Structural displacement model of chitooligosaccharide transport through chitoporin.

VhChiP is a chitooligosaccharide-specific porin identified in the outer membrane of Vibrio campbellii type strain American Type Culture Collection BAA 1116. VhChiP contains three identical subunits, and in each subunit, the 19-amino acid N-terminal segment serves as a molecular plug (the "N-plug") that controls the closed/open dynamics of the neighboring pores. In this study, the crystal structures of VhChiP lacking the N-plug were determined in the absence and presence of chitohexaose. Binding studies of sugar-ligand interactions by single-channel recordings and isothermal microcalorimetry experiments suggested that the deletion of the N-plug peptide significantly weakened the sugar-binding affinity due to the loss of hydrogen bonds around the central affinity sites. Steered molecular dynamic simulations revealed that the movement of the sugar chain along the sugar passage triggered the ejection of the N-plug, while the H-bonds transiently formed between the reducing end GlcNAc units of the sugar chain with the N-plug peptide may help to facilitate sugar translocation. The findings enable us to propose the structural displacement model, which enables us to understand the molecular basis of chitooligosaccharide uptake by marine Vibrio bacteria.

A three-step "ping-pong" mechanism of a GH20 ß-N-acetylglucosaminidase from Vibrio campbellii belonging to a major Clade A-I of the phylogenetic tree of the enzyme superfamily.

ß-N-acetylglucosaminidase (GlcNAcase) is an essential biocatalyst in chitin assimilation by marine Vibrio species, which rely on chitin as their main carbon source. Structure-based phylogenetic analysis of the GlcNAcase superfamily revealed that a GlcNAcase from Vibrio campbellii, formerly named V. harveyi, (VhGlcNAcase) belongs to a major clade, Clade A-I, of the phylogenetic tree. Pre-steady-state and steady-state kinetic analysis of the reaction catalysed by VhGlcNAcase with the fluorogenic substrate 4-methylumbelliferyl N-acetyl-ß-D-glucosaminide suggested the following mechanism: (1) the Michaelis-Menten complex is formed in a rapid enzyme-substrate equilibrium with a Kd of 99.1 ± 1 µM. (2) The glycosidic bond is cleaved by the action of the catalytic residue Glu438, followed by the rapid release of the aglycone product with a rate constant (k2) of 53.3 ± 1 s-1. (3) After the formation of an oxazolinium ion intermediate with the assistance of Asp437, the anomeric carbon of the transition state is attacked by a catalytic water, followed by release of the glycone product with a rate constant (k3) of 14.6 s-1, which is rate-limiting. The result clearly indicated a three-step "ping-pong" mechanism for VhGlcNAcase.

Non-cholera Vibrio spp. invasive infections in the summer following May 2023 flood disaster in Romagna, Italy: a case series.

Non-cholera Vibrio spp. includes ubiquitous organisms living in aquatic environments. Their occurrence is associated with global warming and meteorological disasters. In May 2023 the Romagna region, Italy, was affected by severe floods. In the following 15 weeks we observed 5 patients with invasive infections caused by V. vulnificus (3/5) and V. harveyi (2/5). All patients (median age 77 years) had medical comorbidities and shared exposure to seawater. Two patients needed surgery; 2 died. In conclusion, we observed an increased burden of Vibrio spp. invasive infections after May 2023 floods, affecting old patients with predisposing medical conditions.

Smelly shark, smelly ray: what is infecting you?

AIMS: Although elasmobranchs are consumed worldwide, bacteriological assessments for this group are still sorely lacking. In this context, this study assessed bacteria of sharks and rays from one of the most important landing ports along the Rio de Janeiro coast. METHODS AND RESULTS: Bacteria were isolated from the cloacal swabs of the sampled elasmobranchs. They were cultured, and Vibrio, Aeromonas, and Enterobacterales were isolated and identified. The isolated bacteria were then biochemically identified and antimicrobial susceptibility assays were performed. Antigenic characterizations were performed for Salmonella spp. and Polymerase Chain Reaction (PCR) assays were performed to identify Escherichia coli pathotypes. Several bacteria of interest in the One Health context were detected. The most prevalent Enterobacterales were Morganella morganii and Citrobacter freundii, while Vibrio harveyi and Vibrio fluvialis were the most prevalent among Vibrio spp. and Aeromonas allosacharophila and Aeromonas veronii bv. veronii were the most frequent among Aeromonas spp. Several bacteria also displayed antimicrobial resistance, indicative of Public Health concerns. A total of 10% of Vibrio strains were resistant to trimethoprim-sulfamethoxazole and 40% displayed intermediate resistance to cefoxitin. Salmonella enterica strains displayed intermediate resistance to ciprofloxacin, nalidixic acid and streptomycin. All V. cholerae strains were identified as non-O1/non-O139. The detected E. coli strains did not exhibit pathogenicity genes. This is the first study to perform serology assessments for S. enterica subsp. enterica isolated from elasmobranchs, identifying the zoonotic Typhimurium serovar. Salmonella serology evaluations are, therefore, paramount to identify the importance of elasmobranchs in the epidemiological salmonellosis chain. CONCLUSIONS: The detection of several pathogenic and antibiotic-resistant bacteria may pose significant Public Health risks in Brazil, due to high elasmobranch consumption rates, indicating the urgent need for further bacteriological assessments in this group.

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.

Sedimentary Vibrio Blooms in the Xisha Islands May Associate with the 2020 Coral Bleaching Event.

Coral reefs are among the most biodiverse ecosystems, providing habitats for various organisms. Studies on coral bleaching have been increasing recently, but little is known about the distribution and community assembly of coral pathogenic bacteria (e.g., several Vibrio species). We elucidated the distribution pattern and interaction relationships of total bacteria and Vibrio spp. in sediments from the Xisha Islands, which are characterized by their high coverage and diversity of coral resources. Vibrio spp. showed significantly higher relative abundance values in the Xisha Islands (1.00 × 108 copies/g) than in other areas (approximately 1 × 104 to 9.04 × 105 copies/g), indicating that the coral bleaching event of 2020 may have promoted the bloom of vibrios. A spatial shift in community composition was observed between the northern (Photobacterium rosenbergii and Vibrio ponticus) and southern (Vibrio ishigakensis and Vibrio natriegens) sites, accompanied by a clear distance-decay pattern. The spatial distance and coral species (e.g., Acroporidae and Fungiidae) had much greater correlations with the Vibrio community than did environmental factors. However, complex mechanisms may exist in the community assembly of Vibrio spp. due to the large proportion of unexplained variation. Stochastic processes may play an important role, as shown by the neutral model. Vibrio harveyi had the highest relative abundance (77.56%) and niche breadth, compared to other species, and it was negatively correlated with Acroporidae, likely reflecting its strong competitive ability and adverse effects on specific corals. Our study provides insights into the bloom and underlying assembly mechanisms of sedimentary vibrios in the Xisha Islands, thereby contributing to identify the potential indicator of coral bleaching and provide inspiration for the environmental management of coral reef areas. IMPORTANCE Coral reefs exert important roles in maintaining the sustainability of marine ecosystems but decline worldwide due to various drivers, especially pathogenic microorganisms. Here, we investigated the distribution pattern and interactions of total bacteria and Vibrio spp. in the sediments from Xisha Islands during the coral bleaching event of 2020. Our results showed that the abundances of Vibrio (1.00 × 108 copies/g) were high across the whole sites, indicating the bloom of sedimentary Vibrio spp. Coral pathogenic Vibrio species were abundant in the sediments, likely reflecting adverse effects on several kinds of corals. The compositions of the Vibrio spp. were separated by geographical location, which was mainly attributable to the spatial distance and coral species. Overall, this work contributes by providing evidence for the outbreak of coral pathogenic vibrios. The pathogenic mechanism of the dominant species (especially V. harveyi) should be comprehensively considered by laboratory infection experiments in the future.

When Vibrios Take Flight: A Meta-Analysis of Pathogenic Vibrio Species in Wild and Domestic Birds.

Of the over 100 species in the genus Vibrio, approximately twelve are associated with clinical disease, such as cholera and vibriosis. Crucially, eleven of those twelve, including Vibrio cholerae and Vibrio vulnificus, have been isolated from birds. Since 1965, pathogenic Vibrio species have been consistently isolated from aquatic and ground-foraging bird species, which has implications for public health, as well as the One Health paradigm defined as an ecology-inspired, integrative framework for the study of health and disease, inclusive of environmental, human, and animal health. In this meta-analysis, we identified 76 studies from the primary literature which report on or examine birds as hosts for pathogenic Vibrio species. We found that the burden of disease in birds was most commonly associated with V. cholerae, followed by V. metschnikovii and V. parahaemolyticus. Meta-analysis wide prevalence of our Vibrio pathogens varied from 19% for V. parahaemolyticus to 1% for V. mimicus. Wild and domestic birds were both affected, which may have implications for conservation, as well as agriculturally associated avian species. As pathogenic Vibrios become more abundant throughout the world as a result of warming estuaries and oceans, susceptible avian species should be continually monitored as potential reservoirs for these pathogens.

Management and Mitigation of Vibriosis in Aquaculture: Nanoparticles as Promising Alternatives.

Vibriosis is one of the most common diseases in marine aquaculture, caused by bacteria belonging to the genus Vibrio, that has been affecting many species of economically significant aquatic organisms around the world. The prevention of vibriosis in aquaculture is difficult, and the various treatments for vibriosis have their limitations. Therefore, there is an imperative need to find new alternatives. This review is based on the studies on vibriosis, specifically on the various treatments and their limitations, as well as the application of nanoparticles in aquaculture. One of the promising nanoparticles is graphene oxide (GO), which has been used in various applications, particularly in biological applications such as biosensors, drug delivery, and potential treatment for infectious diseases. GO has been shown to have anti-bacterial properties against both Gram-positive and Gram-negative bacteria, but no research has been published that emphasizes its impact on Vibrio spp. The review aims to explore the potential use of GO for treatment against vibriosis.

Antimicrobial Potential of Aqueous Extract of Giant Sword Fern and Ultra-High-Performance Liquid Chromatography-High-Resolution Mass Spectrometry Analysis.

Vibriosis and parasitic leech infestations cause the death of various farmed fish, such as groupers, hybrid groupers, sea bass, etc., in Malaysia and other Southeast Asian countries. In the absence of natural control agents, aquaculture operators rely on toxic chemicals to control Vibrio infections and parasitic leeches, which can have a negative impact on the environment and health. In the present study, we investigated the antivibrio and antiparasitic activities of the aqueous extract of giant sword fern (GSF) (Nephrolepis biserrata, Nephrolepidaceae, locally known as "Paku Pedang") against four Vibrio spp. and the parasitic leech Zeylanicobdella arugamensis, as well as its metabolic composition using the ultra-high-performance liquid chromatography-high-resolution mass spectrometry system (UHPLC-HRMS). The data show that the aqueous extract of GSF at a concentration of 100 mg/mL exhibits potent bactericidal activity against V. parahaemolyticus with a zone of inhibition of 19.5 mm. In addition, the extract showed dose-dependent activity against leeches, resulting in the complete killing of the parasitic leeches within a short period of 11-43 min when tested at concentrations ranging from 100 to 25 mg/mL. The UHPLC-HRMS analysis detected 118 metabolites in the aqueous extract of GSF. Flavonoids were the primary metabolites, followed by phenolic, aromatic, fatty acyl, terpenoid, vitamin and steroidal compounds. Notably, several of these metabolites possess antibacterial and antiparasitic properties, including cinnamaldehyde, cinnamic acid, apigenin, quercetin, cynaroside, luteolin, naringenin, wogonin, 6-gingerol, nicotinamide, abscisic acid, daidzein, salvianolic acid B, etc. Overall, our study shows the significant antibacterial and antiparasitic potential of the GSF aqueous extract, which demonstrates the presence of valuable secondary metabolites. Consequently, the aqueous extract is a promising natural alternative for the effective control of Vibrio infections and the treatment of parasitic leeches in aquaculture systems.

Anti-infective potential of plant-derived quorum sensing inhibitors against multi-drug resistant human and aquatic bacterial pathogens.

The present study intended to decipher the anti-infective potential of bioactive phytocompounds, such as rosmarinic acid, morin, naringin, chlorogenic acid, and mangiferin, against aquatic and human bacterial pathogens using Artemia spp. nauplii and Caenorhabditis elegans as animal models, respectively. Initially, the test compounds were screened against the QS traits in Vibrio spp., such as bioluminescence production and biofilm formation. The test compounds effectively inhibited the bioluminescence in V. harveyi. Further, the confocal laser scanning microscopic analysis revealed that these natural compounds could efficiently reduce the clumping morphology, a characteristic biofilm formation in Vibrio spp., without inhibiting bacterial growth. The results of in vivo analysis showed a significant increase in the survival of Artemia spp. nauplii infected with Vibrio spp. upon exposure to these compounds. Moreover, the compounds used in this study were already proven and reported for their quorum sensing inhibitory efficacy against Pseudomonas aeruginosa. Hence, the anti-infective efficacy of these compounds against P. aeruginosa (PAO1) and its clinical isolates (AS1 and AS2) was studied using C. elegans as a live animal model system. The results of time-killing assay deciphered that rosmarinic acid and naringin are being the most effective ones in rescuing the animals from P. aeruginosa infection followed by morin, mangiferin, and chlorogenic acid. Further, the toxicity results revealed that these compounds did not show any lethal effect on C. elegans and Artemia spp. nauplii at the tested concentrations. In conclusion, the phytochemicals used in this study were effective in controlling the QS-regulated virulence traits in Vibrio spp. and P. aeruginosa infections in Artemia spp. nauplii and C. elegans animal model systems, respectively.

A structural model for (GlcNAc)2 translocation via a periplasmic chitooligosaccharide-binding protein from marine Vibrio bacteria.

VhCBP is a periplasmic chitooligosaccharide-binding protein mainly responsible for translocation of the chitooligosaccharide (GlcNAc)2 across the double membranes of marine bacteria. However, structural and thermodynamic understanding of the sugar-binding/-release processes of VhCBP is relatively less. VhCBP displayed the greatest affinity toward (GlcNAc)2, with lower affinity for longer-chain chitooligosaccharides [(GlcNAc)3-4]. (GlcNAc)4 partially occupied the closed sugar-binding groove, with two reducing-end GlcNAc units extending beyond the sugar-binding groove and barely characterized by weak electron density. Mutation of three conserved residues (Trp363, Asp365, and Trp513) to Ala resulted in drastic decreases in the binding affinity toward the preferred substrate (GlcNAc)2, indicating their significant contributions to sugar binding. The structure of the W513A-(GlcNAc)2 complex in a 'half-open' conformation unveiled the intermediary step of the (GlcNAc)2 translocation from the soluble CBP in the periplasm to the inner membrane-transporting components. Isothermal calorimetry data suggested that VhCBP adopts the high-affinity conformation to bind (GlcNAc)2, while its low-affinity conformation facilitated sugar release. Thus, chitooligosaccharide translocation, conferred by periplasmic VhCBP, is a crucial step in the chitin catabolic pathway, allowing Vibrio bacteria to thrive in oceans where chitin is their major source of nutrients.

Fine-Scale Structuring of Planktonic Vibrio spp. in the Chinese Marginal Seas.

Vibrio is ubiquitous in marine environments with high metabolism flexibility and genome plasticity. Studies have investigated the ecological distribution of Vibrio spp. in several narrow zones, but a broad scale pattern of distribution and community assembly is still lacking. Here, we elucidated the distribution of Vibrio spp. in seawater along the Chinese marginal seas with a high spatial range. Comparison of Vibrio abundance between 3- and 0.2-µm-pore-size membranes showed distinction in preferential lifestyle. Vibrio spp. in the Yellow Sea (YS) was low in abundance and adopted a particle-associated lifestyle, whereas that in the East China Sea (ECS) and South China Sea (SCS) was more abundant and was likely in a temporary free-living state as a strategy to cope with nutrient limitation. Vibrio community compositions were also separated by sampling area, with different dominant groups in YS (Vibrio chagasii and Vibrio harveyi), ECS and SCS (Vibrio japonicus and V. chagasii). The community niche breadth was significantly wider in ECS and SCS than that of YS. Among species, V. chagasii and V. harveyi had the largest niche breadths likely reflecting strong competitive positions. Stochastic processes played important roles in shaping the geographical pattern of the vibrionic community. Environmental selection (e.g., temperature, salinity, and dissolved oxygen) had a much greater impact on the community in surface than in bottom water. The large proportions of unexplained variations (78.9%) imply complex mechanisms in their community assembly. Our study provides insights into the spatial distribution patterns and underlying assembly mechanisms of Vibrio at a broad spatial scale. IMPORTANCE Vibrio spp. may exert large impacts on biogeochemical cycling in coastal habitats, and their ecological importance has drawn increasing attention. Here, we investigated the spatial distribution pattern and community assembly of Vibrio populations along the Chinese marginal seas, spanning a wide spatial scale. Our results showed that the abundances of the Vibrio population increased with decreasing latitude and their preferential lifestyle differed among adjacent coastal areas. The compositions of Vibrio spp. were also separated by geographical location, which was mainly attributable to stochastic processes. Overall, this work contributes to the understanding of the ecological distribution patterns and the community assembly mechanisms of marine vibrios at a high spatial range. The large proportion of unexplained variations indicates the existence of complex mechanisms in the assembly of vibrionic community which should be considered comprehensively in future.

Isolation and characterization of four pathogenic strains of Vibrio spp. from farmed sea urchins, Strongylocentrotus intermedius, in China.

The sea urchin Strongylocentrotus intermedius, famous for its gonadal quality, is one of the most important farmed species in the sea area of northern China. Since 2020, outbreaks of black peristomial membrane disease (commonly called black mouth disease) have frequently occurred in spring and winter in cultured S. intermedius. In this study, we isolated the predominant bacteria from different tissues of diseased sea urchins from a North China farm in the spring of 2021. Four pathogenic strains (named SIBMPM01, SIBMPM02, SIBMPM03 and SIBMCF01) were obtained and characterized by Gram staining, morphological observation, artificial infection tests, and metabolic characteristics. Our results showed that: 1) all obtained strains belonged to the genus Vibrio and had morphological differences. Phylogenetic analysis indicated that the four obtained strains might be novel Vibrio species. 2) Laboratory-based artificial infection tests showed that sea urchins infected with either SIBMPM01, SIBMPM02, SIBMPM03 or SIBMCF01 exhibited pathological symptoms of a black peristomial membrane in a dosage-dependent and temperature-dependent manner. The virulence of SIBMCF01 was greater than those of the others. 3) Metabolic characterization data showed that SIBMPM01, SIBMPM02, SIBMPM03 and SIBMCF01 shared similar metabolic characteristics. 4) Antimicrobial susceptibility tests demonstrated that the four obtained strains were all sensitive to ampicillin, doxycycline, norfloxacin, ofloxacin, furazolidone and chloramphenicol. SIBMPM01 was specifically sensitive to neomycin, and SIBMCF01 was specifically sensitive to carboxybenzyl penicillin.

Evaluation of three Bacillus spp. isolated from the gut of giant freshwater prawn as potential probiotics against pathogens causing Vibriosis and Aeromonosis.

Pathogens from the Vibrio and Aeromonas genera often cause detrimental effects to the aquaculture sector. Previously, antibiotics was used to resolve the infections, but this caused the spread of antibiotic resistant bacteria and antibiotic resistance genes into the environment. As an approach to address this issue, probiotic bacteria were introduced to improve the hosts' microbiome, disease protection, health condition, growth efficiency, feed consumption, stress response and general vigour. However, reports showed that some commercially available probiotics were restricted to a small number of microbial species and there are inconsistencies concerning its effectiveness. Hence, the aim of this study was to isolate and evaluate new Bacillus spp. from the gut of giant freshwater prawn as potential probiotics. Three Bacillus spp. isolates, Bacillus subtilis FS6 (MZ960135), Bacillus velezensis FS26 (MZ960133) and Bacillus pumilus FS97 (MZ960136) were characterised, and in vitro testing showed good probiotic properties which can help in dealing with diseases in aquaculture. Among the Bacillus spp., Bacillus velezensis FS26 showed higher antimicrobial activity towards Aeromonas hydrophila LMG 13658 and Aeromonas veronii clone DK-A. veronii-27 at 23.7 mm and 25 mm, respectively. Bacillus subtilis FS6 and Bacillus velezensis FS26 resulted in good adherence to both xylene and chloroform hydrocarbons. The Bacillus spp. isolated displayed high survivability towards 0.3% bile salt and exhibited amylase, protease, and lipase activities. Thus, the isolated Bacillus spp. are considered safe based on the sensitivity analysis towards antibiotics and γ-haemolytic activity.

Aquatic environments: A potential source of antimicrobial-resistant Vibrio spp.

Vibrio spp. are associated with water and seafood-related outbreaks worldwide. They are naturally present in aquatic environments such as seawater, brackish water and freshwater environments. These aquatic environments serve as the main reservoirs of antimicrobial-resistant genes and promote the transfer of antimicrobial-resistant bacterial species to aquatic animals and humans through the aquatic food chain. Vibrio spp. are known as etiological agents of cholera and non-cholera Vibrio infections in humans and animals. Antimicrobial-resistant Vibrio species have become a huge threat in regard to treating Vibrio infections in aquaculture and public health. Most of the Vibrio spp. possess resistance towards the commonly used antimicrobials, including ß-lactams, aminoglycosides, tetracyclines, sulphonamides, quinolones and macrolides. The aim of this review is to summarize the antimicrobial resistance properties of Vibrio spp. isolated from aquatic environments to provide awareness about potential health risks related to Vibrio infections in aquaculture and public health.

Isolation and characterization of Bacillus spp. from aquaculture cage water and its inhibitory effect against selected Vibrio spp.

The aim of this study was to evaluate the probiotic potential of Bacillus strains with antibacterial activity against Vibrio spp. in vitro. 13 Bacillus strains were selected using Bacillus-selective agar. Among the strains elected in the preliminary screening for antagonistic effect, only the strain PJ_11 with better antibacterial ability was selected and used in further experiments. Based on the morphological and biochemical features with phylogenetic analysis of the 16S rRNA sequencing, selected strain was identified as B. pumilus. PJ_11 showed probiotic properties such as able to survive in wide range of temperatures and salinity, tolerance to 0.3% oxgal and pH 2.0, and high cell-adhesion activity. In this study, there was no special feature regarding the inhibitory effect of the isolate against Gram-positive or Gram-negative indicator bacteria. However, the inhibition effect of the culture was found to be greater than that of the supernatant. PJ_11 exhibited strong antibacterial activity against Vibrio vulnificus, Vibrio parahaemolyticus and Vibrio anguillarum that were isolated from fish (Dicentrarchus labrax) with disease symptoms. The inhibition diameter of strain PJ_11 ranged from 17.63 to 26.68 mm. In co-culture assay with the strain PJ_11 culture, the growth of V. vulnificus, V. parahaemolyticus and V. anguillarum was inhibited after 120 h with an initial level of 1.0 × 108 CFU/mL. PJ_11 had antibiotic susceptibility to commonly used antibiotics in aquaculture but only resistant to chloramphenicol. Isolate was non-hemolytic. These findings suggested that the strain PJ_11 is good probiotic candidate and may have potential applications to prevent/control vibriosis in aquaculture.

Simultaneous isolation and enumeration of virulent Vibrio cholerae and Vibrio vulnificus using an advanced MPN-PCR method.

Vibrio cholerae and Vibrio vulnificus are critical foodborne pathogens that need to be intensively controlled for their infection due to the intake and distribution of seafood, especially raw oysters. For this reason, various methods have already been developed for the detection and enumeration of these bacteria. The most probable number (MPN)-PCR (polymerase chain reaction) method is commonly used with the selective-differential medium for the efficiency and convenience of cell enumeration. One of the most frequently used for detecting Vibrio spp. is thiosulfate-citrate-bile salts-sucrose (TCBS) agar. But this selective-differential medium can fail to distinguish between V. cholerae, V. vulnificus, and Vibrio alginolyticus. For this reason, the conventional MPN-PCR method with TCBS medium for the detection of Vibrio spp. has a problem with processing PCR two times. This study suggests a simple and minimized detection method using one-time PCR and non-NaCl Luria-Bertani (LB-0) medium culture. This detection method is based on the difference in salt requirement between V. cholerae and V. vulnificus. Employing the developed methodology, the simultaneous cell enumeration of V. cholerae and V. vulnificus can be possible at a low cost. Furthermore, this study proposes a new specific primer to detect virulence-related genes from V. cholerae and V. vulnificus. This advanced MPN-PCR method was verified using bioaccumulated pacific oysters (Crassostrea gigas) by V. cholerae and V. vulnificus.

Prevalence, antibiogram and virulence characterization of Vibrio isolates from fish and shellfish in Egypt: a possible zoonotic hazard to humans.

AIMS: Infection of seafood with pathogenic species of the genus Vibrio causes human food-borne illnesses. This study was executed to examine the antimicrobial resistance phenotypes, biofilm-forming capability and virulence-associated genes of Vibrio from fish and shellfishes. METHODS AND RESULTS: Three hundred fresh water and marine fish and shellfish samples were collected from wet markets and supermarkets in Mansoura, Egypt. Bacteriological examination and PCR amplification identified 92 Vibrio spp., including 42 Vibrio parahaemolyticus and 50 Vibrio alginolyticus isolates from the examined fish and shellfish (infection rate: 30·67%). However, V. vulnificus was not found in this study. Vibrio spp. exhibited variable frequencies of antimicrobial resistance with higher percentages to ampicillin and penicillin. Multidrug resistance (MDR) was detected in 69·04 and 38% of V. parahaemolyticus and V. alginolyticus respectively. PCR testing of virulence genes, tdh, trh and tlh revealed the presence of tlh and trh in 100 and 11·9% of V. parahaemolyticus isolates respectively and none of V. alginolyticus carried any of these genes. Biofilm-forming capability was displayed by 76% of V. parahaemolyticus and 73·8% of V. alginolyticus isolates. Both V. parahaemolyticus and V. alginolyticus showed nonsignificant weak positive correlations (r < 0·4) between antimicrobial pairs belonging to different classes; however, a significant positive correlation (P <0·05) between trh and resistance to erythromycin (r = 0·45) and imipenem (r = 0·38) was only identified in V. parahaemolyticus. CONCLUSIONS: This study reports the existence of MDR strains of V. parahaemolyticus and V. alginolyticus from the common types of fishes and shellfishes in Egypt. Furthermore, the presence of virulence genes in these isolates and the ability to produce a biofilm in vitro pose potential health hazards to consumers. SIGNIFICANCE AND IMPACT OF THE STUDY: Frequent monitoring of seafood for the presence of Vibrio spp. and their antimicrobial susceptibility, virulence determinants and biofilm-forming capability is important for assessing the risk posed by these organisms to the public and for improving food safety.

Susceptibility variation to the main pathogens of Crassostrea gigas at the larval, spat and juvenile stages using unselected and selected oysters to OsHV-1 and/or V. aestuarianus.

French commercial hatcheries are massively producing Crassostrea gigas selected for their higher resistance to OsHV-1, and soon should also implement selection for increasing resistance to Vibrio aestuarianus. The first objective of this study was to optimize the breeding programs for dual resistance to OsHV-1 and V. aestuarianus to determine the earliest life stage for which oysters are able to develop disease resistance. Wild stocks and selected families were tested using experimental infections by both pathogens at the larval, spat and juvenile stages. Oyster families could be evaluated for OsHV-1 as soon as the larval stage by a bath method, but this only highlighted the most resistant families; those that showed the highest resistance to V. aestuarianus could be determined using the cohabitation method at the juvenile stage. The second objective of this study was to determine if selection to increase/decrease the resistance to OsHV-1 and V. aestuarianus could have an impact on other major pathogens currently detected in hatchery at the larval stage, and in nursery and field at the spat/juveniles stages (V. coralliilyticus, V. crassostreae, V. tasmaniensis, V. neptunius, V. europaeus, V. harveyi, V. chagasi). No relationship was found between mortality caused by V. aestuarianus/OsHV-1 and the mortality caused by the other virulent bacterial strains tested regardless the stages, except between OsHV-1 and V. tasmaniensis at the juvenile stage. Finally, miscellaneous findings were evidenced such as (1) bath for bacterial challenges was not adapted for spat, (2) the main pathogens at the larval stage were OsHV-1 and V. coralliilyticus using bath, while it was V. coralliilyticus, V. europaeus, and V. neptunius at the juvenile stage by injection, and (4) variation in mortality was observed among families/wild controls for all pathogens at larval and juvenile stages, except for V. harveyi for larvae.

Dietary supplementation with probiotic Rhodobacter sphaeroides SS15 extract to control acute hepatopancreatic necrosis disease (AHPND)-causing vibrio parahaemolyticus in cultivated white shrimp.

Cultivation of Penaeus vannamei (Pacific white shrimp) is faced with the serious problem of acute hepatopancreatic necrosis disease (AHPND), caused by Vibrio parahaemolyticus that carries plasmids containing binary toxin genes. The disease is typically moderated by the use of antibiotics. To investigate the control of AHPND and maintenance of water quality without the use of antibiotics, the supplementation of shrimp feed with anti-vibrio compounds from a crude extract of probiotic Rhodobacter sphaeroides SS15 was evaluated. The experimental design comprised four treatments: two that were challenged with AHPND-causing V. parahaemolyticus SR2 at a density of 6.0 × 105 cells mL-1 and two that were not challenged. The unchallenged groups comprised a control group that received commercial feed only (CF) and a group that received CF supplemented with 0.27% (w/w) of the extract of R. sphaeroides SS15 (modified CF: MCF). The treatments challenged with V. parahaemolyticus SR2 comprised a challenge group that received CF only (challenge CF: CF-SR2) and a challenge group that received modified CF (challenge MCF: MCF-SR2). V. parahaemolyticus SR2 was inoculated at the start of cultivation and at day 48 at the same cell density. No significant difference in growth performance was found among all treatments. All water quality parameters were better in the two treatments that received modified CF but excess nitrite, due to overfeeding in low salinity (5-8 ppt), caused shrimp mortality in all treatments. Vibrio populations were much higher in the CF treatments than in the modified CF treatments. After the first challenge, the survival rate was about 67% in both the CF-SR2 and MCF-SR2 treatments, compared with approximately 83% in the unchallenged treatments. One day after the second challenge, mortality in the CF-SR2 treatment was 100%, whereas 16.67% survived in the MCF-SR2 treatment. The survival rate was roughly 27% higher in the MCF treatment than in the CF treatment. The hepatopancreas and gut of both modified CF treatments showed no sign of AHPND. Via better water quality and trained immunity, the anti-vibrio compounds in the modified CF have great potential to increase the survival of cultivated shrimp infected with AHPND-causing strain SR2.