BackgroundVibrio pathogens are causative agents of mid-culture outbreaks, and early mortality syndrome and secondary aetiology of most dreadful viral outbreaks in shrimp aquaculture. Among the pathogenic vibrios group, Vibrio alginolyticus and V. harveyi are considered as the most significant ones in the grow-out ponds of giant black tiger shrimp Penaeus monodon in India. Use of antibiotics was banned in many countries due to the emergence of antibiotic-resistant strains and accumulation of residual antibiotics in harvested shrimp. There is an urgent need to consider the use of alternative antibiotics for the control of vibriosis in shrimp aquaculture. Biofilm formation is a pathogenic and/or establishment mechanism of Vibrio spp. This study aims to develop novel safe antibiofilm and/or antiadhesive process using PHB to contain vibrios outbreaks in shrimp aquaculture.ResultsIn this study a poly-hydroxy butyrate (PHB) polymer producing bacterium Brevibacterium casei MSI04 was isolated from a marine sponge Dendrilla nigra and production of PHB was optimized under submerged-fermentation (SmF) conditions. The effect of carbon, nitrogen and mineral sources on PHB production and enhanced production of PHB by response surface methods were demonstrated. The maximum PHB accumulation obtained was 6.74 g/L in the optimized media containing 25 g/L starch as carbon source, 96 h of incubation, 35°C and 3% NaCl. The highest antiadhesive activity upto 96% was recorded against V. vulnificus, and V. fischeri, followed by 92% against V. parahaemolyticus and V. alginolyticus and 88% inhibition was recorded against V. harveyi.ConclusionIn this study, a thermostable biopolymer was chemically characterized as PHB based on 1HNMR spectra, FT-IR and GC-MS spectra. The NMR spectra revealed that the polymer was an isocratic homopolymer and it also confirmed that the compound was PHB. The antiadhesive activity of PHB was determined in microtitre plate assay and an effective concentration (EC) of PHB (200 μl containing 0.6 mg PHB) was confirmed by confocal laser scanning microscopic analysis of vibrio biofilm on pre-treated glass and polystyrene surfaces. This is a first report on anti-adhesive activity of PHB against prominent vibrio pathogens in shrimp aquaculture.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0114-3) contains supplementary material, which is available to authorized users.

BackgroundIron is an essential element in several pathways of microbial metabolism, and therefore low iron toxicity is expected on the usage of Fe nanoparticles (NPs). This study aims to determine the effect of Fe NPs on biosurfactant production by marine actinobacterium Nocardiopsis sp. MSA13A under solid state culture. Foam method was used in the production of Fe NPs which were long and fiber shaped in nature.ResultsThe SEM observation showed non toxic nature of Fe NPs as no change in the morphology of the filamentous structure of Nocardiopsis MSA13A. The production of biosurfactant by Nocardiopsis MSA13A under solid state culture supplemented with Fe NPs increased to 80% over control. The biosurfactant produced by Nocardiopsis MSA13A was characterized as glycolipid derivative which effectively disrupted the pre-formed biofilm of Vibrio pathogen.ConclusionThe use of metal NPs as supplement would reduce the impact of non-metallic ions of the metal salts in a fermentation process. This would ultimately useful to achieve greener production process for biosurfactants. The present results are first report on the optimization of biosurfactant production under SSC using Fe NPs.

Luminescent vibrios are ubiquitous in the marine environment and are the causative agents of vibriosis and mass mortality in many aquatic animals. In aquatic environments, treatments cannot be limited to the diseased population alone, therefore treatment of the entire aquatic system is the only possible approach. Thus, the use of antibiotics to treat part of the infected animals requires a dose based on the entire biomass, which results in the treatment of uninfected animals as well as non-target normal microbial flora. A treatment method based on anti-virulence or quorum quenching has recently been proposed as an effective treatment strategy for aquatic animals. Polyhydroxy butyrates (PHB) are bacterial storage molecules, which accumulate in cells under nutritional stress. The degradation of PHB releases short-chain β-hydroxy butyric acid, which may act as anti-infective molecule. To date, there is very limited information on the potential anti-infective and anti-virulence mechanisms involving PHB. In this study, we aim to examine the effect of PHB on inhibition of the virulence cascade of Vibrio such as biofilm formation, luminescence, motility behaviour, haemolysin and quorum sensing. A luminescent Vibrio PUGSK8, tentatively identified as Vibrio campbellii PUGSK8 was tested in vitro for production of extracellular virulence factors and then established as a potential shrimp pathogen based on in vivo challenge experiments. The ability of Vibrio PUGSK8 to form biofilms and the effect of PHB on biofilm formation was tested in a 96-well microtitre-plate assay system. The motility behaviour of Vibrio PUGSK8 was evaluated using twitching, swimming and swarming plate assays. Reporter strains such as Chromobacterium violaceum CV026 and Agrobacterium tumefaciens were used to detect quorum-sensing molecules. Gas chromatography–mass spectrometry spectral analysis was performed to elucidate the fragmentation pattern and structure of N-hexanoyl homoserine lactone. PHB depolymerase activity in Vibrio PUGSK8 was quantified as the amount of the enzyme solution to hydrolyse 1 μg of PHB per min. An in vivo challenge experiment was performed using a gnotobiotic Artemia assay. Of the 27 isolates tested, the Vibrio PUGSK8 strain was selected for target-specific assays based on the high intensity of luminescence and production of virulence factors. The virulence cascade detected in Vibrio PUGSK8 include luminescence, motility behaviour, biofilm formation, quorum sensing and haemolysin production. Thus inhibition/degradation of the virulence cascade would be an effective approach to contain Vibrio infections in aquatic animals. In this report, we demonstrate that the degradation intermediate of PHB effectively inhibits biofilm formation, luminescence, motility behaviour, haemolysin production and the N-acyl-homoserine lactone (AHL)-mediated quorum-sensing pathway in PUGSK8. Interestingly, the growth of Vibrio PUGSK8 remains unaffected in the presence of PHB, with PHB degradation being detected in the media. PHB depolymerase activity...

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Amongst the present water treatment technologies membrane separation is gaining focus nowadays. Emulsion liquid membrane is one of the advanced strategies used for pollution removal from waste water in which the use of biosurfactant makes this approach eco-friendly technique. The norfloxacin belongs to the fluoroquinolones class of antibiotic and extensively used for urinary tract infections, which is the model pollutant in this study. Saponin was solvent extracted from soapnuts and used as biosurfactant. The main and interactive effects of parameters such as saponin concentration (0.01-0.03 g/100 mL), NaOH concentration (0.25-0.75 M) and Initial Norfloxacin concentration (25-75 mg/L) were investigated using Response Surface Methodology (RSM)-Box Behnken Design (BBD) and Artificial Neural Networks (ANN) design. The results suggested that developed 3-8-1 ANN model evaluated in terms of performance measure (R 2 : 0.9806, Chi square: 0.02) and error functions (RMSE: 0.11, MAE: 0.09, SEP: 1.16, MPE: 1.76) demonstrated superiority more precisely than the RSM model in terms of both data fitting and prediction capability. The optimal conditions for ELM were found to be: initial norfloxacin concentration 67.65 mg/L, saponin concentration 0.021 mg/100 mL, and NaOH concentration 0.36 M and the extraction efficiency at these parameter settings would be 91.27%.

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Biofilm-forming pathogens are potential threat to indwelling medical devices and infectious diseases. Management of device-associated Candida infections remains to be challenging to the existing drug discovery platforms. The available antifungal drugs are effective on the control of free-living pathogens but not effective on the biofilm-film forming pathogens. Thus antifungal drug synergized with antibiofilm agent would be an effective strategy to treat Candida biofilm.The mature C. albicans biofilms are anchored by complex architecture in terms of distribution of fungal cells stabilized by exocellular polymeric substances. Findings of the present study bring out a new insight on the possible development of enterococci probiotics and/or its expolysaccharide (EPS) as synergistic with existing antifungal drugs to treat biofilm infections.The probiont Enterococcus faecalis MSI12 was picked from 142 seawater isolates screened for EPS production using congo red plate assay. The probiotic characteristics of the isolate MSI12 was evaluated based on the temperature, pH, acid tolerance, autoaggregation, hydrophobicity and antioxidant activity. The biofilm disruption ability of the lyophilized EPS was determined in microtitre plate assay using fluconazole as reference drug. The scanning electron microscope and confocal laser scanning microscope images were used for analysis of antibiofilm activity. The cell viability of E. faecalisMSI12 was very high at higher temperature, acidic pH, bile salt and salt concentration when compared to the reference strain Lactobacillus plantarum. Therefore the strain MSI12 might survive in the niche like human gut without prebiotics. The EPS from Enterococcus sp. MSI12 showed significant reduction of treated Candida biofilm. The antibiofilm potential of EPS was very stronger than standard antifungal drug fluconazole. This study revealed that biofilm disruption/control using a probiontEPS could deliver a synergistic approach as the probiotic strain can colonize in the host to prevent the formation of candida biofilms.