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.

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.

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.

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.

Monohalogenated maleimides as potential agents for the inhibition of Pseudomonas aeruginosa biofilm.

New monohalogenated maleimide derivatives (with bromine, chlorine or iodine) were synthesized to test the effect of halogen atoms in inhibiting the formation of Pseudomonas aeruginosa biofilm. The evaluation of their biological activities clearly defines a structure-activity relationship. In this study, the bactericidal action of the three compounds was observed at the concentration range 0.3-5.0 mM on Luria-Bertani agar plates. The halogen atom of these molecules was critical in modulating the antibacterial activity, with a slightly higher effectiveness for chlorine. Confocal laser scanning microscopy was used to examine P. aeruginosa biofilms cultivated in flow cells. At concentration as low as 40 microM, the bromine and iodine compounds displayed a total inhibition towards the formation of bacterial biofilm. At this concentration, the bacterial attachment to glass surfaces was strongly affected by the presence of bromine and iodine whereas the chlorine derivative behaved as a bactericidal compound. A bioluminescent reporter strain was then used to detect the effect of the chemically synthesized maleimides on quorum sensing (QS) in P. aeruginosa. At the concentration range 10-100 microM, bioluminescence assays reveal that halogenated maleimides were able to interfere with the QS of the bacterium. Although the relationship between the weak inhibition of cell-to-cell communication (15-55% of the signal) and the high inhibition of biofilm formation has not been elucidated clearly, the results demonstrate that bromo- and iodo-N-substituted maleimides bromine and iodine may be used as new potent inhibitors that control bacterial biofilms.

Modification of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) film by chemical graft copolymerization.

The graft copolymerization of 2-hydroxyethylmethacrylate (HEMA) onto poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) films has been investigated. The graft copolymerization was conducted in aqueous media using benzoyl peroxide (BPO) as chemical initiator. PHBHV films were prepared by solvent casting. Different parameters affecting the graft yield were studied such as monomer concentration, initiator concentration, and reaction time. The extent of grafting has been modulated by the preparation conditions, in particular the concentration of HEMA. However, it is interesting to note that the initiator concentration had only a slight influence on the graft yield. Characterization of the grafted PHBHV films assumed that the graft copolymerization not only occurred on the film surface but also took place into the film bulk. Differential scanning calorimetry showed that crystallinity dramatically decreased with increasing graft yield, indicating that graft copolymerization hindered the crystallization process. Wettability has been obviously improved by grafting a hydrophilic monomer such as HEMA for high graft yield (>130%).

Degradation and controlled release behavior of epsilon-caprolactone copolymers in biodegradable antifouling coatings.

Copolymers of caprolactone with delta-valerolactone and L-lactide were synthesized by ring-opening polymerization in the presence of tetrabutoxytitane in order to decrease the crystallinity of polycaprolactone (PCL) and to enlarge its potential applications. The kinetics of degradation and controlled release of bioactive molecules were investigated in aqueous medium at room temperature for 9 months. The influence of the comonomer structures, their molar ratio, and the presence of fillers on these kinetics were examined. Complementary analytical methods were used (i) to quantify the degradation of the copolymers by titration of products of degradation (lactic acid, hydroxycaproic acid, and hydroxypentanoic acid) and (ii) to reveal the degradation processes by determination of molecular weights and thermal characteristics. After aging, films were observed by scanning electronic microscopy and EDX microanalysis to check their capabilities for the release of bioactive agent. The results showed that the incorporation of a comonomer such as L-lactide or delta-valerolactone led to a faster degradation than that of PCL homopolymer. The release of biocides could be correlated with the degradation of copolymer but depended on the structure of the leached molecule.

Maturation by LctT is required for biosynthesis of full-length lantibiotic lacticin 481.

In lantibiotic lacticin 481 biosynthesis, LctT cleaves the precursor peptide and exports mature lantibiotic. Matrix-assisted laser desorption ionization-time of flight mass spectrometry revealed that a truncated form of lacticin 481 is produced in the absence of LctT or after cleavage site inactivation. Production of truncated lacticin 481 is 4-fold less efficient, and its specific activity is about 10-fold lower.

Lipid A components from Pseudomonas aeruginosa PAO1 (serotype O5) and mutant strains investigated by electrospray ionization ion-trap mass spectrometry.

The lipid A components of the Pseudomonas aeruginosa strains PAO1 (wild-type) and derived mutants PAO1 algC::tet and PAO1 PDO100 were isolated after mild acetic acid hydrolysis of LPS. Their structural heterogeneities were characterized using electrospray ionization (ESI) ion-trap mass spectrometry (MS) with direct infusion in the negative ion mode without prior derivatization. The ESI-mass spectra revealed monophosphorylated molecules corresponding to known tetra-, penta- and hexaacylated structures of P. aeruginosa lipid A. The MS/MS fragmentation patterns allowed the location of fatty acyl chains on the disaccharide backbone of lipid A. In addition, a hexaacylated lipid A containing a hexadecanoyl chain was detected for the first time in strain P. aeruginosa PAO1. With multiple stages of fragmentation (MS(n)), the position of this hexadecanoyl chain O-linked to the decanoyl chain at the C-3(') position of the glucosamine backbone was determined. This sensitive method is suitable to reveal lipid A heterogeneity, i.e. the nature, number and distribution of acyl chains, without prior lipopolysaccharide purification. The lipid A from mutant strains were also characterized and significant differences were shown in the abundance of monophosphorylated lipid A components between the wild-type and the mutant strains.

Bacterial biofilm in seawater: cell surface properties of early-attached marine bacteria.

The development of antifouling strategies in seawater requires knowledge of the physico-chemical properties of the cell surfaces of early adherent bacteria. The hydrophilic, electrostatic and the Lewis acid-base cell surface properties of eleven marine bacteria were characterized. Although these bacteria adhered to a hydrophilic support immersed for 3 and 6 h, they presented various physico-chemical properties. Eleven strains possessed a hydrophilic surface and five a hydrophobic surface. Although the majority of the bacteria presented an electron-donating character, some could not generate Lewis acid-base interactions with the support. On the other hand, all strains possessed an isoelectric point ranging from 2.2 to 3.4 and were negatively charged at the pH of seawater. Hydrophilicity was a preponderant property among these bacteria, but other properties should not be ignored. The development of new antifouling paints must take account all the possible interaction levels used by the bacteria to adhere to an immersed surface.

On-line high-performance liquid chromatography-mass spectrometric detection and quantification of N-acylhomoserine lactones, quorum sensing signal molecules, in the presence of biological matrices.

A protocol using reversed-phase liquid chromatography coupled with positive-ion electrospray ionization and ion trap mass spectrometry is described for the identification and quantification of N-acylhomoserine lactones (HSLs) in crude cell-free supernatants of bacterial cultures. The HSLs are produced by gram-negative bacteria and act as intercellular signals inducing density-dependent gene expression. Compared with the multi-step procedures previously reported, which included chemical extraction, purification and the use of Escherichia coli HSL biosensors, this on-line LC-MS-MS method is fast and detects 11 HSLs. Its speed and robustness allow the analysis of a large number of samples without loss of performance (no signal variation for a control sample after 90 chromatographic injections). The selectivity is based on the MS-MS fragment ions of the molecular [M+H]- ions and on their relative intensities. For quantification, the m/z 102 ion, specific for the lactone ring and detected with a good signal-to-noise ratio, allows low detection limits even in complex matrix samples (0.28 up to 9.3 pmol). Moreover, this method allows the quantification of 11 HSLs whatever their chemical structure, substituted or not. The protocol was applied to Vibrio vulnificus, a marine bacterium. Six HSLs were detected and quantified with relative standard deviations for repeatability of < 10%.

Bacteriocin detection from whole bacteria by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Class I bacteriocins (lantibiotics) and class II bacteriocins are antimicrobial peptides secreted by gram-positive bacteria. Using two lantibiotics, lacticin 481 and nisin, and the class II bacteriocin coagulin, we showed that bacteriocins can be detected without any purification from whole producer bacteria grown on plates by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). When we compared the results of MALDI-TOF-MS performed with samples of whole cells and with samples of crude supernatants of liquid cultures, the former samples led to more efficient bacteriocin detection and required less handling. Nisin and lacticin 481 were both detected from a mixture of their producer strains, but such a mixture can yield additional signals. We used this method to determine the masses of two lacticin 481 variants, which confirmed at the peptide level the effect of mutations in the corresponding structural gene.