Search Results (155)

Pathogenic Vibrio species are a major cause of disease outbreaks in aquaculture, leading to substantial economic losses and posing risks to food safety. The increasing prevalence of antibiotic resistance among these pathogens has created an urgent need for alternative antimicrobial strategies. Phage-derived endolysins, which specifically degrade bacterial peptidoglycan, have emerged as promising candidates to replace or supplement conventional antibiotics. In this study, we systematically characterized the endolysin LysV039C from the Vibrio alginolyticus phage phiV039C. Whole-genome analysis identified a 462 bp endolysin gene, which was cloned into the pET-28a vector, expressed in E. coli BL21, and purified. The lytic activity of the recombinant enzyme was evaluated using a turbidity reduction assay. LysV039C showed the strongest activity (58.9%) against host bacteria in the logarithmic phase, with peak activity (60.7–64.1%) achieved at 53 μg/mL, 35 °C, and neutral pH. The addition of 2.5 mM EDTA enhanced activity to 63.8%, whereas 10 mM divalent metal ions strongly inhibited the enzyme (<6.5%). Lytic spectrum analysis demonstrated that LysV039C exhibited a broader lytic spectrum against six Vibrio species than its parent phage phiV039C. Overall, LysV039C combines high environmental adaptability (alkaline tolerance, suitability for aquaculture temperature ranges) with efficient and broadened lytic activity against multiple Vibrio species. These findings provide a foundation for developing eco-friendly agents for the prevention and control of pathogenic Vibrio and offer a valuable reference for the mining of other phage-derived antibacterials against aquatic pathogens. Full article

Vibrio spp., known as typical marine pathogens, pose serious threats to human health worldwide due to their high incidence and prevalence. In this study, a quantitative detection method was developed for Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio cholerae and Vibrio alginolyticus in aquatic products. A set of multiplex chamber-based digital PCR (cdPCR) primers and probes was designed based on new molecular targets of Vibrio species. The conditions of multiplex cdPCR were optimized and compared with those of multiplex qPCR. Under optimal conditions, the performance indicators, including the specificity, linear range, and detection limit, were systematically evaluated. The results indicate that the established method had 100% specificity among all tested strains and did not cross-react with other foodborne pathogens tested in parallel. The linear dynamic range was 10²–10⁶ CFU/mL, and the limits of detection (LODs) for V. parahaemolyticus, V. vulnificus, V. cholerae and V. alginolyticus were 5.40 × 10² CFU/mL, 3.05 × 10² CFU/mL, 4.55 × 10² CFU/mL and 1.32 × 10² CFU/mL, respectively. Furthermore, the feasibility of the proposed method was assessed in contaminated food samples, with a 10-fold improvement in detection sensitivity over that of the qPCR method under the conditions of this study. The entire detection process could be completed within 2 h. Taken together, the results of this study offer a readily adaptable diagnostic method, allowing for the simultaneous and quantitative detection of four pathogenic Vibrio species, which is promising for food safety and environmental monitoring. Full article

Rapid and accurate detection of Vibrio alginolyticus and Vibrio vulnificus is important for seafood safety and foodborne infection control. In this study, we developed a detection platform that combines loop-mediated isothermal amplification (LAMP) with Pyrococcus furiosus Argonaute (PfAgo)-mediated sequence-specific cleavage. The LAMP rapidly amplified pathogen-specific target sequences under isothermal conditions, while PfAgo provides an additional layer of sequence verification through a programmable guide DNA-directed cleavage step to reduce false-positive signals caused by nonspecific amplification. Under optimized conditions, the assay achieved limits of detection of 3 × 10¹ CFU·mL⁻¹ for V. alginolyticus and 10² CFU·mL⁻¹ for V. vulnificus, with no positive signals observed among the tested non-target bacteria. In artificially contaminated salmon samples, both V. alginolyticus and V. vulnificus were detected without enrichment after simple boiling-based DNA extraction, with a practical detection limit of 2.9 × 10² CFU·mL⁻¹ in the final homogenate. Compared with culture-based methods and instrument-intensive molecular assays, the proposed LAMP-PfAgo platform exhibits a rapid two-step workflow with an added sequence-verification layer. These results support LAMP coupled with Argonaute-mediated cleavage as a practical strategy for foodborne pathogen monitoring in seafood. Full article

Macrophage migration inhibitory factor (MIF) is broadly acknowledged as a central pro-inflammatory regulator, owing to its multifaceted functions including immune cell recruitment, initiation and amplification of pro-inflammatory cytokine cascades, enhancement of macrophage viability, facilitation of macrophage polarization toward a pro-inflammatory state, and attenuation of glucocorticoid-mediated immunosuppression. However, functional investigations of MIF in Mytilus coruscus remain limited. In this study, we identified the MIF gene in M. coruscus, and bioinformatic analyses revealed that the gene encodes a 115-amino-acid polypeptide that exhibits close phylogenetic affinity with MIF homologs from other mollusks. McMIF was predominantly expressed in immune-related tissues, with notably high expression levels in the digestive gland. Upon Vibrio alginolyticus infection, both the mRNA and protein levels of McMIF were significantly upregulated, suggesting that McMIF is involved in the antibacterial immune response of M. coruscus. Meanwhile, the m6A modification level of McMIF was markedly reduced following infection, suggesting a potential relationship between m6A modification and the antibacterial immune function of MIF. Furthermore, knockdown of McMIF followed by LPS stimulation led to an increased level of apoptosis in digestive gland cells, suggesting that McMIF is involved in the inhibition of apoptosis induced by immune stimulation. Collectively, these findings provide insights into the immunological characteristics of McMIF in M. coruscus. Full article

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 (${\chi }_{20}^2$ = 238.48), species (${\chi }_{74}^2$ = 440.34), genus (${\chi }_{51}^2$ = 414.37), VAlg detection methods (${\chi }_{10}^2$ = 150.43), nation (${\chi }_{30}^2$ = 632.27), and continent (${\chi }_4^2$ = 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 (${\beta }_0=-3.28\pm 3.76, F_{54;125}$ = 3.01, p = 0.001) and additive (${\beta }_0=-1.89\pm 1.47, F_{31;148}$ = 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 ($R^2$ = 46.59%, ${\beta }_0=-1.63\pm 0.83, F_{10;169}=11.16$, p = 0.001), nation ($R^2=37.72$ = 1.89, p = 0.006), medium $R^2$ = 19.42%, ${\beta }_0=2.24\pm 1.85, F_{15;164}$ = 1.69, p = 0.03), shellfish type ($R^2$ = 16.99%, ${\beta }_0=-3.90\pm 1.32, F_{20;159}$ = 1.34, p = 0.12), and continent ($R^2$ = 15.29%, ${\beta }_0=-1.81\pm 0.41, F_{4;175}$ = 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. Full article

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. Full article

Vibrio alginolyticus is a common pathogenic bacterium and can cause diseases in aquaculture animals. Lysine lactylation (Kla) is a novel post-translational modification (PTM) that has been confirmed to play critical roles in key biological processes. However, the modification landscape and functions of Kla in V. alginolyticus remain unclear. In this study, lactylation modification profiles of the bacterial pathogen V. alginolyticus were first systematically characterized; a total of 9308 lactylation sites on 2155 proteins were successfully identified. The lactylation of cAMP receptor protein (CRP) and triosephosphate isomerase (TPI) was verified by Co-immunoprecipitation (Co-IP) and Western blot to validate the lactylome data. Bioinformatic analysis of the Kla sites revealed 32 conserved sequence motifs surrounding the modified residues. Kla proteins were mainly involved in central metabolic pathways, including glycolysis/gluconeogenesis and ribosome biogen regulators were found to contain Kla modification sites. To investigate crosstalk among lysine acylations in V. alginolyticus, we integrated Kla, lysine acetylation (Kac), and lysine succinylation (Ksuc) profiles and identified 337 co-modified proteins and 5 co-modified sites. Additionally, phylogenetic analysis of Vibrio alginolyticus CobQ based on protein sequence alignment revealed no homology to the known delactylase CobB. Combined in vitro and in vivo functional validation identified VaCobQ as a candidate delactylase with potential NAD⁺-independent activity. This study establishes a lysine lactylation landscape in V. alginolyticus, providing a resource for exploring Kla functions in bacterial metabolism and its possible connections to virulence. Full article

Silver nanoparticle (AgNP)-based products have been increasingly applied in aquaculture due to their antimicrobial properties and capacity to modulate host immunity. This study investigated the biological activities of synthesized silver nanowires (AgNWs), with particular emphasis on their anti-Vibrio efficacy and immunomodulatory effects, to evaluate their potential application in shrimp aquaculture. Antibacterial activity was assessed using nonlinear regression analysis to determine minimum inhibitory concentrations (MICs) against three major Vibrio pathogens, while cytotoxicity and immune responses were evaluated using white shrimp hemocytes through cell viability assays and in vitro gene expression analysis, respectively. AgNWs exhibited antibacterial effects on Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio harveyi, with MIC values of 873.7, 58.78, and 672.1 μg/mL, respectively. Hemocyte viability remained above 90% at AgNW concentrations of up to 1000 mg/L, indicating good biocompatibility. AgNWs significantly upregulated immune-related lipopolysaccharide and β-1,3-glucan-binding protein (LGBP) and Toll gene expression at specific concentrations, indicating immunostimulation. These results suggest that AgNWs possess antibacterial activity and immunomodulatory potential with low cytotoxicity, supporting their promise as a novel functional agent for shrimp disease management. Full article

Vibrio alginolyticus is a foodborne pathogen commonly found in seafood and freshwater products, causing human illness through the consumption of tainted seafood. Small non-coding RNAs (sRNAs) take effect on the stability and translation of their target mRNAs by base-pairing, thereby quickly altering bacterial physiology and pathogenicity at the post-transcriptional level. This work constructed a label-free in-frame deletion mutant and a complement strain of micX, a cell-density-associated sRNA in V. alginolyticus. The ΔmicX mutant exhibited reduced growth and a reduction in the synthesis of exopolysaccharides, biofilm, and alkaline serine protease. A TMT-based quantitative proteomic analysis comparing ΔmicX with the wild-type strain identified 900 differentially expressed proteins, comprising 376 that were upregulated and 524 that were downregulated. The upregulated proteins are primarily associated with porin activity, transmembrane signaling receptor function, and the two-component system. The downregulated proteins are mainly engaged in processes including biofilm formation, cellular communication, and transmembrane transport activity. Of note, the expression levels of proteins involved in the type VI secretion system, exopolysaccharide synthesis, mannose-sensitive hemagglutinin type IV pili (MSHA), and biofilm formation were significantly reduced in the absence of micX. Furthermore, the expression levels of proteins associated with quorum sensing (particularly LuxR and AphA) changed significantly in the ΔmicX vs. WT comparison. These findings strengthened comprehension of the novel sRNA regulatory network and established a theoretical foundation for additional investigations into the virulence of V. alginolyticus. Full article

This pioneering study investigates bacteria isolated from marine pelagic fish, fishing vessels, and gear surfaces, focusing on the variability in biofilm formation across different substrates, media, and cultivation conditions. Bacteria from fish intestines, skin, and gills, including spoilage organisms and potential fish and human pathogens, can contaminate vessel surfaces, gear, and containers and may act as microbial reservoirs and transmission vectors. In this study, biofilm formation was evaluated at air–liquid interfaces and on submerged plastic, metal, and glass surfaces under various incubation temperatures and media. Vibrio spp. were isolated both from fishing nets and fish gills, particularly Vibrio alginolyticus, V. gigantis, and V. pelagius. Although V. harveyi was examined as a representative vibrio, it did not form a biofilm on polypropylene. Photobacterium damselae subsp. damselae, Pseudomonas fragi, P. gessardii, Psychrobacter spp., and Rothia endophytica showed a strong affinity for stainless steel. Overall adhesion regardless of media type was highest for P. gessardii, followed by P. damselae and Aeromonas veronii, which adhered strongly to steel, glass, and polypropylene; however, only P. gessardii also adhered well to polystyrene, an important finding because these are known fish and human pathogens. These results highlight species-dependent biofilm triggers and their substantial variability and provide guidance for standardized marine biofilm protocols. Full article

Vibrio alginolyticus is an important antibiotic-resistant pathogen in aquaculture that can cause mortality in a wide range of aquatic animals and infect humans. It is urgently necessary to discover and develop effective antibiotic alternatives. Citral, a key antibacterial component of lemongrass oil, can be used as a food flavoring and additive. Although the antimicrobial activity and antibiofilm effect of citral against V. alginolyticus have been noted in our previous study, the potential lipidome influence of citral remains unclear. Accordingly, a non-targeted lipidomics approach was employed to investigate citral-induced lipidome disturbances and reveal potential regulated targets of citral against V. alginolyticus. We found that the citral exposure triggered substantial lipidome alterations (i.e., composition, contents, and structure) in V. alginolyticus. Specifically, the content of most phospholipids (e.g., phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), phosphatidylserines (PSs), phosphatidylinositols (PIs), and phosphatidylglycerols (PGs)) decreased with the increase in citral concentration, while ceramides (Cers) and lysophospholipids (LPLs) (e.g., lyso-PAs, lyso-PCs, lyso-PEs, and lyso-PGs) showed concentration-dependent accumulation under citral treatment. Notably, the critical lipid remodeling in response to citral exposure mainly involved the phospholipid and sphingolipid metabolic pathways. Collectively, our study reveals the bacterial lipidome response to citral exposure and highlights pivotal metabolic pathways, potentially offering a novel perspective for future investigations into lipid-centric antibacterial targets. Full article

Background/Objectives: Candidozyma auris is the most frequent multidrug-resistant fungal infection in the Arabian Peninsula, with high mortality rates; therefore, new medications are in high demand. Microbes in marine habitats have genetically evolved to survive under a variety of adverse conditions, including severe temperatures, salinity, pH, and other stress factors, by generating various bioactive metabolites. These bioactive secondary metabolites have strong potential for use as antifungal agents. Due to the shortage of antifungal medications and the emergence of treatment resistance in C. auris, identifying new therapeutics from synthetic bacterial components or natural materials has become a necessity. Natural molecules have numerous advantages over synthetic substances, including structural variation and low toxicity. Few next-generation sequence-based investigations have been carried out on anti-Candidozyma auris bacterial species to identify potential therapeutic candidates. Therefore, the aim of this study is to identify biosynthetic gene clusters from marine bacteria using next-generation sequencing to discover novel drug compounds against multidrug-resistant C. auris. Methods: More than 68 isolates were collected from various marine environments using standard techniques. All isolates were tested against the multidrug-resistant C. auris. Scanning electron microscopy was utilized to investigate the cell membrane rupture caused by defused metabolites of the IRMCESH58L bacterium in C. auris. The Vibrio sp. IRMCESH58L genome was sequenced using long-read nanopore sequencing technology. Results: The bacterial strain IRMCESH58L, isolated from a fish liver sample, showed the highest and most constant activity against C. auris. An in vitro toxicity test found that IRMCESH58L had no cell cytotoxicity against HFF-1 cells. The assembled plasmid-free genome is 6,556,025 bp (48.93% G+C), with an N50 of 909243. Comparative analysis confirmed its relation to Vibrio alginolyticus. Conclusions: Whole-genome analysis of the native bacterial strain IRMCESH58L revealed various biosynthetic gene clusters, including those involved in surfactin’s biosynthesis of putative natural anti-C. auris chemicals, but no pathogenic protein-coding genes, emphasizing the importance of marine bacteria in the fight against C. auris. Following this in vivo study, therapeutic targets will later be selected for further pre-clinical studies. Full article

Background: Bacteriophages and phage-derived lytic enzymes are increasingly considered to be targeted antimicrobial tools in aquaculture; however, their compatibility with non-target microbial communities under hatchery-relevant conditions remains insufficiently characterized. Objectives This study evaluates the impact of a lytic phage (CH20) and a phage-derived lysin (LysVp1), applied under previously validated conditions for rapid Vibrio control, on the microbiota associated with seawater, rotifers, and zebrafish larvae challenged with Vibrio alginolyticus GV09. Methods: Treatments were independently applied to each biological matrix using short exposure times representative of hatchery practices, intentionally capturing the critical window during which microbial transfer from live feed to larvae occurs. Microbial communities were analyzed using 16S rRNA gene sequencing, with DNA- and RNA-derived datasets evaluated separately. Results: Alpha diversity indices were compared using appropriate statistical tests, while beta diversity was assessed using Aitchison distance, PERMANOVA, and dispersion analyses, and differential abundance was evaluated using ANCOM-BC2. Alpha diversity metrics showed no significant differences among treatments across all matrices, indicating the preservation of microbial richness and diversity. Beta diversity patterns differed according to the nucleic acid source, with RNA-based analyses revealing treatment-associated shifts in rotifer and water microbiota that were not consistently detected at the DNA level. In zebrafish larvae, neither phage nor lysin treatment significantly altered overall community structure, although dispersion effects reflected limitations related to sample size. Conclusions: Overall, these results indicate that phage CH20 and lysin LysVp1 exert minimal impact on alpha diversity and limited, context-dependent effects on microbial community structure, supporting their microbiota-safe potential for aquaculture applications. Full article

The misuse of antibacterial agents has contributed to the growing prevalence of antibiotic resistance, highlighting an urgent need to explore alternative anti-infection therapeutic strategies. Antimicrobial peptides (AMPs) are naturally occurring molecules. They exhibit broad-spectrum antimicrobial activity and represent promising candidates for the development of novel therapeutics. A cysteine-rich antimicrobial peptide was identified and characterized from the genome of Tigriopus japonicus and designated “TjRcys1”. The precursor form of TjRcys1 comprises 96 amino acids. Structural analyses of TjRcys1 revealed random coils, two α-helices, and two β-strands. Recombinant TjRcys1 had inhibitory effects upon Staphylococcus aureus and Bacillus sp. T2, with a minimum inhibitory concentration of 64 μM for both. TjRcys1 did not show complete inhibition against Vibrio alginolyticus, Klebsiella pneumoniae, or Aeromonas hydrophila at 64 μM, but it did slow their growth rate. TjRcys1 could disrupt the permeability of the cell membrane of S. aureus. Transcriptomic analyses indicated that TjRcys1 could interfere with the ribosome biosynthesis and nucleotide metabolism of K. pneumoniae. Our results provide a valuable reference for the development of new AMPs and optimization of their design. Full article

During four outbreaks in 2023 and 2024, samples from pond-reared Nile tilapia were taken from different farms located in Kafr Elsheikh governorate, Egypt. Samples were submitted for laboratory examinations. Diseased fish exhibited bacterial septicemia and some cases died without showing any clinical signs. A total of 30 bacterial isolates were isolated and identified. Of these isolates, 57% were identified as Gram-positive bacteria, whereas the remaining 43% were identified as Gram-negative bacteria. PCR targeting the 16S rRNA gene and genome sequencing confirmed five bacterial isolates as Aeromonas veronii (30%), Vibrio alginolyticus (13.3%), Enterococcus faecalis (23.3%), Aerococcus viridans (16.7%), and Staphylococcus epidermidis (16.7%). The NCBI GenBank accession numbers of these strains were (PV018985) for A. veronii, (PV016854) for V. alginolyticus, (PV013413) for E. faecalis, (PV032005) for A. viridans, and (PV012491) for Staph. epidermidis. The antibiogram revealed that the bacterial strains showed resistance to most of the antibiotics tested. A. viridans exhibited resistance to nearly all the antibiotics except for intermediate sensitivity to ciprofloxacin and amoxycillin/clavulanic acid. However, A. veronii showed high sensitivity to amoxycillin/clavulanic acid, oxytetracycline, kanamycin, and trimethoprim/sulfamethoxazole and intermediate susceptibility to ciprofloxacin. Similarly, E. faecalis showed high susceptibility to amoxycillin/clavulanic acid, ciprofloxacin, and trimethoprim/sulfamethoxazole in addition to intermediate sensitivity to ampicillin and kanamycin. Furthermore, Staph. epidermidis strain was highly susceptible to ampicillin, amoxycillin/clavulanic acid, oxytetracycline, novobiocin, and trimethoprim/sulfamethoxazole and was partially sensitive to kanamycin and ciprofloxacin. To conclude, summer mortalities recorded in farmed tilapia were closely related to a multifactorial bacterial origin with different sensitivity to antibiotic discs. Full article