Multilocus sequence typing of antimicrobial-resistant zoonotic bacteria from shrimp aquaculture environments in Selangor, Malaysia.

Breaking paradigms: Reconsidering the role of harmless vibrios in shrimp health.

Metabarcoding detection of Vibrio species in Black-tailed Gulls reveals seabirds as overlooked sentinels of coastal zoonotic risk in South Korea.

Background: Coastal waters around Tripoli, Libya, are severely polluted by urban expansion, industrial discharges, and untreated sewage, creating conditions that threaten marine organisms and human consumers. Objectives: This preliminary study aimed to isolate and identify aquatic bacterial species from the liver of Mugil cephalus, determine the antibiotic susceptibility profiles of the isolated bacteria, and investigate histopathological lesions associated with bacterial stress in Mugil cephalus. Results: Gross examination revealed mild lordosis, gill marbling, severe hepatic congestion, and hemorrhages involving the liver, swim bladder, and kidneys. Histological analysis demonstrated pronounced vascular congestion, widespread vacuolar degeneration of hepatocytes, nuclear pyknosis, leukocyte infiltration, and activation of melano-macrophage centers in 13 out of 15 specimens (87%); unidentified parasitic structures were observed in 12 out of 15 specimens (80%). Bacteriological examination of liver tissue yielded Gram-negative isolates from all 15 samples, predominantly Aeromonas and Vibrio species, of which 12 isolates (80%) exhibited multidrug resistance. These findings suggest an association between environmental contamination, bacterial colonization, and progressive liver damage, underscoring the species’ potential role as a sentinel for antimicrobial resistance and highlighting possible zoonotic risks. Conclusion: The observed hepatic lesions may serve as valuable bioindicators for ongoing ecosystem monitoring and underscore the need for improved wastewater management and antimicrobial stewardship within a One Health framework.

Vibrio harveyi is an important bacterial pathogen in marine aquaculture, responsible for outbreaks affecting a wide range of fish species. Early detection of potentially virulent isolates in fish and environmental samples is essential for effective disease prevention and management, particularly under field conditions where the pathogen may be present at low abundance. In this study, we developed and evaluated the analytical and diagnostic performance of a two-step detection protocol that combines a short alkaline peptone water (APW-1) enrichment step with a duplex PCR assay targeting the species-specific toxR gene and the plasmid-associated fpcrp gene, a putative marker linked to increased virulence in some fish-pathogenic vibrios. This marker was detected in a diverse set of publicly available genomes from virulent V. harveyi isolates as well as in virulent isolates used in this study. This integrated approach enables reliable detection of V. harveyi in complex biological and environmental matrices while simultaneously providing virulence-associated screening. The duplex PCR showed high analytical specificity for V. harveyi based on toxR amplification when tested against a panel of closely related Vibrio species and allowed the identification of V. harveyi isolates carrying the putative virulence-associated marker. Without enrichment, the limit of detection was approximately 105 CFU/g or CFU/mL in fish tissues and water samples, whereas incorporation of an 8 h APW-1 enrichment step improved the analytical sensitivity to around 102 CFU/g or CFU/mL. The diagnostic performance of the complete two-step protocol was evaluated using experimentally spiked samples and field samples from Mediterranean aquaculture settings, including fish tissues and aquatic environments. Overall, this enrichment-duplex PCR assay provides a rapid, sensitive and practical tool for the early detection and risk-oriented monitoring of potentially virulent V. harveyi isolates in aquaculture environments, supporting surveillance programmes and control strategies.

The significant regulatory role of cytochrome P450 in the innate immune response of the Zhikong scallop (Chlamys farreri).

Advances in Quorum-Sensing Inhibitors as Novel Therapeutic Targets.

Vibrio parahaemolyticus is a major zoonotic pathogen. Its outer membrane proteins (OMPs) are key targets for developing vaccines. This study aimed to evaluate the potential of OmpU and VP0802 as subunit vaccine candidates against Vibrio species. Unlike prior studies relying on qualitative survival endpoints and narrow strain panels, we employed serum bactericidal assays and an expanded strain panel to quantify cross-protective efficacy. The recombinant proteins OmpU and VP0802 were cloned, expressed, and purified. Their native abundance, surface exposure, immunogenicity, and protective efficacy were evaluated. Both recombinant OMPs exhibited strong binding to whole-cell murine antisera in an indirect enzyme-linked immunosorbent assay (titer 1:81 000). Confocal microscopy further confirmed high surface availability, with specific anti-OmpU and anti-VP0802 sera binding to bacterial cells. Immunization results showed that OmpU induced high-titer antisera (up to 1:243 000), accompanied by broad cross-reactivity and bactericidal activity against different V. parahaemolyticus serotypes and multiple Vibrio species, with a potent bactericidal antibody titer of 1:512. In contrast, VP0802 generated lower antibody titers (up to 1:9000), reduced immunoblot band, weaker immunofluorescence signals, and significantly reduced bactericidal activity (1:32). OmpU represents a promising cross-protective subunit vaccine candidate against multiple Vibrio species.

Pathogenic Vibrio bacteria employ many virulence factors to cause disease in marine animals, including biofilms, proteases, and the secretion of toxins. Here, we identify four secreted toxins encoded by V. campbellii that are toxic to eukaryotic cells. These toxins are secreted by the type III secretion system, a needle-like apparatus used by many bacteria to inject toxins into host cells. The toxins identified in this study are regulated by quorum sensing, a method bacteria use to communicate and control virulence genes required for pathogenesis. The results of this study indicate that different Vibrio species employ distinct sets of toxins that possibly facilitate their interaction with a host.

The gut microbiota influence many aspects of their host’s health and physiology including the digestion of food, and food intake in turn influences the composition of the gut microbiome. However, the ways in which food can alter the behavior of intestinal bacteria remain largely unknown, due in large part to the difficulty of assessing behavior in situ. Larval zebrafish provide a model for addressing this gap because of their optical transparency and their ability to be prepared germ-free and then associated with specific microbial species. Using light sheet fluorescence microscopy to visualize bacteria inside the intestines of live zebrafish larvae, we examine the properties of two commensal strains with markedly different physical characteristics. One is a zebrafish-commensal Enterobacter species that forms large aggregates in unfed larvae, and the other is a pathobiont Vibrio species, capable of damaging intestinal tissue, that is motile and planktonic. We use recently developed ultraviolet irradiation methods to dramatically lower the microbial content in rotifers, a common live food for larval fish, thereby enabling the assessment of feeding effects independent of the introduction of new microbes. Following host consumption of rotifers, Enterobacter clusters disintegrate into motile individuals. Vibrio remains planktonic in fed larvae but decreases the activity of its Type VI Secretion System, as revealed by a fluorescent fusion protein comprising one of the secretion apparatus proteins and green fluorescent protein, leading to a strong decrease in damage to host tissue. Our results reveal that feeding can have major impacts on bacterial behavior that should be considered in models of normal gut microbiome dynamics as well as pathogenesis.

Comparative persistence of opportunistic Vibrio alginolyticus and major foodborne Vibrio species (Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus) in ready-to-eat seafood under different storage conditions.

Extracellular vesicles (EVs) are involved in diverse functions in nature, from biogeochemical cycles to pathogenesis. Here, we investigate whether taxonomy represents a boundary that constrains the uptake of vesicles between different marine species. We focus on Vibrio spp. since they are ubiquitous and play different roles, including in diseases affecting humans and marine organisms, such as corals. Spectral flow cytometry data showed intra- and inter-species uptake of EVs from the pathogenic Vibrio kanaloae and Vibrio coralliilyticus by different Vibrio strains and species, including other marine and non-marine Gram-negative and Gram-positive recipient bacteria. EV fusion efficiencies with recipient cells ranged from 53% to 99%, although these values may vary depending on the nutrient status of the cells. Data suggest that EV fusion between cells from different taxa can occur regardless the phylogenetic distance between the EV donor and recipient cell and the quantity of EVs added. Data revealed that nutrient conditions did not play a significant role in the number of EVs released by the analyzed Vibrio spp., although differences were observed in the amount of DNA exported in EVs (p-value = 0.02), with more bulk vesicular DNA cargo in diluted nutrient conditions. Nearly the entire genome of the targeted Vibrio spp. was detected in the EV fraction. Our data suggest that taxonomic distance between the EV donor and recipient cell is not a major boundary.

Uncovering how major extracellular proteins drive virulence and functional compensation in the mollusk pathogen Vibrio europaeus.

Catfish spine injuries are a frequent but under-recognized source of soft-tissue trauma, particularly in the Gulf South, where contact with native catfish species is common. These injuries carry risks of retained foreign bodies, venom-associated inflammation, and secondary infection from aquatic pathogens. Factors associated with complications and the microbiologic spectrum of infection remain poorly characterized. We performed a retrospective case series of patients presenting with catfish-related injuries to a large Gulf South health system between January 2018 and December 2024. Demographics, injury characteristics, timing of presentation, interventions, complications, and wound culture results were collected. Complicated outcomes were defined as infection, need for surgical intervention, or prolonged follow-up. Univariate Poisson regression with log link was used to estimate risk ratios (RRs) for complications. A total of 155 patients were included; 59 (38%) experienced a complicated clinical course. Delayed presentation was the only factor significantly associated with increased risk. Compared with presentation within 6 hours, presentation at 24 to 72 hours (RR 2.70, 95% CI 1.20-6.07, P = 0.015) and beyond 72 hours (RR 2.25, 95% CI 1.11-4.77, P = 0.028) significantly increased complication risk. No associations were observed for age, sex, body mass index, diabetes, liver disease, or injury location. Among 49 patients undergoing culture testing, 17 had positive cultures, most commonly Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Aeromonas species, Vibrio species, and 1 case of Mycobacterium marinum. Delayed presentation significantly increases the risk of complications following catfish spine injuries. Early evaluation and empiric antimicrobial coverage targeting aquatic and skin flora may improve outcomes.

Vibrio parahaemolyticus, a gram-negative marine bacterium, is a major cause of seafood-borne gastroenteritis worldwide. This pathogen relies on type III secretion system 2 (T3SS2), which is encoded on a pathogenicity island, for its enteropathogenicity. Expression of T3SS2 is activated by a regulatory pathway centered on the transcriptional activator VtrB, which is antagonized by the xenogeneic silencer, histone-like nucleoid-structuring protein (H-NS). However, the complete transcriptional network is not yet fully understood. In this study, we identified TsrA as a negative regulator of T3SS2 gene expression. TsrA is a small protein conserved among Vibrio species that lacks a putative DNA-binding motif but has been implicated in the regulation of virulence genes in Vibrio cholerae. In V. parahaemolyticus, deletion of tsrA increased VtrB production and T3SS2 secretion, thereby enhancing T3SS2-dependent pathogenicity. Transcription of vtrB occurs via a two-step activation process, in which TsrA affects the primary activation step, thereby modulating VtrB production. We further provide experimental evidence that TsrA physically interacts with H-NS via its C-terminal region, which correlates with its regulatory activity on vtrB expression. A systematic mutational analysis of the C-terminal 26 residues revealed several residues critical for TsrA regulatory activity. Moreover, the regulatory effect of TsrA on T3SS2 gene expression was dependent on H-NS, demonstrating that TsrA functions in concert with H-NS. Thus, our findings provide new insights into the regulatory mechanisms of virulence gene expression in V. parahaemolyticus by defining the role of TsrA in this network, while also placing TsrA among H-NS co-regulators.IMPORTANCENucleoid-associated proteins (NAPs) play key roles in virulence gene regulation in bacteria. The best-studied NAP is H-NS, which often functions with co-regulators to fine-tune gene expression. TsrA, a small protein lacking a DNA-binding motif conserved among Vibrio species, has been suggested to be functionally related to H-NS in Vibrio cholerae, although its mechanism remains unknown. Here, we demonstrate that TsrA negatively regulates the expression of type III secretion system 2 (T3SS2), a major virulence determinant of Vibrio parahaemolyticus, an important seafood-borne pathogen. TsrA modulates the transcription of vtrB, which encodes the essential activator for T3SS2 expression, through direct physical interaction with H-NS. Our findings reveal a molecular link between TsrA and H-NS, providing mechanistic insights into NAP- and TsrA-mediated regulation of virulence in Vibrio.

While eukaryotes employ alternative splicing to diversify protein functions, analogous strategies in bacteria remain underexplored. Here we identify a conserved intragenic coding mechanism in Vibrio cholerae that generates two isoforms of the essential scaffold Fha and show that these isoforms cooperate through liquid-liquid phase separation to promote the assembly of the type VI secretion system (T6SS). The full-length isoform, FhaL, seeds assembly by engaging the membrane complex, whereas an internally translated isoform, FhaS, enhances secretion efficiency by strengthening specific interactions with baseplate components. This isoform partitioning is ecologically critical; a mutant producing only FhaL is impaired in bacterial competition, susceptible to eukaryotic predation, and defective in host colonization. Both isoforms form condensates, and a single residue change within a C-terminal helix abolishes condensate formation and significantly reduces T6SS activities. The internal translation and condensate-forming residues are strictly conserved across >10,000 V. cholerae isolates and active in diverse Vibrio species. These findings define a translational-biophysical mechanism that tunes a widespread contractile protein nanomachine for ecological success.

Vibrio cholerae (V. cholerae), causing deadly cholera, which is responsible for significant public health problems in endemic regions, should be monitored in contaminated environments. Here, we report an integrated protocol incorporating alkaline lysis DNA extraction and loop-mediated isothermal amplification (LAMP) targeting the V. cholerae rpoB gene and producing a colorimetric reading within 1 h. This protocol yielded 100% specificity for V. cholerae after testing three non-cholerae Vibrio species and four Enterobacteriaceae as negative controls. A positive signal was detected across a range of 3 × 108 to 3 × 103 cells/mL for four V. cholerae isolates within 30-60 min. Following the inoculation of 3 × 104 V. cholerae cells into five simulated environmental matrices (soil, sand, seawater, tap water, and anthropogenic effluents), positive results were similarly obtained between 3 × 104 and 3 × 103 cells/mL over the same time interval. In contrast, at the same conditions, Vibrio alginolyticus (V. alginolyticus) and Escherichia coli mock-infected matrices, which were used as negative controls, remained negative. These data suggested that this LAMP protocol could be implemented in the field to monitor V. cholerae in environmental matrices at the point of interest in countries affected by this deadly pathogen.IMPORTANCECholera is an intestinal infection caused by the bacterium Vibrio cholerae, resulting in vomiting and acute watery diarrhea. If left untreated, it can cause severe dehydration and death. Although various detection tests have been developed for different types of samples, cholera remains endemic in several countries, particularly where access to safe drinking water is limited and sanitation infrastructure remains inadequate. The objective of our research is to explore different environmental matrices to design a screening system for V. cholerae in environmental samples, thus contributing to the development of a system that can be used in the environment.

Vibrio vulnificus is a halophilic marine bacterium widely distributed in coastal environments and seafood products and is responsible for severe human infections and foodborne disease outbreaks. Rapid and sensitive detection of V. vulnificus is therefore critical for clinical diagnosis and seafood safety monitoring. The objective of this study was to develop and evaluate a rapid, sensitive, and specific nucleic acid detection method for V. vulnificus suitable for application in complex biological and food matrices. An ERA-CRISPR/Cas13a-based nucleic acid detection assay was established by coupling enzymatic recombinase amplification (ERA) with Cas13a-mediated collateral cleavage of fluorescent RNA reporters. The analytical performance of the assay was systematically evaluated using V. vulnificus standard plasmids, artificially contaminated human serum samples, and spiked oyster homogenates. The assay exhibited high specificity for V. vulnificus, with no cross-reactivity observed for other common Vibrio species. The limit of detection (LOD) was 1 × 10-1 copies/μL for standard plasmid templates. In complex matrices, including human serum and oyster samples, the assay achieved an LOD of 1 × 101 CFU/mL. The entire detection process was completed under isothermal conditions within 45 min. A sensitive and specific ERA-CRISPR/Cas13a-based assay for the detection of V. vulnificus was successfully developed and validated. The method demonstrates strong analytical performance in both biological and seafood matrices. A rapid isothermal ERA-CRISPR/Cas13a assay for V. vulnificus detection was developed. High specificity was achieved without cross-reactivity to other Vibrio species. The assay enables sensitive detection in serum and oyster samples. The method shows potential for routine seafood safety surveillance and marine pathogen monitoring.

V. alginolyticus (VAlg) is one of the three Vibrio species causing human vibriosis. Its presence in shellfish constitutes a potential health risk. Thus, this investigation aimed to determine the prevalence and distribution of VAlg in 18,544 shellfish across geographies using standard protocols and random intercepts/mixed-effects regressions. The global VAlg prevalence in shellfish was 19.70% (95% CI: 13.54–27.75). VAlg pooled prevalence was significantly different (p < 0.0001) by shellfish type ( = 238.48), species ( = 440.34), genus ( = 414.37), VAlg detection methods ( = 150.43), nation ( = 632.27), and continent ( = 33.81). Europe and South America showed the highest pooled VAlg contaminated rates in shellfish (27% and 32%, respectively) but Asia had a low rate (~4%). By shellfish type, gastropods (snails) topped the list with 50%, followed by bivalves (29%). Among shellfish species, the VAlg rate declined from 67% to 5% (k ≤ 6), but with a more stable prevalence in Litopenaeus vannamei (19.02%, k = 11) and Crassostrea gigas (15.18%, k = 9). Edible oysters and clams had lower VAlg pooled rates (4–15%). Targeted culture with MALDI-TOF or species-specific qPCR detected VAlg in 100% of tested shellfish samples. By contrast, conventional phenotypic tests detected less VAlg in shellfish yielding 20% by general biochemical/API tests, 8.8% by generic PCR, and near-zero (0.3–3%) by multiplex PCR or MALDI-TOF/PCR. Both the multiplicative ( = 3.01, p = 0.001) and additive ( = 3.83, p = 0.001) interactions of nation and sample size explained 73.14% and 58.04% of the true variance in VAlg prevalence in shellfish. Other factors include detection techniques ( = 46.59%, , p = 0.001), nation ( = 1.89, p = 0.006), medium = 19.42%, = 1.69, p = 0.03), shellfish type ( = 16.99%, = 1.34, p = 0.12), and continent ( = 15.29%, = 5.90, p = 0.001). In conclusion, the study reveals substantial occurrence of VAlg in shellfish worldwide, with notable regional and species-specific hotspots. Harmonized and molecular-based surveillance of VAlg in shellfish linked to food safety criteria will be essential to manage its emerging threat.

Expanded polystyrene litter in marine environments harbors diverse and distinct microbial communities, referred to as the plastisphere. This study aimed to investigate the monthly dynamics of bacterial and potentially pathogenic bacterial (PPB) communities on expanded polystyrene over one year. Vibrio species dominated the PPB community, cooccurring at consistently higher abundances on expanded polystyrene than in the surrounding seawater, particularly under higher temperatures and low dissolved organic carbon (DOC) levels. At a temperature threshold of 16 °C, the abundance of zoonotic species, such as Vibrio parahaemolyticus and Vibrio alginolyticus, increased significantly. Some psychrotrophic Vibrio spp. were detected under moderately eutrophic conditions, suggesting that expanded polystyrene may also serve as a dispersal vector facilitating their transport to more favorable habitats. Multivariate analyses, including partial least squares path modeling, revealed temperature and DOC as the primary environmental factors influencing PPB community composition. However, environmental responses varied by taxonomic groups, with different preferences observed under varying eutrophic conditions. In conclusion, these findings demonstrate that expanded polystyrene litter supports a selective and environmentally responsive bacterial population, highlighting the potential role of plastic debris in promoting pathogenic bacterial persistence and spread in marine ecosystems, particularly under conditions associated with climate change, including warming and eutrophication.