Unveiling the pathogenic and multidrug-resistant profiles of Vibrio alfacsensis: A potential identified threat in turbot (Scophthalmus maximus) aquaculture.

Vibrio alfacsensis is traditionally seen as an environmental symbiont within its genus, with no detailedly documented pathogenicity in marine aquaculture to date. This study delves into the largely unexplored pathogenic potential and emerging antibiotic resistance of V. alfacsensis. The VA-1 strain, isolated from recirculating aquaculture system (RAS) effluent of cultured turbot (Scophthalmus maximus), underwent comprehensive analysis including biochemical identification, antibiotic susceptibility testing and reinfection trials. The results confirmed VA-1's pathogenicity and significant multiple antibiotic resistance. VA-1 could induce systemic infection in turbot, with symptoms like kidney enlargement, exhibiting virulence comparable to known Vibrio pathogens, with an LD50 around 2.36 × 106 CFU/fish. VA-1's remarkable resistance phenotype (14/22) suggested potential for genetic exchange and resistance factor acquisition in aquaculture environments. Phylogenetic analysis based on 16S rDNA sequences and whole-genome sequencing has firmly placed VA-1 within the V. alfacsensis clade, while genome-wide analysis highlights its similarity and diversity in relation to strains from across the globe. VA-1 contained numerous replicons, indicating the possibility for the spread of resistance and virulence genes. This study suggests V. alfacsensis may acquire and transfer pathogenic and resistant traits through horizontal gene transfer, a likelihood intensified by changing environmental and aquaculture conditions, highlighting the need for vigilant pathogen monitoring and new non-antibiotic treatments.

Spotlight on the epidemiology and antimicrobial susceptibility profiles of Vibrio species in the MENA region, 2000-2023.

Recent cholera outbreaks in many countries in the Middle East and North Africa (MENA) region have raised public health concerns and focused attention on the genus Vibrio. However, the epidemiology of Vibrio species in humans, water, and seafood is often anecdotal in this region. In this review, we screened the literature and provided a comprehensive assessment of the distribution and antibiotic resistance properties of Vibrio species in different clinical and environmental samples in the region. This review will contribute to understanding closely the real burden of Vibrio species and the spread of antibiotic-resistant strains in the MENA region. The overall objective is to engage epidemiologists, sanitarians and public health stakeholders to address this problem under the One-health ethos.

The Vibrio genus contains many bacterial species normally found in freshwater, estuaries and marine environments. Some of these species can be transmitted by water and food and can make people severely ill. For instance, some groups of the bacterium Vibrio cholerae (serogroups O1 and O139) can cause serious watery diarrhea called cholera. Other pathogenic Vibrio bacteria can cause other types of infections such as gastroenteritis and wound infections. Some of these bacteria are becoming increasingly resistant to antibiotics, which will threaten and complicate therapy. This review discusses the occurrence and antibiotic resistance of different important Vibrio species in the Middle East and North Africa (MENA) region.

The coral pathogen Vibrio coralliilyticus uses a T6SS to secrete a group of novel anti-eukaryotic effectors that contribute to virulence.

Vibrio coralliilyticus is a pathogen of coral and shellfish, leading to devastating economic and ecological consequences worldwide. Although rising ocean temperatures correlate with increased V. coralliilyticus pathogenicity, the specific molecular mechanisms and determinants contributing to virulence remain poorly understood. Here, we systematically analyzed the type VI secretion system (T6SS), a contact-dependent toxin delivery apparatus, in V. coralliilyticus. We identified 2 omnipresent T6SSs that are activated at temperatures in which V. coralliilyticus becomes virulent; T6SS1 is an antibacterial system mediating interbacterial competition, whereas T6SS2 mediates anti-eukaryotic toxicity and contributes to mortality during infection of an aquatic model organism, Artemia salina. Using comparative proteomics, we identified the T6SS1 and T6SS2 toxin arsenals of 3 V. coralliilyticus strains with distinct disease etiologies. Remarkably, T6SS2 secretes at least 9 novel anti-eukaryotic toxins comprising core and accessory repertoires. We propose that T6SSs differently contribute to V. coralliilyticus's virulence: T6SS2 plays a direct role by targeting the host, while T6SS1 plays an indirect role by eliminating competitors.

Investigation of vibriosis caused by Vibrio anguillarum in rainbow trout (Oncorhynchus mykiss).

Identifying pathogenic microorganisms causing disease is important for epidemiological research, antimicrobial therapy, and control. The current study was carried out to use different methods for the identification of Vibrio anguillarum from diseased rainbow trout (Oncorhynchus mykiss) obtained from Türkiye (Mugla-Fethiye), the damage caused by the pathogenic microorganism in the tissues and organs, and the determination of the antibiotic effective against the pathogen. Hemorrhagic and ulcerative skin lesions and diffuse petechial hemorrhage in the internal organs were clinically detected in diseased fish obtained from the rainbow trout farm. Bacteria isolated from diseased fish were subjected to analysis using conventional bacteriological methods, a commercial bacterial identification test kit (API), an automated bacteria identification system known as Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), BD Phoenix™, and 16S rRNA sequence analysis. All isolated bacteria were identified as V. anguillarum by API 20E and conventional bacteriological method. These results have been confirmed with 16S rRNA sequence analysis. However, the isolated bacteria were identified as Grimontia hollisae (syn. Vibrio holisae) with the BD Phoenix system. Histologically, tissue damage such as melano-macrophage centers and necrosis in the kidney and spleen, hyperemia and mononuclear cell infiltration in the liver, as well as mononuclear cell infiltration on muscles, talengectiasis in the gill tissue was observed. In addition, it has been determined that the most effective antibiotic against the pathogen was enrofloxacin. When comparing all identification methods used for this pathogen causing tissue damage, it was demonstrated that the MALDI-TOF MS provides better results than other methods in terms of cost and identification time, and it could be used as an alternative to the conventional method to the identification of V. anguillarum.

Investigating dynamics, etiology, pathology, and therapeutic interventions of Caligus clemensi and Vibrio alginolyticus co-infection in farmed marine fish.

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 ß-lactams, aminoglycosides, and trimethoprim-sulfamethoxazole in V. alginolyticus isolates. Upregulation of the inflammatory marker IL-1ß 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.

Evolutionary dynamics of the successful expansion of pandemic Vibrio parahaemolyticus ST3 in Latin America.

The underlying evolutionary mechanisms driving global expansions of pathogen strains are poorly understood. Vibrio parahaemolyticus is one of only two marine pathogens where variants have emerged in distinct climates globally. The success of a Vibrio parahaemolyticus clone (VpST3) in Latin America- the first spread identified outside its endemic region of tropical Asia- provided an invaluable opportunity to investigate mechanisms of VpST3 expansion into a distinct marine climate. A global collection of VpST3 isolates and novel Latin American isolates were used for evolutionary population genomics, pangenome analysis and combined with oceanic climate data. We found a VpST3 population (LatAm-VpST3) introduced in Latin America well before the emergence of this clone in India, previously considered the onset of the VpST3 epidemic. LatAm-VpST3 underwent successful adaptation to local conditions over its evolutionary divergence from Asian VpST3 isolates, to become dominant in Latin America. Selection signatures were found in genes providing resilience to the distinct marine climate. Core genome mutations and accessory gene presences that promoted survival over long dispersals or increased environmental fitness were associated with environmental conditions. These results provide novel insights into the global expansion of this successful V. parahaemolyticus clone into regions with different climate scenarios.

Biological and genomic characterization of the novel bacteriophage vB_VpM-pA2SJ1, which infects Vibrio parahaemolyticus associated with acute hepatopancreatic necrosis disease.

Vibrio parahaemolyticus is a major seafood-borne zoonotic pathogen that causes gastroenteritis in humans and acute hepatopancreatic necrosis disease (AHPND) in shrimp. In this study, we isolated and characterized Vibrio phage vB_VpM-pA2SJ1, which infects clinical and AHPND-associated strains of V. parahaemolyticus. The phage genome is a linear dsDNA 51,054 bp in length with a G + C content of 43.7%, and it contains 89 open reading frames. Genome comparisons revealed basal similarity to other Vibrio phages, particularly Vibrio phage vB_VpP_1, with 84.2% identity and 46% coverage. Phylogenetic analysis based on the whole genome, the terminase large subunit, and the major capsid protein revealed that phage vB_VpM-pA2SJ1 did not cluster with other known phage families, thus indicating its uniqueness.

LncRNA (BCO1-AS) regulate inflammatory responses in bacterial infection through caspase-1 in turbot (Scophthalmus maximus).

LncRNA plays key role in several biological processes, including transcriptional regulation, post transcriptional control and epigenetic regulation. However, research on the functional roles of lncRNAs in teleost species remains limited. Here, we discovered a lncRNA (BCO1-AS) with a critical role in antibacterial responses. Briefly, the full length of BCO1-AS was 2005 bp. Subsequently, BCO1-AS was distributed throughout the nucleus, where it may either trans- or cis-regulate the nearby genes. In addition, BCO1-AS was widely expressed in all the examined tissues with the highest expression level in intestine, while the lowest expression level was detected in muscle. Moreover, following Vibrio anguillarum challenge, BCO1-AS was significantly down-regulated in intestine, and up-regulated in gill and skin. In CHIRP experiment, BCO1-AS could effectively enrich RNA and might interact with several immune-related genes. Furthermore, we found that LPS could induce the expression of BCO1-AS. Finally, BCO1-AS could positively regulate caspase-1 at the mRNA and protein level. The BCO1-AS was speculated to inhibit the synthesis of inflammatory components. In summary, these results showed the roles of BCO1-AS in the regulation of inflammatory in turbot, which provided valuable information for further understanding the immune regulation network of lncRNA in teleost fish.

Bile acids activate the antibacterial T6SS1 in the gut pathogen Vibrio parahaemolyticus.

The marine bacterium Vibrio parahaemolyticus is a major cause of seafood-borne gastroenteritis in humans and of acute hepatopancreatic necrosis disease in shrimp. Bile acids, produced by the host and modified into secondary bile acids by commensal bacteria in the gastrointestinal tract, induce the virulence factors leading to disease in humans and shrimp. Here, we show that secondary bile acids also activate this pathogen's type VI secretion system 1, a toxin delivery apparatus mediating interbacterial competition. This finding implies that Vibrio parahaemolyticus exploits secondary bile acids to activate its virulence factors and identify the presence of commensal bacteria that it needs to outcompete in order to colonize the host.IMPORTANCEBacterial pathogens often manipulate their host and cause disease by secreting toxic proteins. However, to successfully colonize a host, they must also remove commensal bacteria that reside in it and may compete with them over resources. Here, we find that the same host-derived molecules that activate the secreted virulence toxins in a gut bacterial pathogen, Vibrio parahaemolyticus, also activate an antibacterial toxin delivery system that targets such commensal bacteria. These findings suggest that a pathogen can use one cue to launch a coordinated, trans-kingdom attack that enables it to colonize a host.

Autochthonous probiotic bacteria improve intestinal pathology and histomorphology, expression of immune and growth-related genes and resistance against Vibrio alginolyticus in Asian seabass (Lates calcarifer).

The study isolated two strains of intestinal autochthonous bacteria Lactiplantibacillus plantarum1 (MH155966.1) (L1) and Lactiplantibacillus plantarum2 (MH105076.1) (L2) from the Choobdeh Abadan region. The aim of this study was to investigate the effects of different strains of probiotic bacteria on the growth performance, digestive enzyme activity, histopathologic and histomorphometric characterization of the intestine, expression of immune and growth related genes, and evaluate Lates calcarifer resistance against Vibrio alginolyticus. To achieve this, for each treatment 60 L. calcarifer juveniles (75 ± 12 g) were randomly distributed in three fiberglass tanks (300 L) and fed for 45 days. The treatments were established as Diet 1 (control diet); L1 (diet with Lb. plantarum isolated 1); L2 (diet with Lb. plantarum isolated 2) with a bacterial concentration of 1 × 109 CFU/g. Nine fish from each treatment were sampled and examined, after euthanasia. The fish were placed 2 cm from the beginning of the intestine for microscopic sampling of villi height, villi width and thickness of the epithelium, with 3 treatments: The result showed differences in the mean values of total weight were found at the end of the experiment. After 45 days of culture, the fish fed with L1 had higher (P < 0.05) growth performance than the other treatment groups. But at the end of the trial, in L2, the digestive enzyme activities were higher (P < 0.05) than the other treatment groups. The fishes fed diets supplemented with the L2 group, like the digestive enzyme activities test, presented an increase in the thickness of the epithelium of the intestine, and villus height, and villus width were greatest in L2. Fish feeding with L1 and L2 probiotics induced higher transcription levels of interleukin-10 (IL-10), granulocyte-macrophage colony-forming cells (GMCFC), epidermal growth factor (EGF), and Transforming Growth Factor Beta (TGF-ß) genes in the gut, which may correlate with better immune and hematological parameters in these groups. The results of the challenge test revealed that the percentage of survival was significantly higher in L1 (76.2%) and L2 (80.95%) treatments than in the control (P < 0.05). These results indicate that host-derived probiotics (Lb. plantarum) have significant potential as important probiotics to enhance nutrient utilization, Digestive enzymes, and metabolism by increasing the gut surface area of Lates calcarifer juveniles at 45 days of culture.

Novel Vibrio vulnificus sequence type 540 infection in a hepatitis patient.

Vibrio vulnificus infection is associated with high morbidity and mortality in high-risk patients. Poor prognoses could lead to >50% mortality rate. The present report describes a case of V. vulnificus bacteremia in a cirrhotic patient with underlying hepatitis C. He presented with generalised abdominal pain associated with distention and could not ambulate for one week. He also complained of fever for six days and pruritus for 10 days. Tea-coloured urine was noted in continuous bag drainage. The abdomen was distended but soft, with mild tenderness palpated over the left lumbar and iliac region. Blood investigation indicated ongoing infection and inflammation. The aerobic blood culture was identified using the matrix-assisted laser desorption/ionisation-time of flight mass spectrometry and confirmed via 16S rDNA sequencing as V. vulnificus. Multilocus sequence typing of the isolated V. vulnificus revealed a novel sequence type, ST540. The patient responded well to the intravenous cefoperazone and was then discharged with a four day-course of oral ciprofloxacin, 500 mg twice daily after completing the intravenous cefoperazone for 10 days. Clinical history and physical examination are important for early antibiotic therapy initiation and appropriate surgical intervention. Furthermore, bacterial strain typing is also essential for epidemiological surveillance and potentially anticipating the pathogen's virulence traits, which are vital in controlling and preventing the spread of infection.

Epidemiological and environmental investigation of the 'big four' Vibrio species, 1994 to 2021: a Baltic Sea retrospective study.

BackgroundThe Vibrio genus comprises several bacterial species present in the Baltic Sea region (BSR), which are known to cause human infections.AimTo provide a comprehensive retrospective analysis of Vibrio-induced infections in the BSR from 1994 to 2021, focusing on the 'big four' Vibrio species - V. alginolyticus, V. cholerae non-O1/O139, V. parahaemolyticus and V. vulnificus - in eight European countries (Denmark, Estonia, Finland, Germany, Latvia, Lithuania, Poland and Sweden) bordering the Baltic Sea.MethodsOur analysis includes data on infections, Vibrio species distribution in coastal waters and environmental data received from national health agencies or extracted from scientific literature and online databases. A redundancy analysis was performed to determine the potential impact of several independent variables, such as sea surface temperature, salinity, the number of designated coastal beaches and year, on the Vibrio infection rate.ResultsFor BSR countries conducting surveillance, we observed an exponential increase in total Vibrio infections (n = 1,553) across the region over time. In Sweden and Germany, total numbers of Vibrio spp. and infections caused by V. alginolyticus and V. parahaemolyticus positively correlate with increasing sea surface temperature. Salinity emerged as a critical driver of Vibrio spp. distribution and abundance. Furthermore, our proposed statistical model reveals 12 to 20 unreported cases in Lithuania and Poland, respectively, countries with no surveillance.ConclusionsThere are discrepancies in Vibrio surveillance and monitoring among countries, emphasising the need for comprehensive monitoring programmes of these pathogens to protect human health, particularly in the context of climate change.

Effects of a phytobiotic-based additive on the growth, hepatopancreas health, intestinal microbiota, and Vibrio parahaemolyticus resistance of Pacific white shrimp, Litopenaeus vannamei.

Vibrio genus is a common pathogen in aquaculture and causes acute hepatopancreatic necrosis disease (AHPND) and massive mortality of shrimp. Many studies have suggested that a single functional ingredient such as plant extract or organic acid can reduce the dependence on antibiotics and promote the growth and immunity of aquatic animals. In this study, we evaluated the effects of a phytobiotic-based compound additive (Sanacore® GM, SNGM), which had a successful trajectory of commercial application in fish farming. However, its effects on the hepatopancreas health and intestinal microbiota of shrimp after Vibrio challenge have not been well evaluated. In the present study, Pacific white shrimp were fed diets with or without supplementation of SNGM, and the SNGM grades were 0-g/kg (CON), 3-g/kg (SNGM3), and 5-g/kg (SNGM5) diets. The feed trial lasted 60 days, after which a Vibrio parahaemolyticus challenge was performed. The results showed that compared to the CON group, both the SNGM3 and SNGM5 groups had a significantly higher weight gain and a lower feed conversion ratio as well as higher survival after Vibrio parahaemolyticus challenge. In the growth trial, the SNGM3 group had a significantly increased total protein, albumin concentration, and acid phosphatase activity in hemolymph compared to the CON group. In the challenge experiment, the SNGM3 and SNGM5 groups had increased albumin and glucose contents as well as the activities of phenoloxidase, lysozyme, alkaline phosphatase, and superoxide dismutase in hemolymph. Both the SNGM3 and SNGM5 groups had improved morphology of the hepatopancreas and intestine. The SNGM5 group had alleviated gut microbiota dysbiosis induced by Vibrio infection by increasing the potential probiotic bacterium abundance (Shewanella) and decreasing the potential pathogenic bacteria abundance (Vibrio, Photobacteriuma, Pseudoalteromonas, and Candidatus_Bacilloplasma). In conclusion, the dietary phytobiotic-based additive at 3-g/kg level increased the growth and Vibrio parahaemolyticus resistance of Pacific white shrimp by promoting immune-related enzyme activities and improving the morphological structure of the hepatopancreas and intestine and the intestinal microbiota composition.

Characteristics and pathogenicity of Vibrio alginolyticus SWS causing high mortality in mud crab (Scylla serrata) aquaculture in Hong Kong.

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.

Potential therapeutic use of dermaseptin S4 from the frog Phyllomedusa sauvagii and its derivatives against bacterial pathogens in fish.

AIM: Dermaseptins are one of the main families of antimicrobial peptides (AMPs) derived from the skin secretions of Hylidae frogs. Among them, dermaseptin S4 (DS4) is characterized by its broad-spectrum of activity against bacteria, protozoa, and fungi. In this study, the physicochemical properties of the native peptide DS4 (1-28) and two derivatives [DS4 (1-28)a and DS4 (1-26)a] isolated from the skin of the frog Phyllomedusa sauvagii were investigated and their antimicrobial properties against two marine pathogenic bacteria (Vibrio harveyi and Vibrio anguillarum) were examined. METHODS AND RESULTS: The results indicate that the peptide DS4 (1-26)a has high-antibacterial activity against the tested strains and low-hemolytic activity (<30% lysis at the highest tested concentration of 100 µg/mL) compared to the other two peptides tested. In addition, all three peptides affect the membrane and cell wall integrity of both pathogenic bacteria, causing leakage of cell contents, with DS4 (1-26)a having the most severe impact. These skills were corroborated by transmission electron microscopy and by the variation of cations in their binding sites due to the effects caused by the AMPs. CONCLUSIONS: These results suggest that DS4 and its derivatives, in particular the truncated and amidated peptide DS4 (1-26)a could be effective in the treatment of infections caused by these marine pathogenic bacteria. Future studies are required to validate the use of DS4  in vivo for the prevention of bacterial diseases in fish.

The virulence plasmid associated with AHPND in shrimp appears to have originated from Vibrio owensii through a process of homologous recombination of parental plasmids and the transposable insertion of two large fragments.

Acute hepatopancreatic necrosis disease (AHPND) is a highly contagious and lethal disease of shrimp caused by Vibrio strains carrying the virulence plasmid (pAHPND) containing the pirAB virulence genes. Through analysis of plasmid sequence similarity, clustering, and phylogeny, a horizontal transfer element similar to IS91 was discovered within the pAHPND plasmid. Additionally, two distinct clades of plasmids related to pAHPND (designated as pAHPND-r1 and pAHPND-r2) were identified, which may serve as potential parental plasmids for pAHPND. The available evidence, including the difference in G+C content between the plasmid and its host, codon usage preference, and plasmid recombination event prediction, suggests that the formation of the pAHPND plasmid in the Vibrio owensii strain was likely due to the synergistic effect of the recombinase RecA and the associated proteins RecBCD on the pAHPND-r1 and pAHPND-r2, resulting in the recombination and formation of the precursor plasmid for pAHPND (pre-pAHPND). The emergence of pAHPND was found to be a result of successive insertions of the horizontal transfer elements of pirAB-Tn903 and IS91-like segment, which led to the deletion of one third of the pre-pAHPND. This plasmid was then able to spread horizontally to other Vibrio strains, contributing to the epidemics of AHPND. These findings shed light on previously unknown mechanisms involved in the emergence of pAHPND and improve our understanding of the disease's spread.

Characterization of Vibrio parahaemolyticus isolated from clinical specimens and oysters in Thailand.

INTRODUCTION: Vibrio parahaemolyticus is a common pathogen that can cause seafood-borne gastroenteritis in humans. We determined the prevalence and characteristics of V. parahaemolyticus isolated from clinical specimens and oysters in Thailand. METHODOLOGY: Isolates of V. parahaemolyticus from clinical specimens (n = 77) and oysters (n = 224) were identified by biochemical testing, polymerase chain reaction (PCR) assays, and serotyping. The toxin genes, antimicrobial resistance, and ß-lactamase production were determined. RESULTS: A total of 301 isolates were confirmed as V. parahaemolyticus by PCR using specific primers for the toxR gene. The majority of clinical isolates carried the tdh+/trh- genotype (82.1%), and one of each isolate was tdh-/trh+ and tdh+/trh+ genotypes. One isolate from oyster contained the tdh gene and another had the trh gene. Twenty-six serotypes were characterized among these isolates, and O3:K6 was the most common (37.7%), followed by OUT:KUT, and O4:K9. In 2010, most clinical and oyster isolates were susceptible to antibiotics, with the exception of ampicillin. In 2012, clinical isolates were not susceptible to cephalothin (52.4%), streptomycin (95.2%), amikacin (66.6%), kanamycin (61.9%), and erythromycin (95.2%), significantly more frequently than in 2010. More than 95% of isolates that were not susceptible to ampicillin produced ß-lactamase enzymes. CONCLUSIONS: We found toxin genes in two oyster isolates, and the clinical isolates that were initially determined to be resistant to several antibiotics. Toxin genes and antimicrobial susceptibility profiles of V. parahaemolyticus from seafood and environment should be continually monitored to determine the spread of toxin and antimicrobial resistance genes.

Low salinity stress increases the risk of Vibrio parahaemolyticus infection and gut microbiota dysbiosis in Pacific white shrimp.

BACKGROUND: Extreme precipitation events often cause sudden drops in salinity, leading to disease outbreaks in shrimp aquaculture. Evidence suggests that environmental stress increases animal host susceptibility to pathogens. However, the mechanisms of how low salinity stress induces disease susceptibility remain poorly understood. METHODS: We investigated the acute response of shrimp gut microbiota exposed to pathogens under low salinity stress. For comparison, shrimp were exposed to Vibrio infection under two salinity conditions: optimal salinity (Control group) and low salinity stress (Stress group). High throughput 16S rRNA sequencing and real-time PCR were employed to characterize the shrimp gut microbiota and quantify the severity level of Vibrio infection. RESULTS: The results showed that low salinity stress increased Vibrio infection levels, reduced gut microbiota species richness, and perturbed microbial functions in the shrimp gut, leading to significant changes in lipopolysaccharide biosynthesis that promoted the growth of pathogens. Gut microbiota of the bacterial genera Candidatus Bacilliplasma, Cellvibrio, and Photobacterium were identified as biomarkers of the Stress group. The functions of the gut microbiota in the Stress group were primarily associated with cellular processes and the metabolism of lipid-related compounds. CONCLUSIONS: Our findings reveal how environmental stress, particularly low salinity, increases shrimp susceptibility to Vibrio infection by affecting the gut microbiota. This highlights the importance of avoiding low salinity stress and promoting gut microbiota resilience to maintain the health of shrimp.

In vivo examination of pathogenicity and virulence in environmentally isolated Vibrio vulnificus.

Human exposure to Vibrio vulnificus, a gram-negative, halophilic environmental pathogen, is increasing. Despite this, the mechanisms of its pathogenicity and virulence remain largely unknown. Each year, hundreds of infections related to V. vulnificus occur, leading to hospitalization in 92% of cases and a mortality rate of 35%. The infection is severe, typically contracted through the consumption of contaminated food or exposure of an open wound to contaminated water. This can result in necrotizing fasciitis and the need for amputation of the infected tissue. Although several genes (rtxA1, vvpE, and vvhA) have been implicated in the pathogenicity of this organism, a defined mechanism has not been discovered. In this study, we examine environmentally isolated V. vulnificus strains using a zebrafish model (Danio rerio) to investigate their virulence capabilities. We found significant variation in virulence between individual strains. The commonly used marker gene of disease-causing strains, vcgC, did not accurately predict the more virulent strains. Notably, the least virulent strain in the study, V. vulnificus Sept WR1-BW6, which tested positive for vcgC, vvhA, and rtxA1, did not cause severe disease in the fish and was the only strain that did not result in any mortality. Our study demonstrates that virulence varies greatly among different environmental strains and cannot be accurately predicted based solely on genotype.

Regulatory role of VvsB protein on serine protease activity of VvsA in Vibrio vulnificus.

BACKGROUND: Vibrio vulnificus NCIMB2137, a Gram-negative, metalloprotease negative estuarine strain was isolated from a diseased eel. A 45 kDa chymotrypsin-like alkaline serine protease known as VvsA has been recently reported as one of the major virulence factor responsible for the pathogenesis of this strain. The vvsA gene along with a downstream gene vvsB, whose function is still unknown constitute an operon designated as vvsAB. OBJECTIVE: This study examines the contribution of VvsB to the functionality of VvsA. METHOD: In this study, VvsB was individually expressed using Rapid Translation System (RTS system), followed by an analysis of its role in regulating the serine protease activity of VvsA. RESULT: The proteolytic activity of VvsA increased upon the addition of purified VvsB to the culture supernatant of V. vulnificus. However, the attempts of protein expression using an E. coli system revealed a noteworthy observation that protein expression from the vvsA gene exhibited higher protease activity compared to that from the vvsAB gene within the cytoplasmic fraction. These findings suggest an intricate interplay between VvsB and VvsA, where VvsB potentially interacts with VvsA inside the bacterium and suppress the proteolytic activity. While outside the bacterial milieu, VvsB appears to stimulate the activation of inactive VvsA. CONCLUSION: The findings suggest that Vibrio vulnificus regulates VvsA activity through the action of VvsB, both intracellularly and extracellularly, to ensure its survival.