glycosidic surfaces with respect to initial surface. This activity was further compared with a surface presenting a simple hydroxyl residue. Exploration of specific interactions through Lectin A deficient Pseudomonas aeruginosa mutant strain provided new evidences that Lectin A was involved in biofilm maturation, rather than bacterial attachment. Subsequently, the determination of surface free energy and the adhesion free energy between surfaces and bacterial cell wall showed that the adhesion was thermodynamically unfavourable.

Additives for Efficient Biodegradable Antifouling Paints.

The evolution of regulations concerning biocidal products aims to increase protection of the environment (e.g., EU Regulation No 528/2012) and requires the development of new non-toxic anti-fouling (AF) systems. The development of these formulations implies the use of ingredients (polymers, active substances, additives) that are devoid of toxicity towards marine environments. In this context, the use of erodable antifouling paints based on biodegradable polymer and authorized biocides responds to this problem. However, the efficiency of paints could be improved by the use of specific additives. For this purpose, three additives acting as surface modifiers were studied (Tween 80, Span 85 and PEG-silane). Their effects on parameters involved in antifouling efficiency as hydrophobicity, hydration and copper release were studied. Results showed that the addition of 3% of additives modulated hydrophobicity and hydration without an increase of copper release and significantly reduced microfouling development. Efficient paints based on biodegradable polymer and with no organic biocide could be obtained by mixing copper thiocyanate and additives.

Metabarcoding and metabolomics offer complementarity in deciphering marine eukaryotic biofouling community shifts.

Metabarcoding and metabolomics were used to explore the taxonomic composition and functional diversity of eukaryotic biofouling communities on plates with antifouling paints at two French coastal sites: Lorient (North Eastern Atlantic Ocean; temperate and eutrophic) and Toulon (North-Western Mediterranean Sea; mesotrophic but highly contaminated). Four distinct coatings were tested at each site and season for one month. Metabarcoding showed biocidal coatings had less impact on eukaryotic assemblages compared to spatial and temporal effects. Ciliophora, Chlorophyceae or Cnidaria (mainly hydrozoans) were abundant at Lorient, whereas Arthropoda (especially crustaceans), Nematoda, and Ochrophyta dominated less diversified assemblages at Toulon. Seasonal shifts were observed at Lorient, but not Toulon. Metabolomics also showed clear site discrimination, but these were associated with a coating and not season dependent clustering. The meta-omics analysis enabled identifications of some associative patterns between metabolomic profiles and specific taxa, in particular those colonizing the plates with biocidal coatings at Lorient.

Anti-Bacterial Adhesion Activity of Tropical Microalgae Extracts.

The evolution of regulations concerning biocidal products aimed towards an increased protection of the environment (e.g., EU Regulation No 528/2012) requires the development of new non-toxic anti-fouling (AF) systems. As the marine environment is an important source of inspiration, such AF systems inhibiting the adhesion of organisms without any toxicity could be based on molecules of natural origin. In this context, the antibiofilm potential of tropical microalgal extracts was investigated. The tropics are particularly interesting in terms of solar energy and temperatures which provide a wide marine diversity and a high production of microalgae. Twenty microalgal strains isolated from the Indian Ocean were studied. Their extracts were characterized in terms of global chemical composition by high resolution magic angle spinning (HR-MAS) and nuclear magnetic resonance (NMR) spectroscopy, toxicity against marine bacteria (viability and growth) and anti-adhesion effect. The different observations made by confocal laser scanning microscopy (CLSM) showed a significant activity of three extracts from Dinoflagellate strains against the settlement of selected marine bacteria without any toxicity at a concentration of 50 µg/mL. The Symbiodinium sp. (P-78) extract inhibited the adhesion of Bacillus sp. 4J6 (Atlantic Ocean), Shewanella sp. MVV1 (Indian Ocean) and Pseudoalteromonas lipolytica TC8 (Mediterranean Ocean) at 60, 76 and 52%, respectively. These results underlined the potential of using microalgal extracts to repel fouling organisms.

Non-Leachable Hydrophilic Additives for Amphiphilic Coatings.

Amphiphilic surfaces are particularly effective at inhibiting the adhesion of microorganisms (bacteria, cells, microalgae, etc.) in liquid media. The aim of this study is to determine the best hydrophilic linker to promote bonding between poly(ethylene glycol) (PEG) as a hydrophilic additive and poly(dimethyl siloxane) (PDMS) as the hydrophobic matrix. Various parameters have been studied (molecular weight, linker type, and polymer end-group), as well as the efficiency of the linking, the capacity of PEG to access to the surface of the film, and overall film homogeneity. According to the results, a PDMS linker paired with a PEG moiety allows for compatibilization of the compounds during cross-linking. This compatibilization seems to provide a good bonding with the matrix and a good surface access to the hydrophilic moiety. Therefore, this structure comprising a linking function attached to the PDMS⁻PEG copolymer has high potential as a non-releasable additive for amphiphilic coating applications.

Anti-Biofilm Effect of Biodegradable Coatings Based on Hemibastadin Derivative in Marine Environment.

Dibromohemibastadin-1 (DBHB) is an already known potent inhibitor of blue mussel phenoloxidase (which is a key enzyme involved in bioadhesion). Within this study, the potentiality of DBHB against microfouling has been investigated. The activity of DBHB was evaluated on key strains of bacteria and microalgae involved in marine biofilm formation and bioassays assessing impact on growth, adhesion and biofilm formation were used. To assess the efficiency of DBHB when included in a matrix, DBHB varnish was prepared and the anti-microfouling activity of coatings was assessed. Both in vitro and in situ immersions were carried out. Confocal Laser Scanning Microscopy (CLSM) was principally used to determine the biovolume and average thickness of biofilms developed on the coatings. Results showed an evident efficiency of DBHB as compound and varnish to reduce the biofilm development. The mode of action seems to be based principally on a perturbation of biofilm formation rather than on a biocidal activity in the tested conditions.

Sponge-Inspired Dibromohemibastadin Prevents and Disrupts Bacterial Biofilms without Toxicity.

Since the banning of several families of compounds in antifouling (AF) coatings, the search for environmentally friendly AF compounds has intensified. Natural sources of AF compounds have been identified in marine organisms and can be used to create analogues in laboratory. In a previous study, we identified that dibromohemibastadin-1 (DBHB) is a promising AF molecule, leading to the inhibition of the activity of phenoloxidase, an enzyme involved in the attachment of mussels to surfaces. This paper describes the activity of the DBHB on biofilm formation and its detachment and on bacterial adhesion and communication: quorum sensing. DBHB has an anti-biofilm activity without affecting adhesion of marine and terrestrial bacteria at a dose of 10 µM. Moreover, DBHB activity on quorum sensing (QS) is demonstrated at doses of 8 and 16 µM. The activity of DBHB on QS is compared to kojic acid, a quorum sensing inhibitor already described. This compound is a promising environmentally friendly molecule potentially useful for the inhibition of microfouling.

Spatio-Temporal Variations of Marine Biofilm Communities Colonizing Artificial Substrata Including Antifouling Coatings in Contrasted French Coastal Environments.

Surface colonization in seawater first corresponds to the selection of specific microbial biofilm communities. By coupling flow cytometry, microscopy and high throughput sequencing (HTS, 454 pyrosequencing) with artificial surfaces and environmental analyses, we intend to identify the contribution of biofilm community drivers at two contrasted French sites, one temperate and eutrophic (Lorient, Atlantic coast) and the other at a mesotrophic but highly contaminated bay (Toulon, North-Western Mediterranean Sea). Microbial communities were shaped by high temperatures, salinity and lead at Toulon by but nutrients and DOC at Lorient. Coatings including pyrithione exhibited a significant decrease of their microbial densities except for nanoeukaryotes. Clustering of communities was mainly based on the surface type and secondly the site, whereas seasons appeared of less importance. The in-depth HTS revealed that γ- and α-proteobacteria, but also Bacteroidetes, dominated highly diversified bacterial communities with a relative low ß-diversity. Sensitivity to biocides released by the tested antifouling coatings could be noticed at different taxonomic levels: the percentage of Bacteroidetes overall decreased with the presence of pyrithione, whereas the α/γ-proteobacteria ratio decreased at Toulon when increased at Lorient. Small diatom cells (Amphora and Navicula spp.) dominated on all surfaces, whereas site-specific sub-dominant taxa appeared clearly more sensitive to biocides. This overall approach exhibited the critical significance of surface characteristics in biofilm community shaping.

Influence of Biodegradable Polymer Properties on Antifouling Paints Activity.

The development of new antifouling paints requires understanding the parameters involved in antifouling activity and to develop new analytical tools for their evaluation. A series of biodegradable poly(ε-caprolactone-co-δ-valerolactone) copolymers varying by molecular weight and composition were synthesized, characterized and formulated as antifouling paints. The physico-chemical properties such as hydration, degradation, erosion and lixiviation of paints were studied. Microfouling (bacteria and microalgae) was observed by microscopic observations in a short delay, whereas macrofouling colonization was observed by visual inspection during one year. The antifouling activity of paints was modified by varying the composition and molecular weight of copolymer. The crystallinity appears to play a major role in antifouling activity, however the involvement of other properties such as hydration, degradation or erosion remains difficult to understand. Confocal laser scanning and scanning electron microscopes were used for the evaluation of antifouling paints. Results show that microalgae seem to be a pertinent indicator of antifouling activity.

Rapid identification of osmolytes in tropical microalgae and cyanobacteria by (1)H HR-MAS NMR spectroscopy.

In this study, we report the chemical characterization of 47 tropical microalgae and cyanobacteria by HR-MAS. The generated data confirm the interest of HR-MAS as a rapid screening technique with the major advantage of its easiness. The sample is used as powder of freeze-dried microalgae without any extraction process before acquisition. The spectral fingerprints of strains are then tested as variables for a chemotaxonomy study to discriminate cyanobacteria and dinoflagellates. The individual factor map generated by PCA analysis succeeds in separating the two groups, essentially thanks to the presence of specific carbohydrates. Furthermore, more resolved signals enable to identify many osmolytes. More precisely the characteristics δ of 2-O-alpha-D-glucosylglycerol (GG) are observed in all 21 h-MAS spectra of tropical cyanobacteria. After specific extraction, complementary analysis by 1D and 2D-NMR spectroscopies validates the identification of this osmolyte.

Bioaccumulation of metallic trace elements and organic pollutants in marine sponges from the South Brittany Coast, France.

The purpose of this study was to compare the accumulation of metallic and organic pollutants in marine sponges with the oyster Crassostrea gigas used as sentinel species. The concentrations of 12 Metallic Trace Elements (MTEs), 16 Polycyclic Aromatic Hydrocarbons (PAHs), 7 PolyChlorinated Biphenyls (PCBs), and 3 organotin derivatives were measured in 7 marine sponges collected in the Etel River (South Brittany, France). Results indicated Al, Co, Cr, Fe, Pb, and Ti particularly accumulated in marine sponges such as Hymeniacidon perlevis and Raspailia ramosa at higher levels compared to oysters. At the opposite, Cu and Zn accumulated significantly at higher concentrations in oysters. Among PAHs analyzed, benzo(a)pyrene bioaccumulated in H. perlevis at levels up to 17-fold higher than in oysters. In contrast, PCBs bioaccumulated preferentially in oysters. Significant differences exist in the abilities of marine phyla and sponge species to accumulate organic and metallic pollutants however, among the few sponge species studied, H. perlevis showed impressive bioaccumulation properties. The use of this species as bioindicator and/or bioremediator near shellfish farming areas is also discussed.

Bacteria and diatom resistance of silicones modified with PEO-silane amphiphiles.

Silicone coatings with enhanced antifouling behavior towards bacteria, diatoms, and a diatom dominated slime were prepared by incorporating PEO-silane amphiphiles with varied siloxane tether lengths (a-c): α-(EtO)3Si(CH2)2-oligodimethylsiloxanen-block-poly(ethylene oxide)8-OCH3 [n = 0 (a), 4 (b), and 13 (c)]. Three modified silicone coatings (A-C) were prepared by the acid-catalyzed sol-gel cross-linking of a-c, respectively, each with a stoichiometric 2:3 M ratio of α, ω-bis(Si-OH)polydimethylsiloxane (Mn = 3,000 g mol(-1)). The coatings were exposed to the marine bacterium Bacillus sp.416 and the diatom (microalga) Cylindrotheca closterium, as well as a mixed community of Bacillus sp. and C. closterium. In addition, in situ microfouling was assessed by maintaining the coatings in the Atlantic Ocean. Under all test conditions, biofouling was reduced to the highest extent on coating C which was prepared with the PEO-silane amphiphile having the longest siloxane tether length (c).

Joint-action of antifouling substances in copper-free paints.

Due to the environmentally harmful impact of tributyltin self-polishing paints, there is a critical need of more ecological alternatives. The aim of the present work is to study the joint-action of three molecules chosen in order to combine the two modes of prevention: chemical and physical repelling of biofouling. This "hybrid" system is principally dedicated to disturb durable settlement of microfouling. Each component was chosen according to its specific properties: chlorhexidine is a bisdiguanide antiseptic with antibacterial activity, zinc peroxide is an inorganic precursor of high instable entities which react with seawater to create hydrogen peroxide, Tween 85 is a non ionic surfactant disturbing interactions between colonizing organisms and surface. Obtained results highlighted the interest on mixing such molecules to get additive action on antifouling efficiency.

Evaluation of antibacterial activity against Salmonella Enteritidis.

Salmonella enterica serovar Enteritidis is a well-known pathogenic bacterium responsible for human gastrointestinal enteritis mainly due to the consumption of eggs and egg-products. The first aim of this work was to study several virulence factors of a strain isolated from egg content: SEovo. First, bacterial growth was studied at several temperatures and cell morphology was observed by scanning electronic microscopy. These experiments showed Salmonella's ability to grow at low temperatures and to produce exoproducts. Next, Salmonella motility was observed performing swimming, twitching, and swarming tests. Results indicated a positive flagellar activity and the cell ability to differentiate and become hyperflagellated under specific conditions. Moreover, SEovo adherence and biofilm formation was carried out. All of these tests enabled us to conclude that SEovo is a potential pathogen, thus it can be used as a model to perform antibacterial experiments. The second part of the study was dedicated to the evaluation of the antibacterial activity of different molecules using several methods. The antibacterial effect of silver and copper aluminosilicates was tested by two different kinds of methods. On the one hand, the effect of these two antibacterial agents was determined using microbiological methods: viable cell count and agar-well diffusion. And on the other hand, the antibacterial activity was evaluated using CLSM and SYTO Red/SYTOX Green dyeing. CLSM allowed for the evaluation of the biocide on sessile cells, whereas the first methods did not. Results showed that adhered bacteria were more resistant than planktonic counterparts and that CLSM was a good alternative to evaluate antibacterial activity on fixed bacteria without having to carry out a removing step.

C-type natriuretic peptide modulates quorum sensing molecule and toxin production in Pseudomonas aeruginosa.

Pseudomonas aeruginosa coordinates its virulence expression and establishment in the host in response to modification of its environment. During the infectious process, bacteria are exposed to and can detect eukaryotic products including hormones. It has been shown that P. aeruginosa is sensitive to natriuretic peptides, a family of eukaryotic hormones, through a cyclic nucleotide-dependent sensor system that modulates its cytotoxicity. We observed that pre-treatment of P. aeruginosa PAO1 with C-type natriuretic peptide (CNP) increases the capacity of the bacteria to kill Caenorhabditis elegans through diffusive toxin production. In contrast, brain natriuretic peptide (BNP) did not affect the capacity of the bacteria to kill C. elegans. The bacterial production of hydrogen cyanide (HCN) was enhanced by both BNP and CNP whereas the production of phenazine pyocyanin was strongly inhibited by CNP. The amount of 2-heptyl-4-quinolone (HHQ), a precursor to 2-heptyl-3-hydroxyl-4-quinolone (Pseudomonas quinolone signal; PQS), decreased after CNP treatment. The quantity of 2-nonyl-4-quinolone (HNQ), another quinolone which is synthesized from HHQ, was also reduced after CNP treatment. Conversely, both BNP and CNP significantly enhanced bacterial production of acylhomoserine lactone (AHL) [e.g. 3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) and butanoylhomoserine lactone (C4-HSL)]. These results correlate with an induction of lasI transcription 1 h after bacterial exposure to BNP or CNP. Concurrently, pre-treatment of P. aeruginosa PAO1 with either BNP or CNP enhanced PAO1 exotoxin A production, via a higher toxA mRNA level. At the same time, CNP led to elevated amounts of algC mRNA, indicating that algC is involved in C. elegans killing. Finally, we observed that in PAO1, Vfr protein is essential to the pro-virulent effect of CNP whereas the regulator PtxR supports only a part of the CNP pro-virulent activity. Taken together, these data reinforce the hypothesis that during infection natriuretic peptides, particularly CNP, could enhance the virulence of PAO1. This activity is relayed by Vfr and PtxR activation, and a general diagram of the virulence activation cascade involving AHL, HCN and exotoxin A is proposed.