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NorR, as a single-target regulator, has been demonstrated to be involved in NO detoxification in bacteria under anaerobic conditions. Here, the norR gene was identified and deleted in the genome of Vibrio alginolyticus. The results showed that deletion of norR in Vibrio alginolyticus led to lower swarming motility and more biofilm formation on aerobic condition. Moreover, we proved that NorR from E. coli had a similar function in controlling motility. NorR overexpression led to increased resistance to oxidative stress and tetracycline. We also observed a reduced ability of the NorR-overexpressing strain to adapt to iron limitation condition. Transcriptome analysis showed that the genes responsible for bacterial motility and biofilm formation were affected by NorR. The expressions of several sigma factors (RpoS, RpoN, and RpoH) and response regulators (LuxR and MarR) were also controlled by NorR. Furthermore, Chip-qPCR showed that there is a direct binding between NorR and the promoter of rpoS. Based on these results, NorR appears to be a central regulator involved in biofilm formation and swarming motility in Vibrio alginolyticus. [ABSTRACT FROM AUTHOR]

Understanding the efficacy of antimicrobials against pathogens from clinical samples is critical for their responsible use. The manuscript presents in vitro efficacy and antimicrobial resistance (AMR) genes in seven species of fish pathogens from the disease outbreaks of Indian aquaculture against oxytetracycline, florfenicol, oxolinic acid, and enrofloxacin. In vitro efficacy was evaluated by minimum inhibitory concentration and minimum bactericidal concentration. The gene-specific PCR screened AMR genes against quinolones (qnrA, qnrB , and qnrS) and tetracyclines (tetM, tetS, tetA, tetC, tetB, tetD, tetE, tetH, tetJ, tetG , and tetY). The results showed that Aeromonas veronii (45%) showed the maximum resistance phenotype, followed by Streptococcus agalactiae (40%), Photobacterium damselae (15%), Vibrio parahaemolyticus (10%), and Vibrio vulnificus (5%). There was no resistance among Vibrio harveyi and Vibrio alginolyticus against the tested antimicrobials. The positive association between tetA, tetB, tetC, tetM , or a combination of these genes to oxytetracycline resistance and qnrS to quinolone resistance indicated their potential in surveillance studies. The prevalence of resistance phenotypes (16.43%) and evaluated AMR genes (2.65%) against aquaculture antimicrobials was low. The resistance phenotype pattern abundance was 0.143. All the isolates showed susceptibility to florfenicol. The results help with the appropriate drug selection against each species in aquaculture practices. [ABSTRACT FROM AUTHOR]

Seven new oleanane- type triterpene saponins, lysimaponins A-G, were isolated from aerial parts of Lysimachia laxa Baudo. Their chemical structures have been elucidated by analysis of spectroscopic and chemical methods. All compounds were evaluated for their antibacterial effects against Microcystis aeruginosa, Vibrio parahaemolyticus, V. harveyi, V. vulinificus, V. cholerae , and V. alginolyticus. All compounds showed potent antibacterial activities against the cyanobacteria M. aeruginosa with IC50 values ranging from 14.4 ± 1.2 to 35.3 ± 2.2 µg/mL. Compounds 1, 2, 4 - 7 inhibited V. parahaemolyticus with MIC values ranging from 64 to 256 µg/mL. The results suggested that saponins from L. laxa could be potential anti-cyanobacteria agents. [ABSTRACT FROM AUTHOR]

Immunosaccharides such as β-glucans and fructooligosaccharide (FOS) strengthen the host's immune responses. This study examined the influences of three levels of the β-1, 3 glucan-FOS combination on Red tilapia performance. Four diets were prepared: T0 as a control, while T0.5, T1, and T1.5 indicate the addition of β-1, 3 glucan-FOS mixture at 0.5%, 1%, and 1.5%, respectively. Then, 240 red Tilapia (Oreochromis niloticus x O. mossambicus) (weight 6.1 ± 0.07gm) were allocated into four groups, and every group was fed tested diets for 56 days. After the feeding trial, growth parameters, erythrogram profile, liver and kidney function testes, glucose, histopathological analysis, and gene expressions for antioxidants, catalase (CAT) and glutathione peroxidase (GPX) besides growth hormone (GH) and insulin-like growth factor 1 IGF1-related markers were assessed. Red tilapia fed T0.5, T1, and T1.5 exhibited consistent growth, survival rate, and homeostasis compared with the control group. Different supplement concentrations displayed varying levels of responses. The hepatorenal biomarkers (alanine transaminase (ALT = SGPT) and aspartate transaminase (AST = SGOT), alkaline phosphatase (ALP = ALK), urea, and creatinine) and glucose showed a significant reduction in the supplemented groups compared with the control, especially in the T1 and T1.5 groups. The intestinal morphometric study revealed that fish group fed on T1 represented the best result, whereas group T1.5, followed by group T0.5, was moderately treated, compared to the control. The real-time quantitative reverse transcription PCR (qRT-PCR) analysis displayed up-regulated expression of antioxidant and growth-correlated genes in the T1.5 groups. After 56 days, the β-1,3 glucan-FOS fed groups also exhibited an increase in survival rates compared to the control when challenged with a pathogenic Vibrio alginolyticus. Current findings suggest that inclusion of β-1,3 glucan-FOS in diets could enhance red tilapia biochemical parameters, growth, and protection against pathogenic V. alginolyticus infection. [ABSTRACT FROM AUTHOR]

Recently, medical plants have been widely used as replacements for antibiotics in disease treatment. Because of its multiple medicinal uses, peppermint (Mentha piperita, MP) is a common herbal remedy. In the present study, MP powder was used as a feed additive to assess growth performance; hematological; biochemical and immune parameters; intestinal histology; and interleukin 1β (IL-1β) gene expression, as well as protection against Vibrio alginolyticus infection in Oreochromis niloticus. O. niloticus (n = 120, 25.66 ± 0.16 g) were fed diets containing 0 (CTR), 2, 3, or 4% MP for 60 days. The results revealed that the inclusion of 2% MP significantly improved the growth indices, intestinal morphological parameters, and reduced the feed conversion ratio. The 2% MP treatment significantly (P < 0.05) increased hematological parameters (red blood cell (RBC) count, white blood cell (WBC) count, packed cell volume% (PCV%), hemoglobin) compared with those of the CTR (P < 0.05). Additionally, feeding fish 2% MP diets decreased the levels of cholesterol and LDL (low-density lipoprotein). There were significant increases in immune responses (serum protein and phagocytic activity and index) and non-significant increases in the expression of IL-1β in the 2% MP group comparing with the other groups and the CTR group (P < 0.05). At the end of the feeding trial (60 days), fish were challenged with a virulent strain of Vibrio alginolyticus and the results showed that the mortality rate decreased in the 2% MP treatment group, followed by the 3% and 4% MP groups. Overall, the results revealed that the dietary inclusion of 2% MP can exhibit growth-promoting and immunostimulant effects for sustainable aquaculture. [ABSTRACT FROM AUTHOR]

The RNA binding protein is crucial for gene regulation at the post transcription level. In this study, functions of the DUF1127-containing protein and ProQ, which are RNA-binding proteins, were revealed in Vibrio alginolyticus. DUF1127 deletion increased the ability of biofilm formation, whereas ProQ deletion reduced the amount of biofilm. Moreover, extracellular proteinase secretion was significantly reduced in the DUF1127 deletion strain. ProQ, not DUF1127-containing protein, can help the cell to defense oxidative stress. Deletion of DUF1127 resulted in a higher ROS level in the cell, however, ProQ deletion showed no difference. RNA-seq unveiled the expression of genes involved in extracellular protease secretion were significantly downregulated and biofilm synthesis-related genes, such as rbsB and alsS, were differentially expressed in the DUF1127 deletion strain. ProQ affected the expression of genes involved in biofilm synthesis (flgC and flgE), virulence (betB and hutG), and oxidative stress. Moreover, the DUF1127-containing and ProQ affected the mRNA levels of various regulators, such as LysR and BetI. Overall, our study revealed that the DUF1127-containing protein and ProQ have crucial functions on biofilm formation in V. alginolyticus. [ABSTRACT FROM AUTHOR]

Acetylation modification has become one of the most popular topics in protein post-translational modification (PTM) research and plays an important role in bacterial virulence. A previous study indicated that the virulence-associated caseinolytic protease proteolytic subunit (ClpP) is acetylated at the K165 site in Vibrio alginolyticus strain HY9901, but its regulation regarding the virulence of V. alginolyticus is still unknown. We further confirmed that ClpP undergoes lysine acetylation (Kace) modification by immunoprecipitation and Western blot analysis and constructed the complementation strain (C-clpP) and site-directed mutagenesis strains including K165Q and K165R. The K165R strain significantly increased biofilm formation at 36 h of incubation, and K165Q significantly decreased biofilm formation at 24 h of incubation. However, the acetylation modification of ClpP did not affect the extracellular protease (ECPase) activity. In addition, we found that the virulence of K165Q was significantly reduced in zebrafish by in vivo injection. To further study the effect of lysine acetylation on the pathogenicity of V. alginolyticus, GS cells were infected with four strains, namely HY9901, C-clpP, K165Q and K165R. This indicated that the effect of the K165Q strain on cytotoxicity was significantly reduced compared with the wild-type strain, while K165R showed similar levels to the wild-type strain. In summary, the results of this study indicate that the Kace of ClpP is involved in the regulation of the virulence of V. alginolyticus. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of this study is to provide ideas and theoretical guidance for green, environmentally friendly and efficient "bacteriostasis with bacteria" technology. Design/methodology/approach: In this paper, a beneficial strain of bacteria was extracted and purified from marine mud. Weight-loss test, morphological observation and electrochemical test were used to systematically study the effect of sulfate-reducing bacteria (SRB)-induced corrosion inhibition on X65 steel in simulated offshore oil field production water. Findings: The results showed that a beneficial strain was selected and identified as Vibrio alginolyticus. Under the condition of co-culture of SRB, the average corrosion rate of X65 steel was significantly reduced. In the mixed bacterial system, the surface of X65 steel samples was relatively flat, and the structure of biofilm and corrosion product film was dense. The number of corrosion pits, the average diameter and depth of corrosion pits were significantly reduced. The localized corrosion of X65 steel was significantly inhibited. Originality/value: The complex and changing marine environment makes the corrosion problem of marine steel increasingly severe, and the microbiologically influenced corrosion (MIC) caused by SRB is particularly serious. The research and development of environmentally friendly corrosion protection technology is a long-term and difficult problem. The use of beneficial microorganisms to control MIC is a green and efficient anticorrosion measure. Compared with terrestrial microorganisms, marine microorganisms can adapt to complex environments, and their metabolites exhibit special biological activities. The use of marine beneficial bacteria can inhibit SRB activity to achieve the corrosion inhibition effect. [ABSTRACT FROM AUTHOR]

Abstract NorR, as a single-target regulator, has been demonstrated to be involved in NO detoxification in bacteria under anaerobic conditions. Here, the norR gene was identified and deleted in the genome of Vibrio alginolyticus. The results showed that deletion of norR in Vibrio alginolyticus led to lower swarming motility and more biofilm formation on aerobic condition. Moreover, we proved that NorR from E. coli had a similar function in controlling motility. NorR overexpression led to increased resistance to oxidative stress and tetracycline. We also observed a reduced ability of the NorR-overexpressing strain to adapt to iron limitation condition. Transcriptome analysis showed that the genes responsible for bacterial motility and biofilm formation were affected by NorR. The expressions of several sigma factors (RpoS, RpoN, and RpoH) and response regulators (LuxR and MarR) were also controlled by NorR. Furthermore, Chip-qPCR showed that there is a direct binding between NorR and the promoter of rpoS. Based on these results, NorR appears to be a central regulator involved in biofilm formation and swarming motility in Vibrio alginolyticus.

Abstract The RNA binding protein is crucial for gene regulation at the post transcription level. In this study, functions of the DUF1127-containing protein and ProQ, which are RNA-binding proteins, were revealed in Vibrio alginolyticus. DUF1127 deletion increased the ability of biofilm formation, whereas ProQ deletion reduced the amount of biofilm. Moreover, extracellular proteinase secretion was significantly reduced in the DUF1127 deletion strain. ProQ, not DUF1127-containing protein, can help the cell to defense oxidative stress. Deletion of DUF1127 resulted in a higher ROS level in the cell, however, ProQ deletion showed no difference. RNA-seq unveiled the expression of genes involved in extracellular protease secretion were significantly downregulated and biofilm synthesis-related genes, such as rbsB and alsS, were differentially expressed in the DUF1127 deletion strain. ProQ affected the expression of genes involved in biofilm synthesis (flgC and flgE), virulence (betB and hutG), and oxidative stress. Moreover, the DUF1127-containing and ProQ affected the mRNA levels of various regulators, such as LysR and BetI. Overall, our study revealed that the DUF1127-containing protein and ProQ have crucial functions on biofilm formation in V. alginolyticus.

Abstract This study investigated a disease outbreak characterized by caligid copepod infestations and subsequent secondary bacterial infections in European seabass (Dicentrarchus labrax) and flathead grey mullet (Mugil cephalus) cultivated at a private facility in the Deeba Triangle region of Egypt. Moribund fish displayed brown spots on the skin, tongue, and gills, along with lethargy and excess mucus. The fish suffered severe infections, exhibiting external hemorrhages, ulcers, and ascites. The fish had pale, enlarged livers with hemorrhaging. Comprehensive parasitological, bacteriological, molecular, immunity and histopathological analyses were conducted to identify the etiological agents and pathological changes. Caligid copepod infestation was observed in wet mounts from the buccal and branchial cavities of all examined fish, and the caligids were identified as Caligus clemensi through COI gene sequencing and phylogenetic analysis. Vibrio alginolyticus was confirmed as a secondary bacterial infection through biochemical tests, recA gene sequencing, and phylogenetic analyses. Antibiotic susceptibility testing revealed resistance to β-lactams, aminoglycosides, and trimethoprim-sulfamethoxazole in V. alginolyticus isolates. Upregulation of the inflammatory marker IL-1β in gill and skin tissues indicated a robust cell-mediated immune response against the pathogens. Histopathological examination revealed severe tissue damage, hyperplasia, hemorrhage, and congestion in the gills, along with hepatocellular degeneration and steatosis in the liver, providing initial insights into this outbreak. A comprehensive therapeutic regimen was implemented, comprising prolonged hydrogen peroxide immersion baths, followed by the application of the nature-identical plant-based compound Lice-less and probiotic Sanolife Pro-W supplementation. This integrated approach effectively eliminated C. clemensi infestations, controlled secondary bacterial infections, and restored fish health, reducing morbidity and mortality rates to minimal levels.

The study examined the effects of different probiotics on growth, survival, and immune gene expression in Macrobrachium rosenbergii post-larvae (PL). The PL were fed with MY-1, Enterotrophotic (ENT), and MY-1 + Enterotrophotic (MY-1 + ENT) enriched artemia for a duration of 20 days, with three feedings per day. Throughout the experiment, water quality parameters were closely monitored. Results showed that the postlarvae fed with MY-1 + ENT exhibited better growth in terms of size and weight compared to the other groups. On the 21st day, both the control and treated PL were exposed to pathogenic Vibrio alginolyticus. The groups administered with MY-1 + ENT had higher survival rates compared to the other treatment and control groups. Moreover, the expression levels of immune-related genes such as cytochrome oxidase (COX), prophenoloxidase (proPO), peroxinectin (PE), antilipopolysaccharide (ALF), and lipopolysaccharide glucan binding protein (LGBP) were examined through qPCR. The results indicated a variable expression pattern suggesting that the application of probiotics enhances the expression and regulation of immune genes in the event of infection by V. alginolyticus. The differences in gene expression highlight the opportunity to develop Immune Gene Expression Finger Printing, which may act as an effective tool for evaluating probiotics and immunostimulants by assessing their ability to modulate immune responses in M. rosenbergii. The study warrants field-level validation of the findings to facilitate the adoption in the hatchery technology for scampi seed production too. [ABSTRACT FROM AUTHOR]

【Objective】The study aims to screen the formula of vacuum freeze-drying protective agent for Vibrio alginolyticus, optimize the freeze-drying process conditions, and explore the influence of freeze-drying on the virulence and drug sensitivity of the strains, with a view to providing a theoretical basis for the application of freeze-drying in the preservation of the strains.【Method】With Vibrio alginolyticus HY9901 as the research object, the vacuum freeze-drying technology was used to freeze-dry the strains. Through single factor test and orthogonal test, the effects of six single cryoprotectants (L-glutamate sodium, trehalose, skim milk, glucose, mannitol and glycine) on the activity of the strains were investigated, and the optimal formula of cryoprotectants was selected. The influences of four factors, namely, concentration of bacterial solution, pre-freezing time, pre-freezing temperature and rehydration medium, on the freeze-drying process of strains were studied, and the freeze-drying process conditions for Vibrio alginolyticus were optimized. At the same time, storage test, LD50 test and antimicrobial susceptibility test were used to investigate the changes in the activity of lyophilized strains and their of biological characteristics.【Result】There were significant differences in the protective effects of 6 cryoprotectants on lyophilized Vibrio alginolyticus, and the effects from strongest to weakest showed as: L-glutamate sodium, trehalose, skim milk, glucose, mannitol and glycine. The optimum freeze-drying compound formula of cryoprotectants was trehalose 50 g/L+ L-glutamate sodium 50 g/L+ skim milk 50 g/L, with a survival rate of 62.12%, which was significantly different from that of single cryoprotectant. The optimal conditions of freeze-drying process were as follows: bacterial solution concentration 1×108-109 CFU/mL, pre-freezing time 3 h, pre-freezing temperature -80 ℃, rehydration medium PBS or 50 g/L skim milk, under which the activity of the strains could reach the maximum of 63.72%. There was no significant difference in the activity of the strains during 60 days of storage under optimal freeze-drying conditions. The LD50 values of the strains before and after freeze-drying were 1.22×108 CFU/mL and 1.20×108 CFU/mL, respectively, with no significant difference in LD50. There was no significant difference in the diameter of the antimicrobial inhibition zone before and after freeze-drying, and the strains were sensitive to florfenicol, chloramphenicol, ciprofloxacin and other drugs. They were moderately sensitive to neomycin and kanamycin, and resistant to drugs like amimcana and piperacillin.【Conclusion】The freeze-drying formula for Vibrio alginolyticus HY9901 is trehalose 50 g/L+ L-glutamate sodium 50 g/L+ skim milk 50 g/L, and the freeze-drying process conditions are: bacterial solution concentration 1×108-109 CFU/mL, pre-freezing time 3 h, pre-freezing temperature -80 ℃, rehydration medium PBS or 50 g/L skim milk. The fomula and process for this strain are reasonable and feasible, with good storage and stability of biological characteristics.

Vibrio alginolyticus causes substantial economic losses in the aquaculture industry. With the rise of multidrug-resistant Vibrio strains, phages present a promising solution. Here, a novel lytic Vibrio phage, vB_ValC_RH2G (RH2G), that efficiently infects the pathogenic strain V. alginolyticus ATCC 17749T, was isolated from mixed wastewater from an aquatic market in Xiamen, China. Transmission electron microscopy revealed that RH2G has the morphology of Siphoviruses, featuring an icosahedral head (73 ± 2 nm diameter) and long noncontractile tail (142 ± 4 nm). A one-step growth experiment showed that RH2G had a short latent period (10 min) and a burst size of 48 phage particles per infected cell. Additionally, RH2G was highly species-specific and was relatively stable at 4–55 °C and pH 4–10. A genomic analysis showed that RH2G has a 116,749 bp double-stranded DNA genome with 43.76% GC content. The intergenomic similarity between the genome sequence of RH2G and other phages recorded in the GenBank database was below 38.8%, suggesting that RH2G represents a new genus. RH2G did not exhibit any virulence or resistance genes. Its rapid lysis capacity, lytic activity, environmental resilience, and genetic safety suggested that RH2G may be a safe candidate for phage therapy in combatting vibriosis in aquaculture settings. [ABSTRACT FROM AUTHOR]

Introduction: Vibrio alginolyticus is a Gram-negative, rod-shaped bacterium belonging to the family of Vibrionaceae, a common pathogen in aquaculture animals, However, studies on its impact on Scylla serrata (mud crabs) are limited. In this study, we isolated V. alginolyticus SWS from dead mud crab during a disease outbreak in a Hong Kong aquaculture farm, which caused up to 70% mortality during summer. Methods: Experimental infection and histopathology were used to investigate the pathogenicity of V. alginolyticus SWS in S. serrata and validate Koch's postulates. Comprehensive whole-genome analysis and phylogenetic analysis antimicrobial susceptibility testing, and biochemical characterization were also performed. Results: Our findings showed that V. alginolyticus SWS caused high mortality (75%) in S. serrata with infected individuals exhibiting inactivity, loss of appetite, decolored and darkened hepatopancreas, gills, and opaque muscle in the claw. Histopathological analysis revealed tissue damage and degeneration in the hepatopancreas, gills, and claw muscle suggesting direct and indirect impacts of V. alginolyticus SWS infection. Conclusions: This study provides a comprehensive characterization of V. alginolyticus SWS as an emerging pathogen in S. serrata aquaculture. Our findings underscore the importance of ongoing surveillance, early detection, and the development of targeted disease management strategies to mitigate the economic impact of vibriosis outbreaks in mud crab aquaculture. [ABSTRACT FROM AUTHOR]

Quorum sensing (QS) orchestrates many bacterial behaviors, including virulence and biofilm formation, across bacterial populations. Nevertheless, the underlying mechanism by which QS regulates capsular polysaccharide (CPS)-dependent phage-bacterium interactions remains unclear. In this study, we report that QS upregulates the expression of CPS-dependent phage receptors, thus increasing phage adsorption and infection rates in Vibrio alginolyticus. We found that QS upregulated the expression of the ugd gene, leading to increased synthesis of Autographiviridae phage receptor CPS synthesis in V. alginolyticus. The signal molecule autoinducer-2 released by Vibrio from different sources can potentially enhance CPS-dependent phage infections. Therefore, our data suggest that inhibiting QS may reduce, rather than improve, the therapeutic efficacy of CPS-specific phages. [ABSTRACT FROM AUTHOR]

Vibriosis in shrimp farms poses potential risks to the industry's sustainability and the consumers' health. Little is known about the dynamics and variation of Vibrio spp--population in shrimp production in Sarawak, Malaysia. The apparent prevalence of three Vibrio species, V. parahaemolyticus, V. cholerae, and V. alginolyticus, were investigated in water (n=12), sediment (n=12), shrimp (n=12), and effluent (n=12) samples collected from two ponds in a coastal shrimp farm during one production cycle. Multiplex-PCR using specific primer sets showed the presence of the three Vibrio species. Based on the results, V. parahaemolyticus was the most prevalent in all four samples, with a contamination rate of 97.92% (95% CI: 89. 10 to 99.89%), while V. cholerae and V. alginolyticus had a contamination rate of 47.92% (95% CI: 34.37 to 61.67%) and 25.0% (95% CI: 14.92 to 38.78%), respectively. High Vibrio load in the shrimp farm is due to favourable environmental factors, such as optimal temperatures, salinity, and pH ranges for the growth of these species. The study's findings offer important preliminary insights into the prevalence and distribution of these pathogenic Vibrio spp., within a shrimp farm in Kuching, Sarawak. This study serves as a potential model for monitoring Vibrio spp. prevalence in other shrimp farms across Sarawak, thereby addressing the scarcity of data on prevalence in the region. [ABSTRACT FROM AUTHOR]

Vibrio vulnificus, a significant marine pathogen, undergoes opaque (Op)-translucent (Tr) colony switching based on whether capsular polysaccharide (CPS) is produced. CPS phase variation is sometime accompanied by genetic variation or down-regulation of particular genes, such as wzb. In addition, CPS prevents biofilm formation and is important to the virulence of V. vulnificus. However, the extent to which there is a difference in gene expression between Tr and Op colonies and the impact of CPS phase variation on other behaviors of V. vulnificus remain unknown. In this work, the data have shown that CPS phase variation of V. vulnificus is affected by incubation time. Tr and Op strains exhibited similar growth rates. However, Tr strains had enhanced biofilm formation capacities but reduced swimming motility compared to Op strains. The RNA-seq assay revealed 488 differentially expressed genes, with 214 downregulated and 274 upregulated genes, between Tr and Op colonies. Genes associated with Tad pili and CPS were downregulated, whereas those involved in flagellum were upregulated, in Tr colonies compared with Op colonies. In addition, 9 putative c-di-GMP metabolism-associated genes and 28 genes encoding putative regulators were significantly differentially expressed, suggesting that CPS phase variation is probably strictly regulated in V. vulnificus. Moreover, 8 genes encoding putative porins were also differentially expressed between the two phenotypic colonies, indicating that bacterial outer membrane was remodeled during CPS phase variation. In brief, this work highlighted the gene expression profiles associated with CPS phase variation, but more studies should be performed to disclose the intrinsic mechanisms in the future. [ABSTRACT FROM AUTHOR]

Introduction: This study characterized Vibrio alginolyticus isolated from seafood and freshwater products in China (2020). Methods and Results: In total, 122 (95.31%) V. alginolyticus isolates were resistant to at least 1 antibiotic category, and 2 (1.56%) isolates were resistant to at least 3 antibiotic categories and belong to multi-drug resistance (MDR) isolates. A high prevalence rate was observed to be blaCARB (98.04%) encoding beta-lactam resistance, followed by tet (97.06%) encoding tetracycline resistance and fos (4.90%) encoding resistance to fosfomycin. Among the 57 V. alginolyticus isolates, the commonest virulence genes were type III secretion system translocated gene vopD, vopB, and vcrH (54.4%, 31/57), type III secretion system regulated gene tyeA (54.39%), followed by vscI and vscF (50.88%) encoded type III secretion system inner rod protein and needle protein, respectively. Multilocus sequence typing (MLST) showed considerable genetic diversity, with 34 distinct sequence types (STs) identified among 55 isolates. ST421 (n = 5), ST166 (n = 4), ST523 (n = 3), ST516 (n = 3), and ST507 (n = 3) were dominant STs among 55 V. alginolyticus isolates. Discussion: These findings highlight the widespread occurrence of V. alginolyticus in both freshwater and seafood products, underscoring the critical need for vigilant monitoring of these bacteria. Such measures are essential for ensuring effective food safety management and safeguarding public health. [ABSTRACT FROM AUTHOR]

Multiple bacterial genera take advantage of the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin to invade host cells. Secretion of the MARTX toxin by Vibrio vulnificus, a deadly opportunistic pathogen that causes primary septicemia, the precursor of sepsis, is a major driver of infection; however, the molecular mechanism via which the toxin contributes to septicemia remains unclear. Here, we report the crystal and cryo-electron microscopy (EM) structures of a toxin effector duet comprising the domain of unknown function in the first position (DUF1)/Rho inactivation domain (RID) complexed with human targets. These structures reveal how the duet is used by bacteria as a potent weapon. The data show that DUF1 acts as a RID-dependent transforming NADase domain (RDTND) that disrupts NAD+ homeostasis by hijacking calmodulin. The cryo-EM structure of the RDTND-RID duet complexed with calmodulin and Rac1, together with immunological analyses in vitro and in mice, provide mechanistic insight into how V. vulnificus uses the duet to suppress ROS generation by depleting NAD(P)+ and modifying Rac1 in a mutually-reinforcing manner that ultimately paralyzes first line immune responses, promotes dissemination of invaders, and induces sepsis. These data may allow development of tools or strategies to combat MARTX toxin-related human diseases. The MARTX toxins secreted by clinical V. vulnificus strains release the DUF1-RID effector duet in infected host cells. Here, the authors show that the duet hijacks calmodulin and Rac1, respectively, and transforms into a potent weapon to promote sepsis. [ABSTRACT FROM AUTHOR]

Transglutaminase (TGase) is a key enzyme that mediates hemolymph coagulation and is thought to contribute to the elimination of pathogenic microorganisms in invertebrates. The objective of this study was to elucidate the involvement of TGase in insect immune responses via functional analysis of this enzyme in the oriental armyworm, Mythimna separata, using recombinant proteins and RNA interference technique. We identified two TGase genes, mystgase1 and mystgase2, in Mythimna separata and found that both genes are expressed in all surveyed tissues in M. separata larvae. Significant changes were induced in hemocytes following Escherichia coli injection. Injection of Gram‐positive bacteria (Micrococcus luteus) and Gram‐negative bacteria (Escherichia coli and Serratia marcescens) into larvae triggered a time‐specific induction of both mystgase1 and mystgase2 in hemocytes. Recombinant MysTGase1 and MysTGase2 proteins bound to both E. coli and M. luteus, localizing within bacterial clusters and resulting in agglutination in a Ca2+‐dependent manner. The hemocytes of larvae injected with recombinant MysTGase1 or MysTGase2 exhibited enhanced phagocytic ability against E. coli, improved in vivo bacterial clearance, and increased resistance to S. marcescens, decreasing larval mortality rate. Conversely, RNA interference targeting mystgase1 or mystgase2 significantly reduced hemocyte phagocytic capability, decreased bacterial clearance, and increased susceptibility to S. marcescens infection, thereby increasing larval mortality rate. The findings of this study are anticipated to expand our understanding of the function of TGases within insect immune responses and may contribute to developing new pest control strategies. [ABSTRACT FROM AUTHOR]

【Objective】In October 2023, skin ulceration, subcutaneous hemorrhage and enteritis appeared in a breeding base of Epinephelus coioides in Shenzhen, China. We clarified the causative organisms that triggered the sudden death of E. coioides and their biological characteristics, and screened the disinfectant that effectively inhibited the causative organisms, with an aim to provide theoretical guidance for the scientific prevention and control of E. coioides infected with Vibrio alginolyticus. 【Method】The pathogen of V. alginolyticus was isolated from the diseased tissues of the dying E. coioides by streak plate method, the morphological characteristics, physiological and biochemical properties, phylogenetic tree, virulence genes and resistance genes as well as drug sensitivity of the pathogen were investigated, and regression infection tests were carried out. 【Result】A dominant strain was isolated from the diseased tissues of E. coioides and named as strain SZGP226. The results of phylogenetic analysis showed that, the sequence of strain SZGP226 was homologous to V. alginolyticus HH101307, IB1 and Xmb015, with a similarity of 99.72%. The strain was identified as positive in physiological and biochemical indexes such as sucrose, mannitol, glucose, urea, oxidase activity and V-P, and it also carried five virulence genes, namely, tlh, toxR, ctsA, sto and vpi. The results of drug sensitivity showed that strain SZGP226 was resistant to 9 antibiotics like ampicillin and benzoxacillin, and carried 9 resistance genes including qnrB, fl oR, tetC, tetM, qnrD, gyrA, Sul1, SHV, and Oxa-24. The screening results of disinfectants showed that, sodium hydroxide had the best bactericidal effect, and its minimal inhibition concentration (MIC) was 8 mg/mL. The regression infection test showed that strain SZGP226 was strongly pathogenic to the E. coioides, with a LC50 of 1.63×107 CFU/mL, and it could cause obvious pathological damage to the liver, kidney and spleen of the E. coioides. 【Conclusion】V. alginolyticus was the main pathogenic bacterium for the sudden death of the E. coioides, and its causative genes included tlh, toxR, ctsA, sto and vpi, and the LC50 was 1.63×107 CFU/mL. Sodium hydroxide (3 mg/mL) showing the best bactericidal effect on strain bactericidal effect could be selected to clean up the culture environment before aquaculture was carried out. [ABSTRACT FROM AUTHOR]

This study delves into the genomic features of 10 Vibrio strains collected from deep-sea hydrothermal vents in the Pacific Ocean, providing insights into their evolutionary history and ecological adaptations. Through sequencing and pan-genome analysis involving 141 Vibrio species, we found that deep-sea strains exhibit larger genomes with unique gene distributions, suggesting adaptation to the vent environment. The phylogenomic reconstruction of the investigated isolates revealed the presence of 2 main clades: The first is monophyletic, consisting exclusively of Vibrio alginolyticus , while the second forms a monophyletic clade comprising both Vibrio antiquarius and Vibrio diabolicus species, which were previously isolated from deep-sea vents. All strains carry virulence and antibiotic resistance genes related to those found in human pathogenic Vibrio species which may play a wider ecological role other than host infection in these environments. In addition, functional genomic analysis identified genes potentially related to deep-sea survival and stress response, alongside candidate genes encoding for novel antimicrobial agents. Ultimately, the pan-genome we generated represents a valuable resource for future studies investigating the taxonomy, evolution, and ecology of Vibrio species. [ABSTRACT FROM AUTHOR]

Vibrio alginolyticus is the causative agent of vibriosis, a common bacterial infection in grouper aquaculture that is associated with the development of haemorrhagic and non‐haemorrhagic ulcerations on the fish. In the present study, comparative proteome analysis was performed on serum samples from Vibrio‐resistant and Vibrio‐susceptible grouper. Samples were analysed using high‐throughput LC‐MS/MS and identified 2770 unique peptides that corresponded to 344 proteins. Subsequent analysis identified 21 proteins that were significantly up‐regulated in the resistant group compared to the control and the susceptible groups. Those proteins are associated with immunostimulatory effects, signalling and binding cascade, metabolism, and maintaining tissue integrity and physiological condition. Besides, potential protein biomarkers related to the immune system were identified, which could be associated with the disease‐resistant phenotype. These data provide insights into the underlying immune mechanism of hybrid groupers upon Vibrio sp. infection. [ABSTRACT FROM AUTHOR]

Objective: The copepod Parvocalanus crassirostris has recently increased its use as live food in marine fish hatcheries. Methods: To investigate the bacterial community of P. crassirostris nauplii and explore the potential use of nauplii as a vector for probiotics, adult copepods were reared with or without a probiotic consortium (Bacillus spp. and Lactobacillus spp.). Nauplii from both treatments were either enriched with probiotics or not, resulting in four treatments: control rearing and nonenriched, control rearing and probiotic enriched, probiotic rearing and nonenriched, and probiotic rearing probiotic enriched. A next‐generation sequencing analysis of the 16S ribosomal RNA gene was performed. Result: The bacterial community of nauplii without probiotics was dominated by the phyla Proteobacteria (mean ± standard deviation = 51.5 ± 8.0%) and Bacteroidetes (27.6 ± 6.7%); from these phyla, the most abundant orders were Rhodobacterales (21.8 ± 2.0%), Flavobacteriales (13.5 ± 9.4%), Chitinophagales (9.7 ± 1.8%), Alteromonadales (9.5 ± 4.0%), and Burkholderiales (3.8 ± 0.8%). Opportunistic pathogenic bacterial genera, such as Vibrio spp. and Pseudomonas spp., were detected at very low relative abundances (<1.2%), indicating that P. crassirostris nauplii could be considered a safe microbiological live feed. Furthermore, P. crassirostris nauplii were a good vector for probiotics delivery, as the adhesion of probiotics on the copepod exoskeleton was successful. Bacillus spp. abundance was 5–6× greater than the abundance of Lactobacillus spp. in P. crassirostris nauplii. The highest mean values of probiotics relative abundance (Bacillus spp. 41 ± 6.0%, Lactobacillus spp. 6.0 ± 3.0%) were observed in nauplii that were reared with probiotics and also enriched with probiotics. Conclusion: Parvocalanus crassirostris nauplii stages I–II appear to be good vectors to provide probiotics to first‐feeding fish larvae. Impact statementMarine fish aquaculture relies on quality live feed for optimal protein production. Assessing copepod nauplii's microbiological safety as fish larvae prey and their role as probiotics vectors is an important contribution to marine fish aquaculture. [ABSTRACT FROM AUTHOR]

Simple Summary: Lactic acid bacteria (LAB) are often used to improve the probiotic content in aquaculture organisms. Although LAB were fed to aquaculture organisms, these LAB were often not detected when analyzing the gut microbiota. While the intestinal environment was indeed improved, this indicates that the effect of LAB on aquaculture organisms may not occur through the bacteria themselves, but rather through substances such as extracellular vesicles involved in these processes. This study will utilize an in vitro cultured intestinal bacteria system to exclude host interference and investigate the potential of L. reuteri-derived EVs for regulating the intestinal microbiota. Extracellular vesicles (EVs) are functional substances secreted by microbes and host cells, and it has been discovered that they participate in the interactions between different microorganisms. Our recent findings indicate that Limosilactobacillus reuteri-derived EVs have the potential to improve the intestinal microbiota of Oplegnathus fasciatus fish and inhibit pathogenic bacteria. Previous research has reported that the host intestinal cells play a regulatory role in the intestinal microbiota. This suggested that to investigate the mechanisms through which L. reuteri-derived EVs regulate the intestinal microbiota, a system that excludes interference from host intestinal cells should be established. In this study, an in vitro cultured intestinal bacteria system, without host factors, was used to simulate the intestinal microbiota of O. fasciatus fish. After adding L. reuteri-derived EVs to the system, the changes in the microbiota were analyzed. The results showed that L. reuteri-derived EVs effectively reduced the abundance of Vibrio spp. In the results of the in vitro experiments, it was also observed that L. reuteri-derived EVs have the ability to inhibit Vibrio alginolyticus. We further sequenced the small RNA contained in L. reuteri-derived EVs and found that these small RNAs can interfere with genes (LysR, pirin, MIpA/OmpV, CatB, and aspartate-semialdehyde dehydrogenase) related to the growth of V. alginolyticus. Taken together, the results indicate that in the absence of host involvement, the small RNAs present in L. reuteri-derived EVs have the function of inhibiting pathogenic bacteria and exhibit the potential to regulate the intestinal microbiota. [ABSTRACT FROM AUTHOR]

The objective of this study was to identify effective strategies for disease prevention and control that circumvent antimicrobial resistance (AMR) issues, thereby facilitating sustainable aquaculture practices. This research focused on isolating probiotic Bacillus strains from the intestinal tract of healthy large yellow croaker (Larimichthys crocea). Among the 11 strains tested, four demonstrated protease and lipase activities. Further testing identified one strain, Lysinibacillus sp. (LYD11), with strong inhibitory activity against Vibrio harveyi and Vibrio alginolyticus, two major pathogens of the large yellow croaker. The screened strains showed more than 45% co-aggregation with five pathogens and were capable of inhibiting pathogen colonization through competition, repulsion, and substitution adhesion of 20.56 to 48.45%, 40.37 to 75.62% and 55.51 to 85.30%, respectively. The hydrophobicity and self-aggregation rates of LYD11 were 82.73% and 49.47%, respectively, indicating a potent ability for adhesion and colonization within the fish intestine. LYD11 demonstrated robust growth under the NaCl 0.5-3.5% and pH range of 6-8 and was unaffected by the gastrointestinal environment. Safety evaluations at high concentrations in grouper, in both injected and fed groups, indicated no adverse effects, as evidenced by the absence of morbidity or mortality. Consequently, Lysinibacillus sp. LYD11 emerges as a potential probiotic candidate for the prevention and control of bacterial diseases in the large yellow croaker. [ABSTRACT FROM AUTHOR]

PhoR is a histidine kinase in a two-component regulatory system that regulates phosphorus metabolic pathways and undertakes the key mission of information transmission in pathogenic bacteria. The full-length phoR gene was successfully cloned from the Vibrio alginolyticus HY9901 strain. A comprehensive analysis of the cloned gene was conducted using bioinformatics. Sequence analysis revealed that the total length of the phoR gene (GenBank accession No.: KJ958404.1) is 1299 bp, with the coding region containing a total of 432 amino acid residues. The phylogenetic tree of PhoR revealed that it belongs to the same subclade as V. diabolicus. The SMART program was employed for the purpose of functional domain prediction, which revealed that PhoR possesses three major functional domains: PAS (amino acids 98 -166), His-KA (amino acids 205-272), and HATPase_c (amino acids 317429). [ABSTRACT FROM AUTHOR]

Vibrio alginolyticus is an opportunistic Gram-negative marine bacterium that infects animals and humans. Its virulence is associated with numerous factors such as cytotoxicity, adhesion and biofilm formation. This review aimed to quantitatively assess the V. alginolyticus's virulence factors to identify the main mechanisms of infection. A systematic literature search was conducted using SCOPUS databases up to 31 December 2022. The bibliometric analysis was performed using VOSviewer software. The results showed that toxins are the most common virulence factor making up more than 50% of virulence-related keywords. This was followed by adhesion/biofilm (12%), antibiotic resistance (9%) and quorum sensing (9%). In terms of cytotoxicity, hemolysins were the most common toxins, while in terms of secretion system, type III secretion system (T3SS) was the most common. Extracellular polysaccharide production was the main parameter related to adhesion and biofilm. In terms of antibiotic resistance, extended-spectrum β-lactamases (ESBLs) genes were the most common, and for quorum sensing, N-acyl homoserine lactone (AHL) was the most common. Vibrio pathogenicity island was also identified as a mechanism for accumulating virulence factors. Toxin-coregulated pilus (Tcp) was identified as the main motility-related virulence factor. This review reports that quorum sensing and kinase inhibitors are promising drug targets. Identifying these mechanisms can help in establishing ideal drug targets and management approaches for V. alginolyticus-related infections. [ABSTRACT FROM AUTHOR]

Simple Summary: As a filter-feeding and sessile invertebrate living in estuaries and intertidal areas, Crassostrea gigas must cope with and adapt to a dynamic and changeable environment. Vibrio alginolyticus is a Gram-negative bacterium that is widespread in oceans and estuaries, and is one of the main Vibrio species that cause oyster disease. In this study, we used transcriptome sequencing to help us better understand how the giant oyster adapts to pathogen-rich environments. By focusing on the gills, which play a crucial role in the immune response, we aimed to shed light on the molecular processes underlying the interaction between the oyster and the pathogen. Over the years, oysters have faced recurring mass mortality issues during the summer breeding season, with Vibrio infection emerging as a significant contributing factor. Tubules of gill filaments were confirmed to be in the hematopoietic position in Crassostrea gigas, which produce hemocytes with immune defense capabilities. Additionally, the epithelial cells of oyster gills produce immune effectors to defend against pathogens. In light of this, we performed a transcriptome analysis of gill tissues obtained from C. gigas infected with Vibrio alginolyticus for 12 h and 48 h. Through this analysis, we identified 1024 differentially expressed genes (DEGs) at 12 h post-injection and 1079 DEGs at 48 h post-injection. Enrichment analysis of these DEGs revealed a significant association with immune-related Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. To further investigate the immune response, we constructed a protein–protein interaction (PPI) network using the DEGs enriched in immune-associated KEGG pathways. This network provided insights into the interactions and relationships among these genes, shedding light on the underlying mechanisms of the innate immune defense mechanism in oyster gills. To ensure the accuracy of our findings, we validated 16 key genes using quantitative RT-PCR. Overall, this study represents the first exploration of the innate immune defense mechanism in oyster gills using a PPI network approach. The findings provide valuable insights for future research on oyster pathogen control and the development of oysters with enhanced antimicrobial resistance. [ABSTRACT FROM AUTHOR]

[Objective] The study aims to screen the formula of vacuum freeze-drying protective agent for Vibrio alginolyticus, optimize the freeze-drying process conditions, and explore the influence of freeze-drying on the virulence and drug sensitivity of the strains, with a view to providing a theoretical basis for the application of freeze-drying in the preservation of the strains. [Method] With Vibrio alginolyticus HY9901 as the research object, the vacuum freeze-drying technology was used to freeze-dry the strains. Through single factor test and orthogonal test, the effects of six single cryoprotectants (L-glutamate sodium, trehalose, skim milk, glucose, mannitol and glycine) on the activity of the strains were investigated, and the optimal formula of cryoprotectants was selected. The influences of four factors, namely, concentration of bacterial solution, pre-freezing time, pre-freezing temperature and rehydration medium, on the freeze-drying process of strains were studied, and the freeze-drying process conditions for Vibrio alginolyticus were optimized. At the same time, storage test, LD50 test and antimicrobial susceptibility test were used to investigate the changes in the activity of lyophilized strains and their of biological characteristics. [Result] There were significant differences in the protective effects of 6 cryoprotectants on lyophilized Vibrio alginolyticus, and the effects from strongest to weakest showed as: L-glutamate sodium, trehalose, skim milk, glucose, mannitol and glycine. The optimum freeze-drying compound formula of cryoprotectants was trehalose 50 g/L+ L-glutamate sodium 50 g/L+ skim milk 50 g/L, with a survival rate of 62.12%, which was significantly different from that of single cryoprotectant. The optimal conditions of freeze-drying process were as follows: bacterial solution concentration 1×108-109 CFU/mL, pre-freezing time 3 h, pre-freezing temperature -80 °C, rehydration medium PBS or 50 g/L skim milk, under which the activity of the strains could reach the maximum of 63.72%. There was no significant difference in the activity of the strains during 60 days of storage under optimal freeze-drying conditions. The LD50 values of the strains before and after freeze-drying were 1.22×108 CFU/mL and 1.20×108 CFU/mL, respectively, with no significant difference in LD50. There was no significant difference in the diameter of the antimicrobial inhibition zone before and after freeze-drying, and the strains were sensitive to florfenicol, chloramphenicol, ciprofloxacin and other drugs. They were moderately sensitive to neomycin and kanamycin, and resistant to drugs like amimcana and piperacillin. [Conclusion] The freeze-drying formula for Vibrio alginolyticus HY9901 is trehalose 50 g/L+ L-glutamate sodium 50 g/L+ skim milk 50 g/L, and the freeze-drying process conditions are: bacterial solution concentration 1×108 - 109 CFU/mL, pre-freezing time 3 h, pre-freezing temperature -80 °C, rehydration medium PBS or 50 g/L skim milk. The fomula and process for this strain are reasonable and feasible, with good storage and stability of biological characteristics. [ABSTRACT FROM AUTHOR]

In September 2023, several hatcheries in Latin America experienced significant mortality rates, up to 90%, in zoea stage 2 of Penaeus vannamei. Observations of fresh mounts revealed structures resembling lipid droplets, similar to those seen in a condition known as "las bolitas syndrome". Routine histopathological examinations identified detached cells and tissues in the digestive tracts of affected zoea, contrasting with the typical algal cell contents seen in healthy zoea. Polymerase chain reaction (PCR) testing for over 20 known shrimp pathogens indicated minimal differences between diseased and healthy batches. Both groups tested negative for acute hepatopancreatic necrosis disease (AHPND) but positive for Vibrio species and Rickettsia-like bacteria in the diseased samples. Histological analyses of the affected zoea revealed characteristic tissue degeneration in the hepatopancreas, forming spheres that eventually migrated into the upper gut, midgut, and midgut caeca, a pathology identified as bolitas syndrome (BS). Microbiological assessments revealed Vibrio species at concentrations of 106 CFU zoea/g in affected zoea, approximately two orders of magnitude higher than in healthy zoea. Bacterial isolation from both healthy and BS-affected zoea on thiosulphate–citrate–bile salts–sucrose (TCBS) agar and CHROMagar™ (Paris, France), followed by identification using API 20E, identified six strains of Vibrio alginolyticus. Despite similarities to "las bolitas syndrome" in fresh mounts, distinct histopathological differences were noted, particularly the presence of sloughed cells in the intestines and variations in hepatopancreatic lobes. This study highlights the critical need for further research to fully understand the etiology and pathology of bolitas syndrome in zoea stage 2 of P. vannamei to develop effective mitigation strategies for hatchery operations. [ABSTRACT FROM AUTHOR]

Climate change is now considered one of the greatest challenges and is expected to have a drastic impact on mariculture. The present study aimed to evaluate the impact of climatic changes on the emergence of bacterial pathogens among cultured marine fish in northern Egyptian provinces. 135 samples of three marine fish species, represented as 45 of Dicentrarchus labrax (700 ±25 g), 45 of Sparus aurata (350 ±25 g), and 45 Argyrosomus regius (1 kg ±50 g) were collected from private marine fish farms located in Deeba Triangle, Shataa Damietta(Damietta governorate) and Ismailia province, Egypt. Moribund fishes exhibited erratic swimming behaviour, haemorrhage, erosion and ulcers on the skin. Necropsy findings of infected fish revealed congested liver or pale with engorged gall bladder, congested kidney and spleen. With the presence of serous to hemorrhagic ascetic fluid. Vibrio alginolyticus, Vibrio parahaemolyticus, and Photobacterium damselae subspecies piscicida were the most retrievable bacterial strains from moribund fish. V. alginolyticus was the most prevalent isolated bacterial strain and represented 50%, 50% and 40% of the total isolates recovered from Sparus aurata, Dicentrarchus labrax and Argyrosomus regius, respectively. Retrieved isolates were morphologically and biochemically identified using the API 20E system, followed by further confirmation by sequencing of 16S rRNA genes. The histopathological examination revealed severe inflammatory reactions together with melanomacrophage center alterations within the examined splenic, hepatic, and renal tissues. Data analysis has shown that poor water quality and severe climatic change, especially during the summer, were implicated in the emergence of bacterial infections among cultured marine fish.

ABSTRACTVibrio alginolyticus, an emergent species of Vibrio genus, exists in aquatic and marine environments. It has undergone genetic diversification, but its detailed genomic diversity is still unclear. Here, we performed a multi-dimensional comparative genomic analysis to explore the population phylogeny, virulence-related genes and potential drug resistance genes of 184 V. alginolyticus isolates. Although genetic diversity is complex, we analysed the population structure using three sub-datasets, including the subdivision for three lineages into sublineages and the distribution of strains in the marine ecological niche. Accessory genes, most of which reclassified V. alginolyticus genomes as different but with relatively close affinities, were nonuniformly distributed among these isolates. We demonstrated that the spread of some post-evolutionary isolates (mainly L3 strains isolated from Chinese territorial seas) was likely to be closely related to human activities, whereas other more ancestral strains (strains in the L1 and L2) tended to be locally endemic and formed clonal complex groups. In terms of pathogenicity, the potential virulence factors were mainly associated with toxin, adherence, motility, chemotaxis, and the type III secretion system (T3SS). We also found five types of antibacterial drug resistance genes. The prevalence of β-lactam resistance genes was 100%, which indicated that there may be a potential risk of natural resistance to β-lactam drugs. Our study reveals insights into genomic characteristics, evolution and potential virulence-associated gene profiles of V. alginolyticus.

Infections by filamentous phages, which are usually nonlethal to the bacterial cells, influence bacterial fitness in various ways. While phage‐encoded accessory genes, for example virulence genes, can be highly beneficial, the production of viral particles is energetically costly and often reduces bacterial growth. Consequently, if costs outweigh benefits, bacteria evolve resistance, which can shorten phage epidemics. Abiotic conditions are known to influence the net‐fitness effect for infected bacteria. Their impact on the dynamics and trajectories of host resistance evolution, however, remains yet unknown. To address this, we experimentally evolved the bacterium Vibrio alginolyticus in the presence of a filamentous phage at three different salinity levels, that is (1) ambient, (2) 50% reduction and (3) fluctuations between reduced and ambient. In all three salinities, bacteria rapidly acquired resistance through super infection exclusion (SIE), whereby phage‐infected cells acquired immunity at the cost of reduced growth. Over time, SIE was gradually replaced by evolutionary fitter surface receptor mutants (SRM). This replacement was significantly faster at ambient and fluctuating conditions compared with the low saline environment. Our experimentally parameterized mathematical model explains that suboptimal environmental conditions, in which bacterial growth is slower, slow down phage resistance evolution ultimately prolonging phage epidemics. Our results may explain the high prevalence of filamentous phages in natural environments where bacteria are frequently exposed to suboptimal conditions and constantly shifting selections regimes. Thus, our future ocean may favour the emergence of phage‐born pathogenic bacteria and impose a greater risk for disease outbreaks, impacting not only marine animals but also humans. [ABSTRACT FROM AUTHOR]

Bacterial α-1,3-glucanase, classified as glycoside hydrolase (GH) family 87, has been divided into 3 subgroups based on differences in gene sequences in the catalytic domain. The enzymatic properties of subgroups 1 and 3 of several bacteria have been previously investigated and reported; however, the chemical characterization of subgroup 2 enzymes has not been previously conducted. The α-1,3-glucanase gene from Paenibacillus alginolyticus NBRC15375 (PaAgl) belonging to subgroup 2 of GH family 87 was cloned and expressed in Escherichia coli. PgAgl-N1 (subgroup 3) and PgAgl-N2 (subgroup 1) from P. glycanilyticus NBRC16188 were expressed in E. coli , and their enzymatic characteristics were compared. The amino acid sequence of PaAgl demonstrated that the homology was significantly lower in other subgroups when only the catalytic domain was compared. The oligosaccharide products of the mutan-degrading reaction seemed to have different characteristics among subgroups 1, 2, and 3 in GH family 87. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The current research focused on the production of silver nanoparticles using dichloro methanemediated Cocos nucifera male flower extract and their minimum inhibitory concentration against ten different bacterial pathogens. C. nucifera male flowers were extracted by 100% DCM, and the phytochemicals present in this extract were studied. These plant phytochemicals act as reducing agents to reduce the silver nitrate (AgNO3) to silver nanoparticles (AgNPs) which was confirmed by UV/Vis spectrophotometer, Fourier transform infra-red spectrum, X-ray diffraction analysis, and scanning electron microscopy. The 15 µL of silver nanoparticles was the minimum inhibitory concentration for Klebsiella pneumonia and Bacillus subtilis. The 20 µL of silver nanoparticles is the MIC for Pseudomonas aeruginosa, Vibrio harveyi, and Plesiomonas shigelloides. 30 µL of plant flower extract concentration highly inhibits the E. coli, Staphylococcus aureus, Salmonella paratyphi, Vibrio mimicus, and Vibrio alginolyticus bacterial pathogens. C. nucifera male flowers extract derived silver nanoparticles are highly stable and have high potential activity against MCF-7 Cell line. [ABSTRACT FROM AUTHOR]

Vibrio species are naturally found in estuarine and marine ecosystems, but are also recognized as significant human enteropathogens, often linked to seafood-related illnesses. In aquaculture settings, Vibrio poses a substantial risk of infectious diseases, resulting in considerable stock losses and prompting the use of antimicrobials. However, this practice contributes to the proliferation of antimicrobial-resistant (AMR) bacteria and resistance genes. Our investigation aimed to explore the potential of biological agents such as bacteriophage CH20 and endolysin LysVPp1 in reducing Vibrio bacterial loads in both rotifer and fish larvae. LysVPp1's lytic activity was assessed by measuring absorbance reduction against various pathogenic Vibrio strains. Phage CH20 exhibited a limited host range, affecting only Vibrio alginolyticus GV09, a highly pathogenic strain. Both CH20 and LysVPp1 were evaluated for their effectiveness in reducing Vibrio load in rotifers or fish larvae through short-setting bioassays. Our results demonstrated the significant lytic effect of endolysin LysVPp1 on strains of Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio splendidus. Furthermore, we have showcased the feasibility of reducing the load of pathogenic Vibrio in live feed and fish larvae by using a non-antibiotic-based approach, such as lytic phage and endolysin LysVPp1, thus contributing to the progress of a sustainable aquaculture from a One Health perspective. [ABSTRACT FROM AUTHOR]

Bacteriophages are currently considered one of the most promising alternatives to antibiotics under the 'One Health' approach due to their ability to effectively combat bacterial infections. This study aimed to characterize Vibrio species in hatchery water samples collected from an aquaculture farm and investigate the biocontrol potential of their bacteriophages. Vibrio spp. (n = 32) isolates confirmed by LNA probe-based qPCR were used as hosts. Three Vibrio phages were isolated. IKEM_vK exhibited a broad host range, infecting V. harveyi (n = 8), V. alginolyticus (n = 2), V. azureus (n = 1), and V. ordalii (n = 1). IKEM_v5 showed lytic activity against V. anguillarum (n = 4) and V. ordalii (n = 1), while IKEM_v14 was specific to V. scophtalmi (n = 4). The morphological appearance of phages and their lytic effects on the host were visualized using scanning electron microscopy (SEM). All three phages remained relatively stable within the pH range of 6–11 and up to 60 °C. The lytic activities and biofilm inhibition capabilities of these phages against planktonic Vibrio cells support their potential applications in controlling vibriosis in aquaculture systems. [ABSTRACT FROM AUTHOR]

The objective of this study is to optimize the ultrasonic-assisted extraction process of Ku Shen (Sophorae Flavescentis Radix) extracts (KSE) against Vibrio parahaemolyticus and explore their anti-biofilm activity and mechanism of action. The ultrasonic-assisted extraction process of KSE optimized by single factor experiment, Box–Behnken design and response surface methodology was as follows: 93% ethanol as solvent, liquid/material ratio of 30  mL/g, ultrasonic power of 500  W, extraction temperature of 80°C and time of 30  min. Under these conditions, the diameter of inhibition circle of KSE was 15.60  ±  0.17  mm, which had no significant difference with the predicted value. The yield of dried KSE is 32.32  ±  0.57% and the content of total flavonoids in KSE was 57.02  ±  5.54%. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of KSE against V. parahaemolyticus were 0.25 and 0.5  mg/mL, respectively. Crystal violet staining, Congo red plate, spectrophotometry, CCK-8 and scanning electron microscopy were used to investigate the activity and mechanism of action of KSE against V. parahaemolyticus biofilm. The results showed that the sub-MIC of KSE could significantly inhibit biofilm formation, reduce the synthesis of polysaccharide intercellular adhesin (PIA) and the secretion of extracellular DNA. In addition, the inhibition rate of biofilm formation and clearance rate of mature biofilm of 1.0  mg/mL KSE were 85.32 and 74.04%, and the reduction rate of metabolic activity of developing and mature biofilm were 77.98 and 74.46%, respectively. These results were confirmed by visual images obtained by scanning electron microscopy. Therefore, KSE has the potential to further isolate the anti-biofilm agent and evaluate it for the preservation process of aquatic products. [ABSTRACT FROM AUTHOR]

Objective: As one of the most important environmental signals, photoperiod plays a crucial role in regulating the growth, metabolism, and survival of organisms. The photoperiod shifts with the transition of the seasons. The difference in photoperiod between summer and winter is the greatest under natural conditions. However, the effect of photoperiod on Huanghe carp (Cyprinus carpio haematopterus) was paid little attention. We investigated the impact of artificial manipulation of seasonal photoperiod on Huanghe carp by integrating growth performance, intestinal flora, and intestinal metabolome. Method: We conducted an 8-week culture experiment with summer photoperiod (14 h light:10 h dark, n = 60) as the control group and winter photoperiod (10 h light:14 h dark, n = 60) based on the natural laws. Results: Winter photoperiod provokes significant weight increases in Huanghe carp. The altered photoperiod contributed to a significant increase in triglyceride and low-density lipoprotein cholesterol levels and the gene expressions of lipid metabolism in the intestine of Huanghe carp. 16s rDNA sequencing revealed that winter photoperiod diminished intestinal flora diversity and altered the abundance. Specifically, the relative abundances of Fusobacteria and Acidobacteriota phyla were higher but Proteobacteria, Firmicutes, and Bacteroidetes phyla were reduced. Analogously, photoperiodic changes induced a significant reduction in the Pseudomonas, Vibrio, Ralstonia, Acinetobacter, and Pseudoalteromonas at the genus level. Additionally, metabolomics analysis showed more than 50% of differential metabolites were associated with phospholipids and inflammation. Microbiome and metabolome correlation analyses revealed that intestinal microbe mediated lipid metabolism alteration. Conclusion: The winter photoperiod induced intestinal flora imbalance and lipid metabolism modification, ultimately affecting the growth of Huanghe carp. This study provides new insights into the effects of seasonal photoperiodic alteration on the well-being of fish. [ABSTRACT FROM AUTHOR]

Benefits of integrated shrimp/seaweed culture systems have been investigated; nevertheless, knowledge of the importance of microorganisms in the systems is limited. In the present study, we evaluated shrimp Litopenaeus vannamei under four experimental conditions: fed pelleted diet in the presence of Ulva lactuca with (UPE) and without water exchange (UPN) and fed only a pelleted diet with (PE) and without water exchange (PN). Bacterial communities present in shrimp, seaweed, and water were analyzed after 4 weeks of an experimental trial. Our results showed significantly lower shrimp final weight, weight gain, specific growth rate (SGR), and higher feed conversion ratio (FCR) with PN treatment compared to the rest of the treatments (P < 0.05). Seaweed resulted in lower relative abundance of Vibrio in the presence of shrimp (UPE and UPN) compared to monoculture (Ulv). Shrimp intestine presented lower Vibrio and significantly increased Sagittula and Lutimonas in treatments without water exchange. In the case of shrimp fed only pellet with water exchange (PE), a significantly higher relative abundance of Lucibacterium and Planctomicrobium genera was found compared to PN, UPE, and UPN treatments. The results suggest that the seaweed in a system with no water exchange is advantageous to promote water bacterial diversity, and that reduces the relative abundance of Vibrio in shrimp gut. Additionally, PERMANOVA analyses revealed that both seaweed presence and water exchange are significant factors in structuring the culture system microbiota (P < 0.001). The present study contributes to understanding the importance of microbial communities in shrimp/seaweed integrated aquaculture systems. [ABSTRACT FROM AUTHOR]

Vibrio alginolyticus and Streptococcus agalactiae are important bacterial pathogens that yielded high losses in Nile tilapia in Egypt. The present study aimed to check the protective efficacy of inactivated whole-cell bivalent vaccines against these pathogens using incomplete Freund's adjuvant and Montanide™ IMS 1312 VG as adjuvants. The antibody titers have been determined at different weeks post-vaccination (WPV). Moreover, the protection levels against the challenged bacterial pathogens have also been examined in relation to the time-dependent protection at different WPV. The results revealed that serum antibodies were generated in all immunized fish at 1st WPV, peaked at 4th WPV, continued, and gradually decreased from 6th WPV to 14th WPV in all vaccinated groups. In addition, vaccines induced significantly higher protection of the immunized tilapia, manifested by higher survival rates. We noticed that the antibody levels and survival rates of the vaccinated fish by a vaccine adjuvanted by Montanide™ IMS 1312 VG were higher than those produced by a vaccine adjuvanted by incomplete Freund's adjuvant at different time points. Moreover, no external clinical signs, visceral adhesions, or internal lesions were recorded in the vaccinated tilapia, demonstrating the safety of the formulated vaccines. According to the aforementioned findings, we could suggest that the prepared bivalent vaccines, using the two adjuvant types, are safe and highly protective and could be utilized as promising candidate vaccines to increase the resistance of Nile tilapia against V. alginolyticus and S. agalactiae infections. Moreover, Montanide™ IMS 1312 VG enhanced the immuno-protectivity and exhibited optimum immune response and earlier protection compared to the vaccine adjuvanted by incomplete Freund's adjuvant, demonstrating its added value during the preparation of tilapia vaccines. [ABSTRACT FROM AUTHOR]

Aims This study investigated phenotypic and genotypic antimicrobial resistance profiles of Vibrio strains identified from Mytilus galloprovincialis farmed for human consumption in the Adriatic Sea Central Italy. Methods and results A total of 475 mussels (M. galloprovincialis) were involved in the present study, and culture-dependent microbiological methods permitted to identify a total of 50 Vibrio strains that were tested for antibiotic susceptibility followed by the genetic determinant detections. Antibiograms showed resistance against ampicillin (36.0%), amoxicillin-clavulanic acid (30.0%), gentamycin (14.0%), and imipenem (18.0%). Biomolecular assays amplified a total of 264 antibiotic resistance genes harbored by both susceptible and resistant Vibrio species. Among resistance genes, aac C2 (62.0%) and aad A (58.0%) for aminoglycosides, bla TEM (54.0%) for beta-lactams, qnr S (24.0%) for quinolones, tet D (66.0%) for tetracyclines, and van B (60.0%) for glycopeptides were mainly amplified by PCR assays. Conclusions Vibrio genus is involved in the antibiotic resistance phenomenon diffusion in the aquatic environments, as demonstrated by the harboring of many genetic determinants representing a kind of genetic " dark world ". [ABSTRACT FROM AUTHOR]

This study examined the dietary effects of Sanacore®GM (SAN) on growth and production, immune-antioxidative status, cytokines, and the resistance of European sea bass to Vibrio alginolyticus infection. Fish (28.5–32.3 g) were fed on 0.0, 1.0, 2.0, 4.0, 6.0, and 8.0 g SAN/kg feed for ten weeks. Following that, fish were intraperitoneally injected with V. alginolyticus, and observed for further ten days. Among different water quality parameters, only unionized ammonia and nitrite values were significantly declined in groups received 4.0–8.0 g SAN/kg feed. Significant increases in fish growth, production, hematological, and immune-antioxidant indices were observed in SAN-fed fish. Compared with the control group, IL-1β and TNF-α genes expressions were down-regulated in groups fed on 4.0–8.0 g SAN/kg feed. After bacterial infection, low fish mortalities (20%–10%) were observed in fish fed with 4.0–8.0 g SAN/feed, respectively. The polynomial fitting curve demonstrates that the optimum SAN level for European sea bass is 5.0 g/kg feed. [ABSTRACT FROM AUTHOR]

Canarium indicum offers the potential of usage in aquaculture. This study aimed to evaluate the utilization of C. indicum on growth performance, health, and resistance of Asian seabass Lates calcalifer challenged with Vibrio alginolyticus. The study employed a completely randomized design with seven treatments, namely positive control (K+), negative control (K-), vitamin control (KVit), antibiotic control (KAnt), doses of 5 g kg-1 (D05), 10 g kg-1 (D10), and 20 g kg-1 C. indicum (D20). The test feed was prepared with a coating method and fed for 30 days. On the 31st day of rearing, a challenge test was conducted by injecting V. alginolyticus intramuscularly at 106 CFU mL-1. The results showed that C. indicum treatment for 30 days had a significantly different effect (P<0.05) on weight gain and average daily growth, while KVit and KAnt treatments were not different from the control. After the challenge test, all doses of C. indicum treatment, as well as KVit and KAnt treatments, showed survival significantly different (P<0.05) from that of the positive control, which ranged from 88.89% to 95.56% and was not significantly different (P>0.05) from the negative control. The survival rate condition is identical to the cumulative survival condition which shows the highest mortality found in the positive control. The administration of C. indicum at a dose of 5 g kg-1 to 20 g kg-1 improved the growth performance and prevented V. alginolyticus infection. [ABSTRACT FROM AUTHOR]

This study aimed to reveal the importance of horizontal gene transfer (HGT) for the agarose‐degrading ability and the related degradation pathway of a deep‐sea bacterium Vibrio natriegens WPAGA4, which was rarely reported in former works. A total of four agarases belonged to the GH50 family, including Aga3418, Aga3419, Aga3420, and Aga3472, were annotated and expressed in Escherichia coli cells. The agarose degradation products of Aga3418, Aga3420, and Aga3472 were neoagarobiose, while those of Aga3419 were neoagarobiose and neoagarotetraose. The RT‐qPCR analysis showed that the expression level ratio of Aga3418, Aga3419, Aga3420, and Aga3472 was stable at about 1:1:1.5:2.5 during the degradation, which indicated the optimal expression level ratio of the agarases for agarose degradation by V. natriegens WPAGA4. Based on the genomic information, three of four agarases and other agarose‐degrading related genes were in a genome island with a G + C content that was obviously lower than that of the whole genome of V. natriegens WPAGA4, indicating that these agarose‐degrading genes were required through HGT. Our results demonstrated that the expression level ratio instead of the expression level itself of agarase genes was crucial for agarose degradation by V. natriegens WPAGA4, and HGT occurred in the deep‐sea environment, thereby promoting the deep‐sea carbon cycle and providing a reference for studying the evolution and transfer pathways of agar‐related genes. [ABSTRACT FROM AUTHOR]

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