Antagonistic and enzymatic activities of Bacillus species isolated from the fish gastrointestinal tract as potential probiotics use in Artemia culture.

Probiotics have been used successfully in aquaculture to enhance disease resistance, nutrition, and/or growth of cultured organisms. Six strains of Bacillus were isolated from the intestinal tracts of fish and recognised by conventional biochemical traits. The six isolated strains were Bacillus cereus and Bacillus subtilis using MALDI-TOF-MS technique. The probiotic properties of these Bacillus strains were studied. The tested bacillus strains exhibit antibacterial activity against the different pathogens. The strain S5 gave the important inhibition zones against most pathogens (20.5, 20.33, 23, and 21 mm against Vibrio alginolyticus, Vibrio parahaemolyticus, Staphylococcus aureus, and Salmonella typhimurium, respectively). According to our results, all Bacillus strains have extracellular components that can stop pathogenic bacteria from growing. The enzymatic characterization showed that the tested strains can produce several biotechnological enzymes such as α-glucosidase, naphtol-AS-BI-Phosphohydrolase, esterase lipase, acid phosphatase, alkaline phosphatase, amylase, lipase, caseinase, and lecithinase. All Bacillus strains were adhesive to polystyrene. The adding Bacillus strains to the Artemia culture exerted significantly greater effects on the survival of Artemia. The challenge test on Artemia culture showed that the protection against pathogenic Vibrio was improved. These findings allow us to recommend the examined strains as prospective probiotic options for the Artemia culture, which will be used as food additives to improve the culture conditions of crustacean larvae and marine fish.

Bacteriophages to control Vibrio alginolyticus in live feeds prior to their administration in larviculture.

AIMS: This study aimed to evaluate the efficiency of two phages [VB_VaC_TDDLMA (phage TDD) and VB_VaC_SRILMA (phage SRI)] alone and in a cocktail to control Vibrio alginolyticus in brine shrimp before their administration in larviculture. METHODS AND RESULTS: Phages were isolated from seawater samples and characterized by host spectrum, growth parameters, adsorption rate, genomic analysis, and inactivation efficiency. Both phages belong to the Caudoviricetes class and lack known virulence or antibiotic-resistance genes. They exhibit specificity, infecting only their host, V. alginolyticus CECT 521. Preliminary experiments in a culture medium showed that phage TDD (reduction of 5.8 log CFU ml-1 after 10 h) outperformed phage SRI (reduction of 4.6 log CFU ml-1 after 6 h) and the cocktail TDD/SRI (reduction of 5.2 log CFU ml-1 after 8 h). In artificial marine water experiments with Artemia franciscana, both single phage suspensions and the phage cocktail, effectively inactivated V. alginolyticus in culture water (reduction of 4.3, 2.1, and 1.9 log CFU ml-1 for phages TDD, SRI, and the phage cocktail, respectively, after 12 h) and in A. franciscana (reduction of 51.6%, 87.3%, and 85.3% for phages TDD, SRI, and the phage cocktail, respectively, after 24 h). The two phages and the phage cocktail did not affect A. franciscana natural microbiota or other Vibrio species in the brine shrimp. CONCLUSIONS: The results suggest that phages can safely and effectively control V. alginolyticus in A. franciscana prior to its administration in larviculture.

Indole-3-acetic acid increases the survival of brine shrimp challenged with vibrios belonging to the Harveyi clade.

Vibrios belonging to the Harveyi clade (including closely related species such as Vibrio campbellii, Vibrio harveyi and Vibrio parahaemolyticus) are important pathogens of aquatic organisms. In this study, we investigated the use of indole-3-acetic acid to control disease caused by Harveyi clade vibrios. Indole-3-acetic acid, which can be produced by various seaweeds and microalgae, was added to the rearing water of brine shrimp larvae challenged with 12 different Harveyi clade Vibrio strains. Indole-3-acetic acid significantly decreased the virulence of 10 of the strains without any effect on their growth. The latter is important as it will minimize the selective pressure for resistance development. The survival rate of brine shrimp larvae increased from 1.2-fold to 4.8-fold upon treatment with 400 µM indole-3-acetic acid. Additionally, indole-3-acetic acid significantly decreased the swimming motility in 10 of the strains and biofilm formation in eight of the strains. The mRNA levels of the pirA and pirB toxin genes were decreased to 46% and 42% by indole-3-acetic acid in the AHPND-causing strain V. parahaemolyticus M0904. Hence, our data demonstrate that indole-3-acetic acid has the potential to be an effective virulence inhibitor to control infections in aquaculture.

Insights into a Pyruvate Sensing and Uptake System in Vibrio campbellii and Its Importance for Virulence.

Pyruvate is a key metabolite in living cells and has been shown to play a crucial role in the virulence of several bacterial pathogens. The bioluminescent Vibrio campbellii, a severe infectious burden for marine aquaculture, excretes extraordinarily large amounts of pyruvate during growth and rapidly retrieves it by an as-yet-unknown mechanism. We have now identified the responsible pyruvate transporter, here named BtsU, and our results show that it is the only pyruvate transporter in V. campbellii. Expression of btsU is tightly regulated by the membrane-integrated LytS-type histidine kinase BtsS, a sensor for extracellular pyruvate, and the LytTR-type response regulator BtsR. Cells lacking either the pyruvate transporter or sensing system show no chemotactic response toward pyruvate, indicating that intracellular pyruvate is required to activate the chemotaxis system. Moreover, pyruvate sensing and uptake were found to be important for the resuscitation of V. campbellii from the viable but nonculturable state and the bacterium's virulence against brine shrimp larvae. IMPORTANCE Bacterial infections are a serious threat to marine aquaculture, one of the fastest growing food sectors on earth. Therefore, it is extremely important to learn more about the pathogens responsible, one of which is Vibrio campbellii. This study sheds light on the importance of pyruvate sensing and uptake for V. campbellii, and reveals that the bacterium possesses only one pyruvate transporter, which is activated by a pyruvate-responsive histidine kinase/response regulator system. Without the ability to sense or take up pyruvate, the virulence of V. campbellii toward gnotobiotic brine shrimp larvae is strongly reduced.

Rearing water microbiomes in white leg shrimp (Litopenaeus vannamei) larviculture assemble stochastically and are influenced by the microbiomes of live feed products.

The development of effective management strategies to reduce the occurrence of diseases in aquaculture is hampered by the limited knowledge on the microbial ecology of these systems. In this study, the dynamics and dominant community assembly processes in the rearing water of Litopenaeus vannamei larviculture tanks were determined. Additionally, the contribution of peripheral microbiomes, such as those of live and dry feeds, to the rearing water microbiome were quantified. The community assembly in the hatchery rearing water over time was dominated by stochasticity, which explains the observed heterogeneity between replicate cultivations. The community undergoes two shifts that match with the dynamics of the algal abundances in the rearing water. Source tracking analysis revealed that 37% of all bacteria in the hatchery rearing water were introduced either by the live or dry feeds, or during water exchanges. The contribution of the microbiome from the algae was the largest, followed by that of the Artemia, the exchange water and the dry feeds. Our findings provide fundamental knowledge on the assembly processes and dynamics of rearing water microbiomes and illustrate the crucial role of these peripheral microbiomes in maintaining health-promoting rearing water microbiomes.

Isolation and characterization of vibriophage vB_Vc_SrVc9: an effective agent in preventing Vibrio campbellii infections in brine shrimp nauplii (Artemia franciscana).

AIMS: This study describes the physicochemical and genomic characterization of phage vB_Vc_SrVc9 and its potential for phage therapy application against a pathogenic Vibrio campbellii strain. METHODS AND RESULTS: A lytic phage vB_Vc_SrVc9 against V. campbellii was isolated from shrimp farm sediment, and characterized physicochemical and genomically. The use of vB_Vc_SrVc9 phage increased the survival in brine shrimp Artemia franciscana and reduced presumptive V. campbellii to nondetectable numbers. Genomic analysis showed a genome with a single contig of 43·15 kb, with 49 predicted genes and no tRNAs, capable of recognizing and generating complete inhibition zones of three Vibrio sp. CONCLUSIONS: To our knowledge vB_Vc_SrVc9 is a lytic phage that could be used against Vibrio infections, reducing vibrio presence without any apparent impact over the natural microbiota at the family level in 28 libraries tested. SIGNIFICANCE AND IMPACT OF THE STUDY: vB_Vc_SrVC9 is a novel phage and ecofriendly alternative for therapeutic applications and biotechnological purposes because is stable at different environmental conditions, has the potential to eliminate several strains, and has a short latent period with a good burst size. Therefore, the use of phages, which are natural killers of bacteria, represents a promising strategy to reduce the mortality of farmed organisms caused by pathogenic bacteria.

Multifunctional carbonized nanogels to treat lethal acute hepatopancreatic necrosis disease.

BACKGROUND: Shrimp aquaculture has suffered huge economic losses over the past decade due to the outbreak of acute hepatopancreatic necrosis disease (AHPND), which is mainly caused by the bacteria Vibrio parahaemolyticus (V. parahaemolyticus) with the virulence pVA1 plasmid, which encodes a secretory photorhabdus insect-related (Pir) toxin composed of PirA and PirB proteins. The Pir toxin mainly attacks the hepatopancreas, a major metabolic organ in shrimp, thereby causing necrosis and loss of function. The pandemic of antibiotic-resistant strains makes the impact worse. METHODS: Mild pyrolysis of a mixture of polysaccharide dextran 70 and the crosslinker 1,8-diaminooctane at 180 â„ƒ for 3 h to form carbonized nanogels (DAO/DEX-CNGs) through controlled cross-linking and carbonization. The multifunctional therapeutic CNGs inherit nanogel-like structures and functional groups from their precursor molecules. RESULTS: DAO/DEX-CNGs manifest broad-spectrum antibacterial activity against Vibrio parahaemolyticus responsible for AHPND and even multiple drug-resistant strains. The polymer-like structures and functional groups on graphitic-carbon within the CNGs exhibit multiple treatment effects, including disruption of bacterial membranes, elevating bacterial oxidative stress, and neutralization of PirAB toxins. The inhibition of Vibrio in the midgut of infected shrimp, protection of hepatopancreas tissue from Pir toxin, and suppressing overstimulation of the immune system in severe V. parahaemolyticus infection, revealing that CNGs can effectively guard shrimp from Vibrio invasion. Moreover, shrimps fed with DAO/DEX-CNGs were carefully examined, such as the expression of the immune-related genes, hepatopancreas biopsy, and intestinal microbiota. Few adverse effects on shrimps were observed. CONCLUSION: Our work proposes brand-new applications of multifunctional carbon-based nanomaterials as efficient anti-Vibrio agents in the aquatic industry that hold great potential as feed additives to reduce antibiotic overuse in aquaculture.

Environmental conditions steer phenotypic switching in acute hepatopancreatic necrosis disease-causing Vibrio parahaemolyticus, affecting PirAVP /PirBVP toxins production.

Bacteria in nature are widely exposed to differential fluid shears which are often a trigger for phenotypic switches. The latter mediates transcriptional and translation remodelling of cellular metabolism impacting among others virulence, antimicrobial resistance and stress resistance. In this study, we evaluated the role of fluid shear on phenotypic switch in an acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio parahaemolyticus M0904 strain under both in vitro and in vivo conditions. The results showed that V. parahaemolyticus M0904 grown at lower shaking speed (110 rpm constant agitation, M0904/110), causing low fluid shear, develop cellular aggregates or floccules. These cells increased levan production (as verified by concanavalin binding) and developed differentially stained colonies on Congo red agar plates and resistance to antibiotics. In addition, the phenotypic switch causes a major shift in the protein secretome. At 120 rpm (M0904/120), PirAVP /PirBVP toxins are mainly produced, while at 110 rpm PirAVP /PirBVP toxins production is stopped and an alkaline phosphatase (ALP) PhoX becomes the dominant protein in the protein secretome. These observations are matched with a very strong reduction in virulence of M0904/110 towards two crustacean larvae, namely, Artemia and Macrobrachium. Taken together, our study provides substantial evidence for the existence of two phenotypic forms in AHPND V. parahaemolyticus strain displaying differential phenotypes. Moreover, as aerators and pumping devices are frequently used in shrimp aquaculture facilities, they can inflict fluid shear to the standing microbial agents. Hence, our study could provide a basis to understand the behaviour of AHPND-causing V. parahaemolyticus in aquaculture settings and open the possibility to monitor and control AHPND by steering phenotypes.

Identification and Characterization of a Newly Isolated Chitinase-Producing Strain Bacillus licheniformis SSCL-10 for Chitin Degradation.

Chitinases or chitinolytic enzymes have different applications in the field of medicine, agriculture, and industry. The present study is aimed at developing an effective hyperchitinase-producing mutant strain of novel Bacillus licheniformis. A simple and rapid methodology was used for screening potential chitinolytic microbiota by chemical mutagenesis with ethylmethane sulfonate and irradiation with UV. There were 16 mutant strains exhibiting chitinase activity. Out of the chitinase-producing strains, the strain with maximum chitinase activity was selected, the protein was partially purified by SDS-PAGE, and the strain was identified as Bacillus licheniformis (SSCL-10) with the highest specific activity of 3.4 U/mL. The induced mutation model has been successfully implemented in the mutant EMS-13 (20.2 U/mL) that produces 5-6-fold higher yield of chitinase, whereas the mutant UV-11 (13.3 U/mL) has 3-4-fold greater chitinase activity compared to the wild strain. The partially purified chitinase has a molecular weight of 66 kDa. The wild strain (SSCL-10) was identified as Bacillus licheniformis using 16S rRNA sequence analysis. This study explores the potential applications of hyperchitinase-producing bacteria in recycling and processing chitin wastes from crustaceans and shrimp, thereby adding value to the crustacean industry.

Characterization of Aquimarina hainanensis isolated from diseased mud crab Scylla serrata larvae in a hatchery.

Mass mortality due to necrosis signs occurred in hatchery-reared zoea stage larvae of the mud crab Scylla serrata in Okinawa, Japan, and a causative bacterium was isolated. In this study, we identified and characterized the bacterium by genome analysis, biochemical properties and pathogenicity. The bacterium was a Gram-negative, non-motile, long rod, forming yellow colonies on a marine agar plate. It grew at 20-33°C (not at 37°C) and degraded chitin and gelatin. Phylogenetic analysis of the 16S rRNA gene sequence identified the bacterium as Aquimarina hainanensis. Genome sequence data obtained from Illumina MiSeq generated 29 contigs with 3.56 Mbp in total length and a G + C content of 32.5%. The predicted 16 chitinase genes, as putative virulence factors, had certain homologies with those of genus Aquimarina. Experimental infection with the bacterium conducted on larvae of four crustacean species, brine shrimp Artemia franciscana, freshwater shrimp Caridina multidentata, swimming crab Portunus trituberculatus and mud crab S. serrata, revealed that this bacterium was highly virulent to these species. The present study suggests that the bacterium caused mass mortality in mud crab seed production was A. hainanensis and can be widely pathogenic to crustaceans.

Structural and functional characterization of a modified legionaminic acid involved in glycosylation of a bacterial lipopolysaccharide.

Nonulosonic acids (NulOs) are a diverse family of α-keto acid carbohydrates present across all branches of life. Bacteria biosynthesize NulOs among which are several related prokaryotic-specific isomers and one of which, N-acetylneuraminic acid (sialic acid), is common among all vertebrates. Bacteria display various NulO carbohydrates on lipopolysaccharide (LPS), and the identities of these molecules tune host-pathogen recognition mechanisms. The opportunistic bacterial pathogen Vibrio vulnificus possesses the genes for NulO biosynthesis; however, the structures and functions of the V. vulnificus NulO glycan are unknown. Using genetic and chemical approaches, we show here that the major NulO produced by a clinical V. vulnificus strain CMCP6 is 5-N-acetyl-7-N-acetyl-d-alanyl-legionaminic acid (Leg5Ac7AcAla). The CMCP6 strain could catabolize modified legionaminic acid, whereas V. vulnificus strain YJ016 produced but did not catabolize a NulO without the N-acetyl-d-alanyl modification. In silico analysis suggested that Leg5Ac7AcAla biosynthesis follows a noncanonical pathway but appears to be present in several bacterial species. Leg5Ac7AcAla contributed to bacterial outer-membrane integrity, as mutant strains unable to produce or incorporate Leg5Ac7AcAla into the LPS have increased membrane permeability, sensitivity to bile salts and antimicrobial peptides, and defects in biofilm formation. Using the crustacean model, Artemia franciscana, we demonstrate that Leg5Ac7AcAla-deficient bacteria have decreased virulence potential compared with WT. Our data indicate that different V. vulnificus strains produce multiple NulOs and that the modified legionaminic acid Leg5Ac7AcAla plays a critical role in the physiology, survivability, and pathogenicity of V. vulnificus CMCP6.

Transmission of Mycobacterium chelonae and Mycobacterium marinum in laboratory zebrafish through live feeds.

The zebrafish (Danio rerio) is a popular vertebrate model organism used in a wide range of research fields. Mycobacteriosis, caused by Mycobacterium species, is particularly concerning because it is a common disease associated with chronic infections in these fish. Infections are also a source of uncontrolled experimental variance that may influence research results. Live feeds for zebrafish are common and include paramecia (Paramecium caudatum), brine shrimp (Artemia franciscana) and rotifers (Branchionus spp.). Although nutritionally beneficial, live feeds may pose a biosecurity risk. In this study, we investigate transmission of Mycobacterium chelonae and Mycobacterium marinum through these three live feeds. We show that all three live feeds ingest both M. marinum and M. chelonae and can transmit mycobacterial infections to zebrafish. This observation emphasizes the need for live feeds to be included in the consideration of potential biosecurity risks. This study is of importance to other beyond the zebrafish community, including those of additional aquatic models and those using live feeds for other types of aquaculture.

Effect of biosurfactant derived from Vibrio natriegens MK3 against Vibrio harveyi biofilm and virulence.

Vibrio harveyi is a marine luminous pathogen, which causes biofilm-mediated infections, pressures the search for an innovative alternate approach to strive against vibriosis in aquaculture. This study anticipated to explore the effect of glycolipid biosurfactant as an antipathogenic against V. harveyi to control vibriosis. In this study, 27 bacterial strains were isolated from marine soil sediments. Out of these, 11 strains exhibited surfactant activity and the strain MK3 showed high emulsification index. The potent strain was identified as Vibrio natriegens and named as V. natriegens MK3. The extracted biosurfactant was purified using high-performance liquid chromatography and it was efficient to decrease the surface tension of the growth medium up to 21 mN/m. The functional group and composition of the biosurfactant were determined by Fourier-transform infrared spectroscopy and nuclear magnetic resonance spectroscopy spectral studies and the nature of the biosurfactant was identified as glycolipid. The surfactant was capable of reducing the biofilm formation, bioluminescence, extracellular polysaccharide synthesis, and quorum sensing in marine shrimp pathogen V. harveyi. The antagonistic effect of biosurfactant was evaluated against V. harveyi-infected brine shrimp Artemia salina. This study reveals that biosurfactant can be considered for the management of biofilm-related aquatic infections.

Interactions among yeast and probiotic bacteria enhance probiotic properties and metabolism offering augmented protection to Artemia franciscana against Vibrio anguillarum.

The interactions of the probiotics Bacillus subtilis, Lactococcus lactis and Lactobacillus plantarum with the yeast Saccharomyces cerevisiae were examined in terms of probiotic and biochemical characteristics. Yeast supernatant had a positive effect on the aggregation biofilm formation capacity and hydrophobicity of probiotics, and resulted in increased lactic acid levels, reduced pH values as well as lower RS and FAN levels of probiotics. The effect of probiotics supernatants on yeast was more complex but best results were obtained in the yeast: probiotic CFS ratio of 1:2 for B. subtilis and of 2:1 for the other probiotics. The observed effects depended on the volume ratio of the cell free supernatant to the culture it was applied on. Best results were obtained by the volume ratio probiotic: yeast of (2:1) for B. subtilis and of (1:2) probiotic: yeast for L. plantarum and L. lactis. These ratios were used for further evaluation in vitro against V. anguillarum, resulting in reduced survival and attachment properties of the pathogen. Moreover, the administration of the corresponding combination of bacteria and yeast to Artemia nauplii greatly improved their survival following a challenge with the pathogen. Our results demonstrate that yeast enhances the protective effect of probiotics in a strain specific manner.

Efficacy of cell free supernatant from Bacillus licheniformis in protecting Artemia salina against Vibrio alginolyticus and Pseudomonas gessardii.

Bacterial diseases are widespread in aquaculture farms and causative agents often adapt to biofilm mode of growth. These biofilms are detrimental to aquaculture species as they resist antibiotics and other agents that are used to control them. Two bacterial pathogens isolated from infected prawn samples were identified as Vibrio alginolyticus and Pseudomonas gessardii on the basis of morphological features, biochemical characteristics, 16S r RNA gene sequencing and phylogenetic analysis. Their pathogenic nature was confirmed by performing in vivo challenge experiments using Artemia salina as a model system. Seven days post infection, the mortality observed with V. alginolyticus and P. gessardii was 97 ±â€¯4.08% and 77.5 ±â€¯5.24%, respectively. The isolates formed extensive biofilms on polystyrene and glass surfaces. These infections could be controlled in an effective manner by using the cell free supernatant (CFS) of a tropical marine epizoic strain of Bacillus licheniformis D1 that is earlier reported to contain an antimicrobial protein (BLDZ1). The CFS inhibited biofilms in an efficient manner (82.35 ±â€¯1.69 and 82.52 ±â€¯1.11% for V. alginolyticus and P. gessardii, respectively) on co-incubation. In addition, pre-formed biofilms of V. alginolyticus and P. gessardii were also removed (84.53 ±â€¯1.26 and 67.08 ±â€¯1.43%, respectively). Fluorescence and scanning electron microscopic studies confirmed the antibiofilm potential of this protein on glass surfaces. The antibiofilm nature was due to the anti-adhesion and antimicrobial properties exhibited by the CFS. Treatment of A. salina with CFS (6 h prior to infections) was effective in protecting larvae against infections by field isolates. This study highlights the significance of marine natural products in providing alternative biofilm controlling agents to tackle infections and decreasing the usage of antibiotics in aquaculture settings.

Detection and quantification of Hepatobacter penaei bacteria (NHPB) by new PCR and quantitative PCR assays.

Necrotizing hepatopancreatitis (NHP) is a bacterial disease caused by a Gram-negative bacterium classified as Hepatobacter penaei. H. penaei affects cultured penaeid shrimp in several countries from the western hemisphere, including the USA, and most Central and South American countries that farm shrimp. The current PCR and quantitative PCR (qPCR) assays based on the amplification of the 16S rRNA gene developed at the University of Arizona Aquaculture Pathology Laboratory (UAZ-APL) are the only techniques recommended in the World Organisation for Animal Health (OIE) manual for H. penaei detection. Although these techniques are quite sensitive and specific to H. penaei detection in shrimp, in recent years, rare non-specific amplifications have been observed in the end-point PCR when screening for H. penaei in Artemia cyst samples submitted to the UAZ-APL. To avoid these non-specific amplifications, new end-point PCR and qPCR assays were developed based on the H. penaei flagella gene, flgE. Unlike the current OIE methods, the new H. penaei PCR assay did not provide any non-specific amplification, and the qPCR assay had a detection limit of 100 copies and a log-linear range up to 108 copies. Because the previous PCR-based assay using the 16S rRNA was showing non-specific amplification, the new non-specific product of around 400 bp was sequenced to determine its identity. A phylogenetic analysis revealed 2 clusters of H. penaei: Ecuador and Central-North America. This information will enable us to determine the genetic diversity and possible origin of H. penaei and emphasizes the need to evaluate H. penaei PCR detection methods to avoid inaccurate detection of H. penaei.

Cyclodepsipeptides and Sesquiterpenes from Marine-Derived Fungus Trichothecium roseum and Their Biological Functions.

On the basis of the 'one strain, many compounds' (OSMAC) strategy, chemical investigation of the marine-derived fungus Trichothecium roseum resulted in the isolation of trichomide cyclodepsipeptides (compounds 1⁻4) from PDB medium, and destruxin cyclodepsipeptides (compounds 5⁻7) and cyclonerodiol sesquiterpenes (compounds 8⁻10) from rice medium. The structures and absolute configurations of novel (compounds 1, 8, and 9) and known compounds were elucidated by extensive spectroscopic analyses, X-ray crystallographic analysis, and ECD calculations. All isolated compounds were evaluated for cytotoxic, nematicidal, and antifungal activities, as well as brine shrimp lethality. The novel compound 1 exhibited significant cytotoxic activities against the human cancer cell lines MCF-7, SW480, and HL-60, with IC50 values of 0.079, 0.107, and 0.149 µM, respectively. In addition, it also showed significant brine shrimp lethality, with an LD50 value of 0.48 µM, and moderate nematicidal activity against Heterodera avenae, with an LC50 value of 94.9 µg/mL. This study constitutes the first report on the cytotoxic and nematicidal potential of trichomide cyclodepsipeptides.

Indole signalling and (micro)algal auxins decrease the virulence of Vibrio campbellii, a major pathogen of aquatic organisms.

Vibrios belonging to the Harveyi clade are major pathogens of marine vertebrates and invertebrates, causing major losses in wild and cultured organisms. Despite their significant impact, the pathogenicity mechanisms of these bacteria are not yet completely understood. In this study, the impact of indole signalling on the virulence of Vibrio campbellii was investigated. Elevated indole levels significantly decreased motility, biofilm formation, exopolysaccharide production and virulence to crustacean hosts. Indole furthermore inhibited the three-channel quorum sensing system of V. campbellii, a regulatory mechanism that is required for full virulence of the pathogen. Further, indole signalling was found to interact with the stress sigma factor RpoS. Together with the observations that energy-consuming processes (motility and bioluminescence) are downregulated, and microarray-based transcriptomics demonstrating that indole decreases the expression of genes involved in energy and amino acid metabolism, the data suggest that indole is a starvation signal in V. campbellii. Finally, it was found that the auxins indole-3-acetic acid and indole-3-acetamide, which were produced by various (micro)algae sharing the aquatic environment with V. campbellii, have a similar effect as observed for indole. Auxins might, therefore, have a significant impact on the interactions between vibrios, (micro)algae and higher organisms, with major ecological and practical implications.

Defense systems in developing Artemia franciscana nauplii and their modulation by probiotic bacteria offer protection against a Vibrio anguillarum challenge.

The alterations of immune responses of Artemia franciscana nauplii as a function of culture time and after a challenge with the pathogen Vibrio anguillarum were studied. The effect of the administration of the probiotic bacteria Bacillus subtilis, Lactobacillus plantarum and Lactococcus lactis either alone or in combination with the pathogen was evaluated. The activity of the antioxidant enzymes superoxide dismutase (SOD), Glutathione reductase (GRed), Glutathione transferase (GST) and Phenoloxidase (PO) presented a significant increase as a function of culture time, appeared elevated following probiotic administration and were depleted 48 h following the experimental challenge. Lipid peroxidation reached peak levels at 48 h of culture, when nauplii start feeding and returned to lower values at 144 h, remaining however significantly higher than control (P < 0.05). The three probiotics significantly reduced lipid peroxidation in comparison with the corresponding control, while challenge with the pathogen resulted in its threefold increase. Survival of nauplii remained high throughout culture and was either increased or remained at control levels following the administration of the probiotics. The challenge with the pathogen resulted in a significantly decreased survival of 15.3% for the positive control, while in the probiotic treated series survival values were not significantly different from the negative control (P > 0.05). Following a combined administration of each probiotic and the pathogen the activities of all enzymes tested were significantly lower (P < 0.001) than the negative control (no treatment), but higher than the positive control (challenge, no probiotic). Lipid peroxidation was significantly lower in the probiotic treated series in comparison to the positive control (P < 0.001). The results of the present study provide evidence that major alterations take place as a function of culture time of Artemia nauplii. In addition the pathogen induces an oxidative stress response. The probiotics B. subtilis, L. plantarum and L. lactis protect Artemia against a V. anguillarum challenge by enhancing its immune responses thus contributing to reduced oxidative damage and increased survival.

In vitro and in vivo exploration of palmitic acid from Synechococcus elongatus as an antibiofilm agent on the survival of Artemia franciscana against virulent vibrios.

Biofilm formation of Vibrio spp. has been demonstrated as a potentially important mechanism contributing antibiotic treatment failure in aquaculture. In the present study, the effect of palmitic acid (PA) identified from Synechococcus elongatus was assessed for the inhibition of quorum sensing (QS) regulated biofilm formation in aquatic bacterial pathogens. The biofilm inhibitory concentration (BIC) of PA against Vibrio spp. was found to be 100µgml-1. In this concentration, PA exhibited a significant inhibition in biofilm biomass of Vibrio harveyi MTCC 3438, V. parahaemolyticus ATCC 17802, V. vulnificus MTCC 1145 and V. alginolyticus ATCC 17749 without hindering their planktonic growth. Also, PA displayed gradual decrease in bioluminescence production of V. harveyi. The results of extracellular polymeric substances quantification, microbial adhesion to hydrocarbons and Fourier transform infrared spectroscopic (FT-IR) analyses suggested that PA positively interferes with the initial adhesion stages of biofilm formation. In addition, confocal and scanning electron microscopic analysis substantiates the antibiofilm efficacy of the PA. The transcriptomic analysis revealed the down-regulation of QS mediated response regulator genes expression in V. harveyi. Concomitantly, PA reduced the intestinal colonization of vibrios in brine shrimp larvae and thereby attenuates the biofilm assemblage and its associated virulence. In vivo studies using brine shrimp larvae manifested the reduction in adherence and virulence, which prompts further investigation about the potential of PA for the treatment of vibriosis.