Acute hepatopancreatic necrosis (AHPND) is a severe bacterial disease affecting farmed shrimp. Although various pathogenic bacteria associated with AHPND-affected shrimp have been described, little is known about the bacterial signatures in the stomachs and intestines when the disease occurs naturally. In this study, we characterized the microbiome of P. vannamei by high-throughput sequencing (HTS). Shrimp samples were collected from a commercial farm and divided into two groups: healthy and affected by AHPND, confirmed by PCR. Stomach and intestine samples were subjected to microbiome analysis targeting the V3-V4 region of the 16S rRNA gene. PERMANOVA analysis revealed a significant disparity in the bacterial diversity between the stomach and intestine microbiomes of these two health conditions. Our results suggest that the significant abundance of Vibrio brasiliensis and V. sinaloensis in the intestines of affected shrimp plays a role in AHPND infection. This imbalance could be mitigated by the presence of Pseudoalteromonas, Gilvimarinus, and other members of the phylum Pseudomonadota such as Cellvibrionaceae, Psychromonadaceae, and Halieaceae, which showed significant abundance in healthy intestines. This study highlights the significance of the microbial community in the differentiation of specific microbial signatures in different organs of P. vannamei. These findings offer a deeper understanding of the intricate dynamics within the shrimp microbiome under these conditions, enriching our view of AHPND progression and paving the way toward future identification of probiotics tailored for more efficient management of this disease.

Nowadays, European seabass (Dicentrarchus labrax) aquaculture is undergoing a significant expansion. Nevertheless, the aquaculture industry is plagued by vibriosis. The spatial and temporal dynamics of Vibrio harveyi were studied on a European seabass farm in northern France during seven months of 2022. Concrete specimens were suspended and water was pumped from different depths (0.3 m, 2.15 m and 4 m deep), providing insights into the biofilm and planktonic V. harveyi dynamics. The abundances of V. harveyi, in the biofilm and free-living forms, were positively correlated. The water parameters revealed seasonal fluctuations in temperature, pH, and salinity, with no significant differences observed across the water column. Quantification of V. harveyi revealed no significant differences between depths, but seasonality, with peak abundances observed in August, correlated with temperature increases. Principal component analysis identified temperature as a primary driver, but also additional parameters, such as salinity and pH. Vibriosis occurred during the sampling period, providing valuable insights into the conditions before, during, and after the outbreaks. This study underscores the importance of understanding V. harveyi behaviour in aquaculture, particularly in the context of global warming, for effective disease management and sustainable practices.

The incidence of Vibrio vulnificus infections, with high mortality rates in humans and aquatic animals, has escalated, highlighting a significant public health challenge. Currently, reliable markers to identify strains with high virulence potential are lacking, and the understanding of evolutionary drivers behind the emergence of pathogenic strains is limited. In this study, we analyzed the distribution of virulent genotypes and phenotypes to discern the infectious potential of V. vulnificus strains isolated from three distinct sources. Most isolates, traditionally classified as biotype 1, possessed the virulence-correlated gene-C type. Environmental isolates predominantly exhibited YJ-like alleles, while clinical and diseased fish isolates were significantly associated with the nanA gene and pathogenicity region XII. Hemolytic activity was primarily observed in the culture supernatants of clinical and diseased fish isolates. Genetic relationships, as determined by multiple-locus variable-number tandem repeat analysis, suggested that strains originating from the same source tended to cluster together. However, multilocus sequence typing revealed considerable genetic diversity across clusters and sources. A phylogenetic analysis using single nucleotide polymorphisms of diseased fish strains alongside publicly available genomes demonstrated a high degree of evolutionary relatedness within and across different isolation sources. Notably, our findings reveal no direct correlation between phylogenetic patterns, isolation sources, and virulence capabilities. This underscores the necessity for proactive risk management strategies to address pathogenic V. vulnificus strains emerging from environmental reservoirs.IMPORTANCEAs the global incidence of Vibrio vulnificus infections rises, impacting human health and marine aquacultures, understanding the pathogenicity of environmental strains remains critical yet underexplored. This study addresses this gap by evaluating the virulence potential and genetic relatedness of V. vulnificus strains, focusing on environmental origins. We conduct an extensive genotypic analysis and phenotypic assessment, including virulence testing in a wax moth model. Our findings aim to uncover genetic and evolutionary factors that drive pathogenic strain emergence in the environment. This research advances our ability to identify reliable virulence markers and understand the distribution of pathogenic strains, offering significant insights for public health and environmental risk management.

Antibiotics are often used to treat severe Vibrio infections, with third-generation cephalosporins and tetracyclines combined or fluoroquinolones alone being recommended by the US Centers for Disease Control and Prevention. Increases in antibiotic resistance of both environmental and clinical vibrios are of concern; however, limited longitudinal data have been generated among environmental isolates to inform how resistance patterns may be changing over time. Hence, we evaluated long-term trends in antibiotic resistance of vibrios isolated from Chesapeake Bay waters (Maryland) across two 3-year sampling periods (2009-2012 and 2019-2022). Vibrio parahaemolyticus (n = 134) and Vibrio vulnificus (n = 94) toxR-confirmed isolates were randomly selected from both sampling periods and tested for antimicrobial susceptibility against eight antibiotics using the Kirby-Bauer disk diffusion method. A high percentage (94%-96%) of V. parahaemolyticus isolates from both sampling periods were resistant to ampicillin and only 2%-6% of these isolates expressed intermediate resistance or resistance to third-generation cephalosporins, amikacin, tetracycline, and trimethoprim-sulfamethoxazole. Even lower percentages of resistant V. vulnificus isolates were observed and those were mostly recovered from 2009 to 2012, however, the presence of multiple virulence factors was observed. The frequency of multi-drug resistance was relatively low (6%-8%) but included resistance against antibiotics used to treat severe vibriosis in adults and children. All isolates were susceptible to ciprofloxacin, a fluoroquinolone, indicating its sustained efficacy as a first-line agent in the treatment of severe vibriosis. Overall, our data indicate that antibiotic resistance patterns among V. parahaemolyticus and V. vulnificus recovered from the lower Chesapeake Bay have remained relatively stable since 2009.IMPORTANCEVibrio spp. have historically been susceptible to most clinically relevant antibiotics; however, resistance and intermediate-resistance have been increasingly recorded in both environmental and clinical isolates. Our data showed that while the percentage of multi-drug resistance and resistance to antibiotics was relatively low and stable across time, some Vibrio isolates displayed resistance and intermediate resistance to antibiotics typically used to treat severe vibriosis (e.g., third-generation cephalosporins, tetracyclines, sulfamethoxazole-trimethoprim, and aminoglycosides). Also, given the high case fatality rates observed with Vibrio vulnificus infections, the presence of multiple virulence factors in the tested isolates is concerning. Nevertheless, the continued susceptibility of all tested isolates against ciprofloxacin, a fluoroquinolone, is indicative of its use as an effective first-line treatment of severe Vibrio spp. infections stemming from exposure to Chesapeake Bay waters or contaminated seafood ingestion.

We report the draft genome of Bacillus velezensis strain 3TSA-3, isolated from Pacific white shrimp Penaeus vannamei postlarvae collected from a hatchery tank with high survival despite the presence of pathogenic Vibrio. The strain possesses genes encoding bacteriocins and lacks virulence factor genes, characteristics for a potential aquaculture probiotic.

BACKGROUND: In this study, the protective immunity and immunogenicity of the monovalent and bivalent Streptococcus iniae and Vibrio harveyi vaccine were evaluated in Asian seabass. To analyze immune responses, 1200 Asian seabass with an average weight of 132.6 ± 25.4 g were divided into eight treatments in triplicates (50 fish per tank) as follows: S. iniae immunized by injection (SI), V. harveyi immunized by injection (VI), bivalent S. iniae and V. harveyi (SVI) immunized by injection, S. iniae immunized by immersion (SIM), V. harveyi (VIM) immunized by immersion, bivalent S. iniae and V. harvei (SVIM) immunized by immersion, phosphate-buffered saline (PBS) by injection (PBSI) and control group without vaccine administration (CTRL). Blood and serum samples were taken at the end of the 30th and 60th days. Then the vaccinated groups were challenged with two bacteria (S. iniae) and (V. harveyi) separately and mortality was recorded for 14 days.
RESULTS: This study reveals that there is no significant difference in the hematological parameters on the 30th and 60th days of the experiment in the vaccine-immunized groups compared to the CTRL group (P > 0.05). Meanwhile, there was no significant difference in the amount of serum albumin level, respiratory burst activity, and serum bactericidal activity in the vaccine-immunized groups compared to the CTRL group on the 30th and 60th days of the experiment (P > 0.05). Total protein on the 60th day (in the VI and SVI groups), globulin on the 30th day (in the VI and SVI groups) and the 60th day (in the VI group) compared to the CTRL and PBSI groups had a significant increase (P < 0.05). Complement activity (in the VI and SVI groups) and lysozyme (in the SI and SVI groups) increased significantly compared to the control group (P < 0.05). Serum antibody titer against S. iniae had a significant increase in the SI, VI, SVI and SVIM groups compared to the CTRL and PBSI groups (P < 0.05). Serum antibody titer against V. harveyi had a significant increase in the groups immunized with the vaccine compared to the CTRL and PBSI groups (P < 0.05). A significant increase in the relative percentage survival (RPS) following challenge with S. iniae in the SVI (86.6%), SI (83.3%,) and VI (73.3%) groups were observed compared to the CTRL (43.3%) and PBSI (40%) groups (P < 0.05). Also, a significant increase in the RPS after challenge with V. harveyi in the SVI group, VI 86.6%, SVI 83.3%, VIM 80% and SVIM 76.6% were observed compared to the CTRL (46.6%) and PBSI (50%) groups (P < 0.05).
CONCLUSIONS: Overall, the results demonstrated that the bivalent vaccine of S. iniae and V. harveywas able to produce significant immunogenicity and RPS in Asian seabass.

Vibrio parahaemolyticus is a gram-negative facultative anaerobic bacterium implicated as the causative agent of several shrimp diseases. As part of the effort to provide biocontrol and cost-effective treatments, this research was designed to elucidate the effect of Morinda citrifolia fruit extract on the immunity of Penaeus vannamei postlarvae (PL) to V. parahaemolyticus. The methanol extract of M. citrifolia was vacuum evaporated, and the bioactive compounds were detected using gas chromatography‒mass spectrometry (GC‒MS). Thereafter, P. vannamei PL diets were supplemented with M. citrifolia at different concentrations (0, 10, 20, 30, 40, and 50 mg/g) and administered for 30 days before 24 h of exposure to the bacterium V. parahaemolyticus. A total of 45 bioactive compounds were detected in the methanol extract of M. citrifolia, with cyclononasiloxane and octadecamethyl being the most abundant. The survival of P. vannamei PLs fed the extract supplement was better than that of the control group (7.1-26.7% survival greater than that of the control group) following V. parahaemolyticus infection. Shrimp fed 50 mg/g M. citrifolia had the highest recorded survival. The activities of digestive and antioxidant enzymes as well as hepatopancreatic cells were significantly reduced, except for those of lipase and hepatopancreatic E-cells, which increased following challenge with V. parahaemolyticus. Histological assessment of the hepatopancreas cells revealed reduced cell degeneration following the administration of the plant extracts (expecially those fed 50 mg/g M. citrifolia) compared to that in the control group. Therefore, the enhanced immunity against V. parahaemolyticus infection in P. vannamei could be associated with the improved hepatopancreas health associated with M. citrifolia fruit extract supplementation.

In this study, three generations of polymerase chain reaction (PCR) assays: (i) conventional PCR, (ii) qPCR and (iii) droplet digital PCR (ddPCR), were systematically tested for their abilities to detect non-pathogenic and pathogenic populations of Vibrio parahaemolyticus. The limit of detection (LOD) for the ddPCR was 1.1 pg/µL of purified DNA, followed by the qPCR (5.6 pg/µL) and the conventional PCR (8.8 pg/µL). Regarding the LOD for V. parahaemolyticus cells, the ddPCR assay was able to detect 29 cells, followed by the conventional PCR assay (58 cells) and the qPCR assay (115 cells). Regarding the sensitivities to detect this pathogen from PCR inhibition prone samples (naturally contaminated mussels), the ddPCR assay significantly outperformed the conventional PCR and qPCR. The ddPCR assay was able to consistently detect non-pathogenic and pathogenic populations of V. parahaemolyticus from naturally contaminated mussels, indicating its tolerance to various PCR inhibitors. This study also revealed the significant difference between conventional PCR and qPCR. The conventional PCR assay showed significantly greater sensitivity than that of the qPCR assay in detecting V. parahaemolyticus in crude samples, whereas the qPCR assay showed better sensitivity in detecting the presence of V. parahaemolyticus in purified DNA samples.

Sponge-associated bacteria are considered a rich source of bioactive compounds particularly to reduce the risk of Vibrio harveyi and Vibrio parahaemolyticus infection. The present study aimed to analyse the effectiveness of 19 isolates to control Vibrio infection in vivo. All 19 isolates displayed a non-pathogenic characteristic on shrimps (cell density of 106 cells/mL) as analysed using the pathogenicity test. The mortality caused by both Vibrio spp. on 50% of the shrimp population (LC50 value) had a cell density of 105 cells/mL as determined using the proportion interval method. On the basis of the challenge test, all isolates improved the survival rate of infected shrimps in diverse effectivities up to 89%, which was nearly 30% higher than the infected control. Two isolates coded as D6.9, and P5.20 reduced shrimp mortality after infection with Vibrio spp. 16S rRNA-based identification showed these isolates were closely similar to different genera of Bacillus and Staphylococcus. The extract derived from the most prospective isolate, D6.9, was dominated by 1-hydroxy-6-(3-isopropenyl-cycloprop-1-enyl)-6-methyl-heptan-2-one, hexadecanoic acid, 4-epicyclomusalenone [(24S)-24-methyl-28-norcycloart-25-en-3-one], and 2,4-dimethyl acetoacetanilide. This observation suggested these isolates characterised by in vivo anti-Vibrio activity need to be further developed as biocontrol candidates.

In the last two decades, episodes of mass mortality in benthic communities have often been associated with climatic anomalies, but the ultimate mechanisms through which they lead to death have rarely been identified. This study reports a mass mortality of wild sponges in the Aegean Sea (Turkey, Eastern Mediterranean), which affected the keratose demosponge Sarcotragus foetidus in September 2021. We examined the occurrence of thermo-dependent bacteria of the genus Vibrio in the sponges, identified through 16S rRNA of colonies isolated from sponge tissue in specific culturing media. Six Vibrio sequences were identified from the sponges, three of them being putatively pathogenic (V. fortis, V. owensii, V. gigantis). Importantly, those Vibrios were isolated from only tissues of diseased sponges. In contrast, healthy individuals sampled in both summer and winter led to no Vibrio growth in laboratory cultures. A 50 years record of sea surface temperature (SST) data for the study area reveals a progressive increase in temperature from 1970 to 2021, with values above 24°C from May to September 2021, reaching an absolute historical maximum of 28.9°C in August 2021. We hypothesize that such elevated SST values maintained for several months in 2021 promoted proliferation of pathogenic Vibrio species (thermo-dependent bacteria) in S. foetidus, triggering or aggravating the course of sponge disease. Thus, vibrioisis emerges as one of the putative mechanisms through which global water warming in the Mediterranean Sea translates into sponge mortality. The historical time course of temperature data for the studied area in the Aegean Sea predicts that recurrent waves of elevated SST are likely to occur in the coming summers. If so, recurrent disease may eventually eliminate this abundant sponge from the sublittoral in the midterm, altering the original bathymetric distribution of the species and compromising its ecological role.