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.

Selective Antagonism of Lactiplantibacillus plantarum and Pediococcus acidilactici against Vibrio and Aeromonas in the Bacterial Community of Artemia nauplii.

Empiric probiotics are commonly consumed by healthy individuals as a means of disease prevention, pathogen control, etc. However, controversy has existed for a long time regarding the safety and benefits of probiotics. Here, two candidate probiotics, Lactiplantibacillus plantarum and Pediococcus acidilactici, which are antagonistic to Vibrio and Aeromonas species in vitro, were tested on Artemia under in vivo conditions. In the bacterial community of Artemia nauplii, L. plantarum reduced the abundance of the genera Vibrio and Aeromonas and P. acidilactici significantly increased the abundance of Vibrio species in a positive dosage-dependent manner, while higher and lower dosages of P. acidilactici increased and decreased the abundance of the genus Aeromonas, respectively. Based on the liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) analyses of the metabolite of L. plantarum and P. acidilactici, pyruvic acid was used in an in vitro test to explain such selective antagonism; the results showed that pyruvic acid was conducive or suppressive to V. parahaemolyticus and beneficial to A. hydrophila. Collectively, the results of this study demonstrate the selective antagonism of probiotics on the bacterial community composition of aquatic organisms and the associated pathogens. IMPORTANCE Over the last decade, the common preventive method for controlling potential pathogens in aquaculture has been the use of probiotics. However, the mechanisms of probiotics are complicated and mostly undefined. At present, less attention has been paid to the potential risks of probiotic use in aquaculture. Here, we investigated the effects of two candidate probiotics, L. plantarum and P. acidilactici, on the bacterial community of Artemia nauplii and the in vitro interactions between these two candidate probiotics and two pathogens, Vibrio and Aeromonas species. The results demonstrated the selective antagonism of probiotics on the bacterial community composition of an aquatic organism and its associated pathogens. This research contributes to providing a basis and reference for the long-term rational use of probiotics and to reducing the inappropriate use of probiotics in aquaculture.

In Silico and In Vitro Investigation of Phytochemicals Against Shrimp AHPND Syndrome Causing PirA/B Toxins of Vibrio parahaemolyticus.

In Southeast Asia, the penaeid shrimp aquaculture production faces a new pandemic bacterial disease called acute hepatopancreatic necrosis disease (AHPND). The highly profitable pacific white shrimp, Litopenaeus vannamei, has become a challenging species due to severe lethal infections. Recent research has identified a critical pathogen, Vibrio parahaemolyticus, which caused significant loss in the shrimp industry. The disease pathway involves a virulence plasmid encoding binary protein toxins (PirA/B) that cause cell death. The protein toxins were inherited and conjugatively transferred to other Vibrio species through a post-segregational killing system. In this study, "in silico" (Glide, 2021) analysis identified four phytocompounds as myricetin (Myr), ( +)-taxifolin (TF), (-)-epigallocatechin gallate (EGCG), and strychnine (STN) which could be most effective against both the toxins concerning its docking score and affinity. The interactions of complexes and the critical amino acids involved in docking were analyzed using the Discovery Studio (version 2016). Molecular dynamic studies showed lower root mean square deviations (RMSD) and improved stabilization of ( +)-taxifolin (TF) and (-)-epigallocatechin-3-gallate (EGCG) against both the protein toxins. The antibacterial potential of all four selected compounds had tested against pathogenic strains of V. parahaemolyticus through minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The best MBC results were observed at concentrations of 1 mg/mL for EGCG and 1.25 mg/mL for TF. Moreover, the complete reduction of viable cell counts in the in vitro bactericidal activity had recorded after 24 h of incubation.

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.

Indole analogues decreasing the virulence of Vibrio campbellii towards brine shrimp larvae.

Indole signalling has been proposed as a potential target for the development of novel virulence inhibitors to control bacterial infections. However, the major structural features of indole analogues that govern antivirulence activity remain unexplored. Therefore, we investigated the impact of 26 indole analogues on indole-regulated virulence phenotypes in Vibrio campbellii and on the virulence of the bacterium in a gnotobiotic brine shrimp model. The results demonstrated that 10 indole analogues significantly increased the fluorescence of indole reporter strain Vibrio cholerae S9149, 21 of them decreased the swimming motility of V. campbellii, and 13 of them significantly decreased the biofilm formation of V. campbellii. Further, we found that 1-methylindole, indene, 2,3-benzofuran, thianaphthene, indole-3-acetonitrile, methyl indole-3-carboxylate, 3-methylindole, and indole-2-carboxaldehyde exhibited a significant protective effect on brine shrimp larvae against V. campbellii infection, resulting in survival rates of challenged brine shrimp above 80%. The highest survival of shrimp larvae (98%) was obtained with indole-3-acetonitrile, even at a relatively low concentration of 20 µM. Importantly, the indole analogues did not affect bacterial growth, both in vitro and in vivo. These results indicate the potential of indole analogues in applications aiming at the protection of shrimp from vibriosis.

Halogenated Indoles Decrease the Virulence of Vibrio campbellii in a Gnotobiotic Brine Shrimp Model.

Indole signaling is viewed as a potential target for antivirulence therapy against antibiotic-resistant pathogens because of its link with the production of virulence factors. This study examined the antimicrobial and antivirulence properties of 44 indoles toward Vibrio campbellii. Based on the results, 17 halogenated indole analogues were selected, as they significantly improved the survival of brine shrimp larvae challenged with V. campbellii. Specifically, 6-bromoindole, 7-bromoindole, 4-fluoroindole, 5-iodoindole, and 7-iodoindole showed a high protective effect, improving the survival of brine shrimp to over 80% even at a low concentration of 10 µM. To explore the impact of selected indole analogues on bacterial virulence phenotypes, swimming motility, biofilm formation, protease activity, and hemolytic activity of V. campbellii were determined. The results showed that all of the 17 selected indole analogues decreased swimming motility at both 10 µM and 100 µM. Most of the indole analogues decreased biofilm formation at a concentration of 100 µM. In contrast, only a slightly decreased protease activity and no effect on hemolytic activity were observed at both concentrations. To our knowledge, this is the first study of the structure-activity relation of halogenated indole analogues with respect to virulence inhibition of a pathogenic bacterium in an in vivo host model system, and the results demonstrate the potential of these compounds in applications aiming at the protection of shrimp from vibriosis, a major disease in aquaculture. IMPORTANCE Bacterial diseases are a major problem in the aquaculture industry. In order to counter this problem, farmers have been using antibiotics, and this has led to the evolution and spread of antibiotic resistance. In order for the aquaculture industry to further grow in a sustainable way, novel and sustainable methods to control diseases are needed. We previously reported that indole signaling is a valid target for the development of novel therapies to control disease caused by Vibrio campbellii and related bacteria, which are among the major bacterial pathogens in aquaculture. In the present study, we identified indole analogues that are more potent in protecting brine shrimp (a model organism for shrimp) from V. campbellii. To our knowledge, this is the first study of the structure-activity relation of halogenated indole analogues with respect to virulence inhibition of a pathogenic bacterium in an in vivo host model system.

Comparative transmission of bacteria from Artemia salina and Brachionus plicatilis to the cnidarian Nematostella vectensis.

The microbial community associated with animals (microbiome) is essential for development, physiology, and health of host organisms. A critical step to understand the assembly of microbiomes is to determine how effectively bacteria colonize and establish within the host. Bacteria commonly colonize hosts through vertical transmission, passively from the environment, or through food consumption. Using the prey feeding method (PFM), we test transmittance of Bacillus velezensis, Pseudoalteromonas spiralis, and Vibrio alginolyticus to Nematostella vectensis using two prey, Artemia salina and Brachionus plicatilis. We compare PFM to a solution uptake method (SUM) to quantify the concentration of bacteria in these host organisms, with plate counts. Larvae had a similar uptake with SUM at 6 h but had greater concentrations at 48 h versus PFM. Juveniles acquired similar concentrations at 6 h for SUM and PFM using B. plicatilis and A. salina. At 2 days, the quantity of bacteria vectored from PFM increased. After 7 days the CFUs decreased 2-fold with B. plicatilis and A. salina relative to the 2-day concentrations, and further decreased after 14 days. Therefore, prey-mediated methods provide greater microbe transplantation than SUM after 24 h, supporting this approach as a more successful inoculation method of individual bacterial species.

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.

Essential Oils Improve the Survival of Gnotobiotic Brine Shrimp (Artemia franciscana) Challenged With Vibrio campbellii.

The halophilic aquatic bacterium Vibrio campbellii is an important aquatic pathogen, capable of causing vibriosis in shrimp and fish resulting in significant economic losses. In a previous work, essential oils (EOs) extracts from Melaleuca alternifolia, Litsea citrata, and Eucalyptus citriodora were found to inhibit the growth of V. campbellii in vitro. This study aimed to determine in vivo EOs' potential protective effect towards gnotobiotic brine shrimp Artemia franciscana, challenged with V. campbellii. The study showed that brine shrimp larvae supplemented with EOs of M. alternifolia (0.0008%) and L. citrata (0.002%) displayed significantly increased survival against V. campbellii. The results indicated that supplementation of these EOs increased the expression of immune-related genes (either in the presence or absence of the pathogen), probably contributing to enhanced protection. Furthermore, in vitro studies indicated that some EOs modulated the expression of virulence factors including swimming motility, biofilm formation, and gelatinase and lipase activity, while flow cytometry data and regrowth assay indicated that these EOs do not exhibit antimicrobial activity as V. campbellii grew at the tested concentrations [M. alternifolia (0.0008%) and L. citrata (0.002%)]. Our findings suggest that EOs extracted from M. alternifolia and L. citrata, can modulate virulence factor production and immunological responses and might hence become part of an intervention strategy to control vibriosis in a fish or shrimp aquaculture setting, a hypothesis that needs to be validated in the future.

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.

Vibrio sp. ArtGut-C1, a polyhydroxybutyrate producer isolated from the gut of the aquaculture live diet Artemia (Crustacea)

BACKGROUND: Vibrio species display variable and plastic fitness strategies to survive and interact with multiple hosts, including marine aquaculture species that are severely affected by pathogenic Vibrios. The culturable Vibrio sp. strain ArtGut-C1, the focus of this study, provides new evidence of such phenotypic plasticity as it accumulates polyhydroxybutyrate (PHB), a biodegradable polymer with anti-pathogen activity, particularly in the marine larviculture phase. The strain was isolated from the gut of laboratory-reared Artemia individuals, the live diet and PHB carrier used in larviculture. Its main phenotypic properties, taxonomic status and genomic properties are reported based on the whole-genome sequencing. RESULTS: Vibrio sp. ArtGut-C1 yielded 72.6% PHB of cells' dry weight at 25 C. The genomic average nucleotide identity (ANI) shows it is closely related to V. diabolicus (ANI: 88.6%). Its genome contains 5,236,997- bp with 44.8% GC content, 3,710 protein-coding sequences, 96 RNA, 9 PHB genes functionally related to PHB metabolic pathways, and several genes linked to competing and colonizing abilities. CONCLUSIONS: This culturable PHB-accumulating Vibrio strain shows high genomic and phenotypic variability. It may be used as a natural pathogen biocontrol in the marine hatchery and as a potential cell factory for PHB production.

Antagonistic Activity Against Pathogenic Vibrio Isolates of Bioflocculant-Producing Bacteria Isolated from Shrimp Ponds.

<b>Background and Objectives:</b> Biofloc culture system has been used in aquaculture as an effective technology for water treatment due to many advantages of being biodegradable and environmentally friendly. This study aims to isolate bioflocculant-producing bacteria antagonistic to pathogenic <i>Vibrio</i> species from Pacific white shrimp ponds in Thua Thien Hue, Vietnam. <b>Materials and Methods:</b> <i>Vibrio</i> isolates were isolated by screening on medium with and without antibiotics. The resistance of <i>Vibrio</i> to antimicrobial agents was assessed by Minimum Inhibitory Concentration (MIC). Bioflocs formed in shrimp cultures were used to screen bioflocculant-producing bacteria. The identification of bacteria was performed by 16S rRNA sequencing. The flocculating activity was measured by a test with kaolin clay suspension. To evaluate the antagonistic activity against <i>Vibrio</i> isolates, an agar well diffusion assay was used. <b>Results:</b> The screening results have found that <i>Vibrio</i> isolates such as <i>V. parahaemolyticus</i> KS02 and <i>V. alginolyticus</i> KS08 from shrimp ponds can be resistant to many antibiotics with the highest resistance rate up to 66.49%. Four bioflocculant-producing isolates were obtained and identified as <i>Bacillus</i> species. Among them, <i>Bacillus velezensis </i>B9 when grown in YPG medium supplemented with 3% sucrose and 0.7% peptone had the highest bioflocculation with an activity of 49.2%. Two isolates of <i>B. subtilis</i> B2 and <i>Bacillus</i> sp. B6 had quite strong antagonistic activities against vibriosis shown in the zones of inhibition on the assay plates with diameters of about 20 mm. <b>Conclusion:</b> The present study has found some <i>Bacillus</i> isolates had bioflocculant-producing efficiency and inhibited pathogenic <i>Vibrio</i> bacteria. These <i>Bacillus</i> isolates will potentially be used as inoculum for bioflocculation to improve shrimp production.

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.

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.

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.

Do acute hepatopancreatic necrosis disease-causing PirABVP toxins aggravate vibriosis?

Gram-negative marine bacterium Vibrio parahaemolyticus is an important aquatic pathogen and has been demonstrated to be the causative agent of acute hepatopancreatic necrotic disease (AHPND) in shrimp aquaculture. The AHPND-causing V. parahaemolyticus strains contain a pVA1 plasmid encoding the binary PirAVP and PirBVP toxins, are the primary virulence factor that mediates AHPND and mortality in shrimp. Since PirABVP toxins are secreted extracellularly, one can hypothesize that PirABVP toxins would aggravate vibriosis in the aquatic environment. To address this, in vivo and in vitro experiments were conducted. Germ-free Artemia franciscana were co-challenged with PirABVP toxins and 10 Vibrio spp. The in vivo results showed that PirABVP toxin interact synergistically with MM30 (a quorum sensing AI-2 deficient mutant) and V. alginolyticus AQ13-91, aggravating vibriosis. However, co-challenge by PirABVP toxins and V. campbellii LMG21363, V. parahaemolyticus CAIM170, V. proteolyticus LMG10942, and V. anguillarum NB10 worked antagonistically, increasing the survival of Artemia larvae. The in vitro results showed that the addition of PirABVP toxins significantly modulated the production of the virulence factors of studied Vibrio spp. Yet these in vitro results did not help to explain the in vivo results. Hence it appears that PirABVP toxins can aggravate vibriosis. However, the dynamics of interaction is strain dependent.

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.

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.

A universal random DNA amplification and labeling strategy for microarray to detect multiple pathogens of aquatic animals.

Diseases caused by bacteria, fungi, and viruses pose a great threat to aquaculture. As DNA microarrays can be used to detect multiple pathogens, here we reported an array with the potential to simultaneously detect 13 bacterial and 11 viral pathogens of aquatic animals. The array included 853 oligonucleotide probes (20-40 mer) complementary to various virus-specific sequences and four chromosomal loci (16S rRNA, gyrB, dnaJ, and recA) of bacteria. Multiplex PCR, phi29 DNA polymerase, and a Klenow fragment-based method were evaluated for amplifying and labeling the nucleic acid of pathogens. While array hybridization signals were most intense using pathogen sequences amplified by multiplex PCR, the phi29 DNA polymerase method was more convenient and ideal since it did not require sequence-specific primers that could bias against detecting novel pathogens. The feasibility of the phi29 DNA polymerase-based microarray strategy was also demonstrated by detecting multiple unknown pathogens from four samples of diseased fish and shrimps.