Sesquiterpenoids and a steroid from the algicolous Trichoderma brevicompactum.

Six new sesquiterpenoids including three bisabolane derivatives, trichobisabolins O1, O2, and P (1-3), two nerolidol derivatives, trichonerolins A and B (4 and 5), one acorane, trichoacorin A (6), along with one new steroid, isoergokonin B (7), were isolated from the culture of Trichoderma brevicompactum A-DL-9-2 obtained from the inner tissue of the red alga Chondria tenuissima. Their structures and relative configurations were assigned by interpretation of 1D/2D NMR and MS data. As acyclic sesquiterpenoids, compounds 4 and 5 were discovered from Trichoderma for the first time. Compounds 1-7 were evaluated for the inhibition of some marine-derived organisms, in which, 3 and 4/5 exhibited potent inhibition against Amphidinium carterae and Chattonella marina with IC50 of 1.8 µg/mL and 1.2 µg/mL, respectively. In addition, compound 7 could inhibit the growth of Pseudoalteromonas citrea with an MIC value of 64 µg/mL.

Use of Quorum Sensing Inhibition Strategies to Control Microfouling.

Interfering with the quorum sensing bacterial communication systems has been proposed as a promising strategy to control bacterial biofilm formation, a key process in biofouling development. Appropriate in vitro biofilm-forming bacteria models are needed to establish screening methods for innovative anti-biofilm and anti-microfouling compounds. Four marine strains, two Pseudoalteromonas spp. and two Vibrio spp., were selected and studied with regard to their biofilm-forming capacity and sensitivity to quorum sensing (QS) inhibitors. Biofilm experiments were performed using two biofilm cultivation and quantification methods: the xCELLigence® system, which allows online monitoring of biofilm formation, and the active attachment model, which allows refreshment of the culture medium to obtain a strong biofilm that can be quantified with standard staining methods. Although all selected strains produced acyl-homoserine-lactone (AHL) QS signals, only the P. flavipulchra biofilm, measured with both quantification systems, was significantly reduced with the addition of the AHL-lactonase Aii20J without a significant effect on planktonic growth. Two-species biofilms containing P. flavipulchra were also affected by the addition of Aii20J, indicating an influence on the target bacterial strain as well as an indirect effect on the co-cultured bacterium. The use of xCELLigence® is proposed as a time-saving method to quantify biofilm formation and search for eco-friendly anti-microfouling compounds based on quorum sensing inhibition (QSI) strategies. The results obtained from these two in vitro biofilm formation methods revealed important differences in the response of biosensor bacteria to culture medium and conditions, indicating that several strains should be used simultaneously for screening purposes and the cultivation conditions should be carefully optimized for each specific purpose.

Spotlight on a novel bactericidal mechanism and a novel SXT/R391-like integrative and conjugative element, carrying multiple antibiotic resistance genes, in Pseudoalteromonas flavipulchra strain CDM8.

Many Pseudoalteromonas strains can produce bioactive compounds with antimicrobial activities. This study focused on a probiotic candidate P.flavipulchra CDM8 to reveal its novel antibacterial mechanism and risks for antibiotic resistance dissemination. Strain CDM8 could form floating biofilm, displayed strikingly broad antibacterial activities against multiple Vibrio and Bacillus species, and decreased the competitor's concentration in their co-cultures in the microtiter plate tests. It could also form vesicle/pilus-like structures on the outer surface, which were indicated to participate in the bactericidal activity and represent a novel antibacterial mechanism of CDM8, according to the scanning electron microscopic observation. However, CDM8 displayed multi-antibiotic resistance, conferred by the multidrug resistance regions in hotspot 4 and variable region III of a novel SXT/R391-like integrative and conjugative element (ICEPflCDM8). Summing up, our results provided a better understanding of the bactericidal mechanism of P. flavipulchra and highlighted the role of SXT/R391-like ICEs in conferring multidrug resistance phenotype of probiotic P. flavipulchra candidates.

Antifouling Activities of Immobilized Ferrocenyl Glucose on a Glassy Carbon Surface.

Polar coatings are used to protect surfaces from marine fouling based on the formation of a hydrated surface layer, which acts as a barrier to marine microorganisms. In this context, we have developed a material with glucose bound to ferrocene to prevent surface absorption. Glucose brings the polarity while ferrocene has the role of varying the state of charge of the surface. We therefore describe the synthesis of 6-deoxy-6-(4-ferrocenyl-1H-1,2,3-triazol-1-yl)-1-(4-aminophenyl)-ß-D-glucopyranose 6 and its immobilization on the surface of a C electrode and develop a methodology used for antibacterial testing. We were able to demonstrate that the immobilization of glucose 6 could be done in an artificial seawater environment by oxidation of an amine. The use of a 96-microwell platform equipped with electrodes for cyclic voltammetry, linked to a potentiostat, allowed the electrical solicitation of the coating in the presence of marine bacteria with a greater number of biological replicates. We have shown that the coating has an antibacterial effect, and this effect is accentuated when the coating is electrically stressed, resulting in the appearance and disappearance of charge on ferrocene, a phenomenon that seems to be conducive to colonization by bacteria.

Metabolomic and proteomic changes induced by growth inhibitory concentrations of copper in the biofilm-forming marine bacterium Pseudoalteromonas lipolytica.

Copper is an essential element for living cells but this metal is present in some marine environments at such high concentrations that it can be toxic for numerous organisms. In polluted areas, marine organisms may develop specific adaptive responses to prevent cell damage. To investigate the influence of copper on the metabolism of a single organism, a dual approach combining metabolomics and proteomics was undertaken on the biofilm-forming bacterial strain Pseudoalteromonas lipolytica TC8. In order to highlight differential adaptation according to the phenotype, the response of P. lipolytica TC8 to copper stress was studied in planktonic and biofilm culture modes under growth inhibitory copper concentrations. As expected, copper exposure led to the induction of defense and detoxification mechanisms. Specific metabolite and protein profiles were thus observed in each condition (planktonic vs. biofilm and control vs. copper-treated cultures). Copper exposure seems to induce drastic changes in the lipid composition of the bacterial cell membrane and to modulate the abundance of proteins functionally known to be involved in copper cell homeostasis in both planktonic and biofilm culture modes. Much more proteins differentially expressed after copper treatment were observed in biofilms than in planktonic cells, which could indicate a more heterogeneous response of biofilm cells to this metallic stress.

Identification and characterization of a LuxI/R-type quorum sensing system in Pseudoalteromonas.

Bacteria usually produce, release and detect quorum sensing (QS)-based signal molecules, and successively orchestrate gene expression in respond to environmental changes. Pseudoalteromonas are typical marine bacteria, but knowledge on their QS systems is extremely fragmentary. In this study, genome sequencing of Pseudoalteromonas sp. R3 was performed. Accordingly, a QS working model including three sets of hierarchically organized QS systems was proposed in strain R3. Among them, the typical LuxI/R-type QS system using acyl-homoserine lactones (AHLs) as signal molecules was characterized. Sequence similarity analysis indicated luxI encoding AHLs synthase is novel. The luxR encoding AHLs receptor is directly adjacent to luxI downstream. Notably, mutagenesis demonstrated LuxI and LuxR affect each other at transcriptional level, and both control the AHLs formation. Interestingly, it was found that LuxI/R-type QS system positively involves resistance to streptomycin. Thin-layer chromatography analysis showed strain R3 can produce 3-OH-C6-HSL and C8-HSL, which was supported by heterologous expression of LuxI in Escherichia coli. Sequence alignment analysis indicated that the N-terminal region of LuxI is more conservative than the C-terminal region, revealing the importance of N-terminal region in AHLs synthesis. The obtained findings enrich our knowledge on LuxI/R-type QS system in Pseudoalteromonas and its regulation on adaptation to marine environments.

The Challenge of the Sponge Suberites domuncula (Olivi, 1792) in the Presence of a Symbiotic Bacterium and a Pathogen Bacterium.

Sponges, which are in close contact with numerous bacteria in prey/predator, symbiotic and pathogenic relationships, must provide an appropriate response in such situations. This starts with a discriminating recognition of the partner either by a physical contact or through secreted molecules or both. We investigated the expression of the Toll-like receptor, Caspase 3/7, Tumor Necrosis Factor receptor-associated factor 6, Bcl-2 homology protein-2 and macrophage expressed genes of axenic sponge cells in the presence of a symbiotic bacterium (Endozoicomonas sp. Hex311), a pathogen bacterium (Pseudoalteromonas sp. 1A1), their exoproducts and lipopolysaccharides. The vast majority of answers are in line with what could be observed with the symbiotic bacterium. The pathogenic bacterium seems to profit from the eukaryotic cell: suppression of the production of the antibacterial compound, inhibition of the apoptosis caspase-dependent pathway, deregulation of bacterial recognition. This work contributes new scientific knowledge in the field of immunology and apoptosis in early branching metazoan harboring within its tissue and cells a large number of symbiotic bacteria.

Towards eco-friendly biocides: preparation, antibiofilm activity of hemibastadin analogues.

The antibiofilm activity of three hemibastadins analogues was evaluated against different marine bacterial strains through mono-species biofilms and through a multi-species model of biofilm. Results showed that compound 3 exhibited interesting antibiofilm efficiencies effective concentrations (EC50 ) in the range of 30-100 µmol l-1 without acute toxicity against bacteria. Toxicity against nontargeted organisms was also considered showing that the compound did not affect the global bacterial community at a concentration of 75-100 µmol l-1 . These results provided baseline data concerning the toxicity of antibiofilm biocides against marine organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports relevant information about antibiofilm activity of original derivatives of hemibastadin alkaloids. The most active compound was shown to act as a specific anti-biofilm inhibitor without affecting viability of the targeted bacteria no more than those of the global bacterial community of a seawater sample. Taken together, these findings indicate the potentiality of such compounds to be used as original nonbiocidal molecules for designing eco-friendly antifouling solutions.

Effect of Pentacyclic Guanidine Alkaloids from the Sponge Monanchora pulchra on Activity of α-Glycosidases from Marine Bacteria.

The effect of monanchomycalin B, monanhocicidin A, and normonanhocidin A isolated from the Northwest Pacific sample of the sponge Monanchora pulchra was investigated on the activity of α-galactosidase from the marine γ-proteobacterium Pseudoalteromonas sp. KMM 701 (α-PsGal), and α-N-acetylgalactosaminidase from the marine bacterium Arenibacter latericius KMM 426T (α-NaGa). All compounds are slow-binding irreversible inhibitors of α-PsGal, but have no effect on α-NaGa. A competitive inhibitor d-galactose protects α-PsGal against the inactivation. The inactivation rate (kinact) and equilibrium inhibition (Ki) constants of monanchomycalin B, monanchocidin A, and normonanchocidin A were 0.166 ± 0.029 min-1 and 7.70 ± 0.62 µM, 0.08 ± 0.003 min-1 and 15.08 ± 1.60 µM, 0.026 ± 0.000 min-1, and 4.15 ± 0.01 µM, respectively. The 2D-diagrams of α-PsGal complexes with the guanidine alkaloids were constructed with "vessel" and "anchor" parts of the compounds. Two alkaloid binding sites on the molecule of α-PsGal are shown. Carboxyl groups of the catalytic residues Asp451 and Asp516 of the α-PsGal active site interact with amino groups of "anchor" parts of the guanidine alkaloid molecules.

Disruption of N-acyl-homoserine lactone-specific signalling and virulence in clinical pathogens by marine sponge bacteria.

In recent years, the marine environment has been the subject of increasing attention from biotechnological and pharmaceutical industries. A combination of unique physicochemical properties and spatial niche-specific substrates, in wide-ranging and extreme habitats, underscores the potential of the marine environment to deliver on functionally novel bioactivities. One such area of ongoing research is the discovery of compounds that interfere with the cell-cell signalling process called quorum sensing (QS). Described as the next generation of antimicrobials, these compounds can target virulence and persistence of clinically relevant pathogens, independent of any growth-limiting effects. Marine sponges are a rich source of microbial diversity, with dynamic populations in a symbiotic relationship. In this study, we have harnessed the QS inhibition (QSI) potential of marine sponge microbiota and through culture-based discovery have uncovered small molecule signal mimics that neutralize virulence phenotypes in clinical pathogens. This study describes for the first time a marine sponge Psychrobacter sp. isolate B98C22 that blocks QS signalling, while also reporting dual QS/QSI activity in the Pseudoalteromonas sp. J10 and ParacoccusJM45. Isolation of novel QSI activities has significant potential for future therapeutic development, of particular relevance in the light of the pending perfect storm of antibiotic resistance meeting antibiotic drug discovery decline.

Extracellular polymeric substances with metal adsorption capacity produced by Pseudoalteromonas sp. MER144 from Antarctic seawater.

The EPS-producing Pseudoalteromonas sp. MER144 was selected among 606 isolates from Antarctic seawater due to its evident slimy appearance on agar plates. The production of EPSs was enhanced by a step-by-step approach varying the carbon source, substrate and NaCl concentrations, temperature, and pH. Optimal conditions for the EPS production resulted at temperature of 4 °C and pH 7, with addition of 2% sucrose (w/v) and 3% NaCl (w/v). EPSs produced under optimal conditions were chemically characterized, resulting in a moderate carbohydrate content (35%), uronic acids (14%), and proteins (12%). Monosaccharide composition was estimated to be Glu:Man:GluN:Ara:GluA:GalA:Gal (1:0.36:0.26:0.06:0.06:0.05:0.03), while the estimated molecular weight was about 250 kDa. The addition of sucrose in the culture medium, by stimulating the EPS production, allowed MER144 to tolerate higher concentrations of mercury and cadmium. This finding was probably dependent on the presence of uronic acids and sulfate groups, which can bind cations, in the extracted EPSs. Monitoring EPS production under optimal conditions at different concentrations of mercury and cadmium revealed that EPS amounts increased at increasing heavy metal concentrations, indicating an adaptation to the stress conditions tested.

Towards smart biocide-free anti-biofilm strategies: Click-based synthesis of cinnamide analogues as anti-biofilm compounds against marine bacteria.

A set of triazole-based analogues of N-coumaroyltyramine was designed to discover potential leads that may help in the control of bacterial biofilms. the most potent compounds act as inhibitors of biofilm development with EC50 closed to ampicillin (EC50 = 11 µM) without toxic effect on bacterial growth even at high concentrations(100 µM).

ISCR2 is associated with the dissemination of multiple resistance genes among Vibrio spp. and Pseudoalteromonas spp. isolated from farmed fish.

58 multiresistant strains representing diverse genera were isolated from farmed fish in an aquaculture facility. Resistant rates of strains harboring ISCR2, an insertion sequence type element, were higher than those in which this element was absent. Full genome sequencing of a Vibrio isolate containing ISCR2 confirmed that it is associated with multiple resistance genes, many of which are of clinical relevance. We describe the structural variation within ISCR2, and its distribution throughout multiple diverse aquatic genera, including Vibrio, Shewenalla, Pseudoalteromonas and Psychrobacter, suggesting the potential role of ISCR2 in disseminating antibiotic resistance. We also observe, and experimentally verify, a novel macrolide resistance gene that is also associated with ISCR2.

Chitosan-zinc oxide nanocomposite coatings for the prevention of marine biofouling.

Marine biofouling is a worldwide problem affecting maritime industries. Global concerns about the high toxicity of antifouling paints have highlighted the need to develop less toxic antifouling coatings. Chitosan is a natural polymer with antimicrobial, antifungal and antialgal properties that is obtained from partial deacetylation of crustacean waste. In the present study, nanocomposite chitosan-zinc oxide (chitosan-ZnO) nanoparticle hybrid coatings were developed and their antifouling activity was tested. Chitosan-ZnO nanoparticle coatings showed anti-diatom activity against Navicula sp. and antibacterial activity against the marine bacterium Pseudoalteromonas nigrifaciens. Additional antifouling properties of the coatings were investigated in a mesocosm study using tanks containing natural sea water under controlled laboratory conditions. Each week for four weeks, biofilm was removed and analysed by flow cytometry to estimate total bacterial densities on the coated substrates. Chitosan-ZnO hybrid coatings led to better inhibition of bacterial growth in comparison to chitosan coatings alone, as determined by flow cytometry. This study demonstrates the antifouling potential of chitosan-ZnO nanocomposite hybrid coatings, which can be used for the prevention of biofouling.

Genetic characterization of plasmid-mediated quinolone resistance gene qnrS2 in Pseudoalteromonas and Shewanella isolates from seawater.

Three qnrS2-containing isolates of Pseudoalteromonas and Shewanella were collected from the seawater samples of Qingdao in China during 2014. They displayed resistance to ampicillin, ciprofloxacin, kanamycin, nalidixic acid and sulfamethoxazole. The qnrS2 genes were identified in the chromosomes of Pseudoalteromonas strains E8 and S16, and in a 140-kb plasmid in Shewanella strain S14, respectively. In addition, two copies of qnrS2 were identified in the strain E8. Sequence analyses revealed that there was an identical DNA segment located in the downstream of qnrS2 in strain S14 and E8, coding for a TetR transcriptional regulator, two putative integrases and a hypothetical protein. However, different genetic structures were identified in the upstream sequences: the terB gene associated with tellurite resistance in the strain S14, and a putative integron with dfrA6 and aadA13 gene cassettes or the Tn7-related gene complex tnsABC in the strain E8. In Pseudoalteromonas strain S16, qnrS2 was bracketed by the endonuclease I and III genes, and the electron transport complex rsxCDGE was located in the upstream sequences. This is the first report of two copies of the qnrS2 gene existing in one bacterial chromosome, and also the first identification of qnrS2 in Shewanella.

Screening of bromotyramine analogues as antifouling compounds against marine bacteria.

Rapid and efficient synthesis of 23 analogues inspired by bromotyramine derivatives, marine natural products, by means of CuSO4-catalysed [3+2] alkyne-azide cycloaddition is described. The final target was then assayed for anti-biofilm activity against three Gram-negative marine bacteria, Pseudoalteromonas ulvae (TC14), Pseudoalteromonas lipolytica (TC8) and Paracoccus sp. (4M6). Most of the synthesised bromotyramine/triazole derivatives are more active than the parent natural products Moloka'iamine (A) and 3,5-dibromo-4-methoxy-ß-phenethylamine (B) against biofilm formation by the three bacterial strains. Some of these compounds were shown to act as non-toxic inhibitors of biofilm development with EC50 < 200 µM without any effect on bacterial growth even at high concentrations (200 µM).

Facile synthesis of BiOI in hierarchical nanostructure preparation and its photocatalytic application to organic dye removal and biocidal effect of bacteria.

Novel BiOI hierarchical nanostructures with porous architecture was prepared at room temperature by an economical, facile and rapid route. The coupling adsorption and degradation efficiency of rhodamine B (RhB), methylene blue (MB), and methyl orange (MO) could achieve 98.7%, 43.2%, 76.9% within 180min in the presence of BiOI, respectively. BiOI hierarchical nanostructures also exhibit excellent effective biocidal performances, and the disinfection rate of the typical biofouling bacteria Bacillus sp. and Pseudoalteromonas sp. could reach 99.9% and 99.8% in 60min, respectively. The circulate photocatalytic degradation of RhB experiment verified the good stability and reusability of BiOI hierarchical nanostructures. Based on active specie trapping experiments, the superoxide radicals (O2(-)) and holes (h(+)) were proved to be the main reactive species in the BiOI-based system. The obviously enhanced coupling adsorption and photocatalytic degradation performance of BiOI hierarchical nanostructures could be mainly attributed to the large surface area (64.5m(2)/g), negatively charged surface and the intrinsic electron structure. This study provides a new strategy to develop novel photocatalysts for water treatment and marine antifouling.

Modulation of violacein production and phenotypes associated with biofilm by exogenous quorum sensing N-acylhomoserine lactones in the marine bacterium Pseudoalteromonas ulvae TC14.

Various phenotypes ranging from biofilm formation to pigment production have been shown to be regulated by quorum sensing (QS) in many bacteria. However, studies of the regulation of pigments produced by marine bacteria in saline conditions and of biofilm-associated phenotypes are scarcer. This study focuses on the demonstration of the existence of a QS communication system involving N-acylhomoserine lactones (AHLs) in the Mediterranean Sea strain Pseudoalteromonas ulvae TC14. We have investigated whether TC14 produces the violacein pigment, and whether intrinsic or exogenous AHLs could influence its production and modulate biofilm-associated phenotypes. Here, we demonstrate that the purple pigment produced by TC14 is violacein. The study shows that in planktonic conditions, TC14 produces more pigment in the medium in which it grows less. Using different approaches, the results also show that TC14 does not produce intrinsic AHLs in our conditions. When exogenous AHLs are added in planktonic conditions, the production of violacein is upregulated by C6-, C12-, 3-oxo-C8 and 3-oxo-C12-HSLs (homoserine lactones), and downregulated by 3-oxo-C6-HSL. In sessile conditions, 3-oxo-C8-HSL upregulates the production of violacein. The study of the biofilm-associated phenotypes shows that oxo-derived-HSLs decrease adhesion, swimming and biofilm formation. While 3-oxo-C8 and 3-oxo-C12-HSLs decrease both swimming and adhesion, 3-oxo-C6-HSLs decrease not only violacein production in planktonic conditions but also swimming, adhesion and more subtly biofilm formation. Therefore, TC14 may possess a functional LuxR-type QS receptor capable of sensing extrinsic AHLs, which controls violacein production, motility, adhesion and biofilm formation.

Sulfonamide inhibition studies of the γ-carbonic anhydrase from the Antarctic bacterium Pseudoalteromonas haloplanktis.

The Antarctic bacterium Pseudoalteromonas haloplanktis encodes for a γ-class carbonic anhydrase (CA, EC 4.2.1.1), which was cloned, purified and characterized. The enzyme (PhaCAγ) has a good catalytic activity for the physiologic reaction of CO2 hydration to bicarbonate and protons, with a k(cat) of 1.4×10(5) s(-1) and a k(cat)/K(m) of 1.9×10(6) M(-1)×s(-1). A series of sulfonamides and a sulfamate were investigated as inhibitors of the new enzyme. Methazolamide and indisulam showed the best inhibitory properties (K(I)s of 86.7-94.7 nM). This contribution shed new light on γ-CAs inhibition profiles with a relevant class of pharmacologic agents.

Polysiloxane-based block copolymers with marine bacterial anti-adhesion properties.

Di- and triblock copolymers based on tert-butyldimethylsilyl methacrylate (MASi) and poly(dimethylsiloxane) (PDMS) macro-RAFT agents were synthesized resulting in copolymers with predictable molar masses and low dispersities (D < 1.2). The block copolymers exhibited two glass transition temperatures, corresponding to the PDMS- and poly(tert-butyldimethylsilyl methacrylate) (PMASi)-enriched phases, respectively. Contact angle measurements revealed the influence of the copolymer composition on their surface free energy, with block copolymers exhibiting surface free energies as low as 15.0 mJ m(-2). A laboratory assay using 96-well plates was used to assess the activity of the block copolymers against two marine bacteria (Pseudoalteromonas sp. and Shewanella sp.) isolated from the Mediterranean Sea. Coatings based on PDMS-based block copolymers demonstrated anti-adhesive performances against the two strains better than that of the coating containing only PMASi-based polymers. Coatings based on diblock copolymers demonstrated antifouling performances in the field that were better than those of the corresponding coatings containing triblock copolymers. Results of both lab and field assays showed that the antifouling properties were related to coatings possessing the highest receding water contact angle.