The widespread presence of a family of fish virulence plasmids in Vibrio vulnificus stresses its relevance as a zoonotic pathogen linked to fish farms.

Vibrio vulnificus is a pathogen of public health concern that causes either primary septicemia after ingestion of raw shellfish or secondary septicemia after wound exposure to seawater. In consequence, shellfish and seawater are considered its main reservoirs. However, there is one aspect of its biology that is systematically overlooked: its association with fish in its natural environment. This association led in 1975 to the emergence of a zoonotic clade within phylogenetic lineage 2 following successive outbreaks of vibriosis in farmed eels. Although this clade is now worldwide distributed, no new zoonotic clades were subsequently reported. In this work, we have performed phylogenetic, genomic and functional studies to show that other zoonotic clades are in fact present in 4 of the 5 lineages of the species. Further, we associate these clades, most of them previously but incompletely described, with the acquisition of a family of fish virulence plasmids containing genes essential for resistance to the immune system of certain teleosts of interest in aquaculture. Consequently, our results provide several pieces of evidence about the importance of this species as a zoonotic agent linked to fish farms, as well as on the relevance of these artificial environments acting as drivers that accelerate the evolution of the species.

An isothermal recombinase polymerase amplification and lateral flow strip combined method for rapid on-site detection of Vibrio vulnificus in raw seafood.

Vibrio vulnificus is an important foodborne pathogenic bacterium that mainly contaminates seafood. Rapid and accurate technologies that suitable for on-site detection are critical for effective control of its spreading. Conventional detection methods and polymerase chain reaction (PCR)-based and qPCR-based approaches have application limitations in on-site scenarios. Application of loop-mediated isothermal amplification (LAMP) technology was a good step towards the on-site detection. In this study, a recombinase polymerase amplification (RPA)-based detection method for V. vulnificus was developed combining with lateral flow strip (LFS) for visualized signal. The method targeted the conservative empV gene encoding the extracellular metalloproteinase, and finished detection in 35 min at a conveniently low temperature of 37 °C. It showed good specificity and an excellent sensitivity of 2 copies of the genome or 10-1 colony forming unit (CFU) per reaction, or 1 CFU/10 g in spiked food samples with enrichment. The method tolerated unpurified templates directly from sample boiling, which added the convenience of the overall procedure. Application of the RPA-LFS method for clinical samples showed accurate and consistent detection results compared to bioassay and quantitative PCR. This RPA-LFS combined method is well suited for on-site detection of V. vulnificus.

Seasonal and Geographical Differences in Total and Pathogenic Vibrio parahaemolyticus and Vibrio vulnificus Levels in Seawater and Oysters from the Delaware and Chesapeake Bays Determined Using Several Methods.

Oyster and seawater samples were collected from five sites in the Chesapeake Bay, MD, and three sites in the Delaware Bay, DE, from May to October 2016 and 2017. Abundances and detection frequencies for total and pathogenic Vibrio parahaemolyticus and Vibrio vulnificus were compared using the standard most-probable-number-PCR (MPN-PCR) assay and a direct-plating (DP) method on CHROMagar Vibrio for total (tlh+ ) and pathogenic (tdh+ and trh+ ) V. parahaemolyticus genes and total (vvhA) and pathogenic (vcgC) V. vulnificus genes. The colony overlay procedure for peptidases (COPP) assay was evaluated for total Vibrionaceae DP had high false-negative rates (14 to 77%) for most PCR targets and was deemed unsatisfactory. Logistic regression models of the COPP assay showed high concordances with MPN-PCR for tdh+ and trh+V. parahaemolyticus and vvhA+V. vulnificus in oysters (85.7 to 90.9%) and seawater (81.1 to 92.7%) when seawater temperature and salinity were factored into the model, suggesting that the COPP assay could potentially serve as a more rapid method to detect vibrios in oysters and seawater. Differences in total Vibrionaceae and pathogenic Vibrio abundances between state sampling sites over different collection years were contrasted for oysters and seawater by MPN-PCR. Abundances of tdh+ and trh+V. parahaemolyticus were ∼8-fold higher in Delaware oysters than in Maryland oysters, whereas abundances of vcgC+V. vulnificus were nearly identical. For Delaware oysters, 93.5% were both tdh+ and trh+, compared to only 19.2% in Maryland. These results indicate that pathogenic V. parahaemolyticus was more prevalent in the Delaware Bay than in the Chesapeake Bay.IMPORTANCE While V. parahaemolyticus and V. vulnificus cause shellfish-associated morbidity and mortality among shellfish consumers, current regulatory assays for vibrios are complex, time-consuming, labor-intensive, and relatively expensive. In this study, the rapid, simple, and inexpensive COPP assay was identified as a possible alternative to MPN-PCR for shellfish monitoring. This paper shows differences in total Vibrionaceae and pathogenic vibrios found in seawater and oysters from the commercially important Delaware and Chesapeake Bays. Vibrio parahaemolyticus isolates from the Delaware Bay were more likely to contain commonly recognized pathogenicity genes than those from the Chesapeake Bay.

Cross-Kingdom Activation of Vibrio Toxins by ADP-Ribosylation Factor Family GTPases.

Pathogenic Vibrio species use many different approaches to subvert, attack, and undermine the host response. The toxins they produce are often responsible for the devastating effects associated with their diseases. These toxins target a variety of host proteins, which leads to deleterious effects, including dissolution of cell organelle integrity and inhibition of protein secretion. Becoming increasingly prevalent as cofactors for Vibrio toxins are proteins of the small GTPase families. ADP-ribosylation factor small GTPases (ARFs) in particular are emerging as a common host cofactor necessary for full activation of Vibrio toxins. While ARFs are not the direct target of Vibrio cholerae cholera toxin (CT), ARF binding is required for its optimal activity as an ADP-ribosyltransferase. The makes caterpillars floppy (MCF)-like and the domain X (DmX) effectors of the Vibrio vulnificus multifunctional autoprocessing repeats-in-toxin (MARTX) toxin also both require ARFs to initiate autoprocessing and activation as independent effectors. ARFs are ubiquitously expressed in eukaryotes and are key regulators of many cellular processes, and as such they are ideal cofactors for Vibrio pathogens that infect many host species. In this review, we cover in detail the known Vibrio toxins that use ARFs as cross-kingdom activators to both stimulate and optimize their activity. We further discuss how these contrast to toxins and effectors from other bacterial species that coactivate, stimulate, or directly modify host ARFs as their mechanisms of action.

Phylogeny and life cycle of the zoonotic pathogen Vibrio vulnificus.

Vibrio vulnificus is a zoonotic pathogen able to cause diseases in humans and fish that occasionally result in sepsis and death. Most reviews about this pathogen (including those related to its ecology) are clearly biased towards its role as a human pathogen, emphasizing its relationship with oysters as its main reservoir, the role of the known virulence factors as well as the clinic and the epidemiology of the human disease. This review tries to give to the reader a wider vision of the biology of this pathogen covering aspects related to its phylogeny and evolution and filling the gaps in our understanding of the general strategies that V. vulnificus uses to survive outside and inside its two main hosts, the human and the eel, and how its response to specific environmental parameters determines its survival, its death, or the triggering of an infectious process.

Whole-genome comparison between reference sequences and oyster Vibrio vulnificus C-genotype strains.

Whole-genome sequences of Vibrio vulnificus clinical genotype (C-genotype) from the CICESE Culture Collection, isolated from oysters, were compared with reference sequences of CMCP6 and YJ016 V. vulnificus C-genotype strains of clinical origin. The RAST web server estimated the whole genome to be ~4.8 Mb in CICESE strain 316 and ~4.7 Mb in CICESE strain 325. No plasmids were detected in the CICESE strains. Based on a phylogenetic tree that was constructed with the whole-genome results, we observed high similarity between the reference sequences and oyster C-genotype isolates and a sharp contrast with environmental genotype (E-genotype) reference sequences, indicating that the differences between the C- and E-genotypes do not necessarily correspond to their isolation origin. The CICESE strains share 3488 genes (63.2%) with the YJ016 strain and 3500 genes (63.9%) with the CMCP6 strain. A total of 237 pathogenicity associated genes were selected from reference clinical strains, where-92 genes were from CMCP6, 126 genes from YJ016, and 19 from MO6-24/O; the presence or absence of these genes was recorded for the CICESE strains. Of the 92 genes that were selected for CMCP6, 67 were present in both CICESE strains, as were as 86 of the 126 YJ016 genes and 13 of the 19 MO6-24/O genes. The detection of elements that are related to virulence in CICESE strains-such as the RTX gene cluster, vvhA and vvpE, the type IV pili cluster, the XII genomic island, and the viuB genes, suggests that environmental isolates with the C-genotype, have significant potential for infection.

Quantitative PCR enumeration of vcgC and 16S rRNA type A and B genes as virulence indicators for environmental and clinical strains of Vibrio vulnificus in Galveston Bay oysters.

Oysters from a reef in Galveston Bay, Texas, USA, were screened for more virulent clinical strains versus less virulent environmental strains of Vibrio vulnificus using a combination of quantitative PCR assays for the virulence correlating gene (clinical variant, vcgC) and 16S rRNA types A and B (type A = environmental, type B = clinical). The combination of vcgC and 16S rRNA type B loci to determine clinical type strains was suitable, as indicated by the strong correlation (R2 = 0.98; p < 0.001) between these gene counts over time and their relative proportion (up to 93.8% and 94.3%, respectively) to vvhA genes used to quantify all strains of V. vulnificus. A strong seasonal shift of V. vulnificus strain types was observed. Environmental strains (16S rRNA type A) predominated from April to mid-June as salinities increased from 22 to 27 PSU (practical salinity unit) and temperatures rose 20 to 28 °C, with peak gene quantities of 16 812 ± 56 CFU/g. As temperatures increased to ≥30 °C from mid-June to September and salinities rose above 27 PSU, clinical strains (16S rRNA type B; vcgC) predominated with peak quantities 31 868 ± 287 and 32 360 ± 178 CFU/g, respectively.

Evolutionary Model of Cluster Divergence of the Emergent Marine Pathogen Vibrio vulnificus: From Genotype to Ecotype.

Vibrio vulnificus, an opportunistic pathogen, is the causative agent of a life-threatening septicemia and a rising problem for aquaculture worldwide. The genetic factors that differentiate its clinical and environmental strains remain enigmatic. Furthermore, clinical strains have emerged from every clade of V. vulnificus In this work, we investigated the underlying genomic properties and population dynamics of the V. vulnificus species from an evolutionary and ecological point of view. Genome comparisons and bioinformatic analyses of 113 V. vulnificus isolates indicate that the population of V. vulnificus is made up of four different clusters. We found evidence that recombination and gene flow between the two largest clusters (cluster 1 [C1] and C2) have drastically decreased to the point where they are diverging independently. Pangenome and phenotypic analyses showed two markedly different lifestyles for these two clusters, indicating commensal (C2) and bloomer (C1) ecotypes, with differences in carbohydrate utilization, defense systems, and chemotaxis, among other characteristics. Nonetheless, we identified frequent intra- and interspecies exchange of mobile genetic elements (e.g., antibiotic resistance plasmids, novel "chromids," or two different and concurrent type VI secretion systems) that provide high levels of genetic diversity in the population. Surprisingly, we identified strains from both clusters in the mucosa of aquaculture species, indicating that manmade niches are bringing strains from the two clusters together. We propose an evolutionary model of V. vulnificus that could be broadly applicable to other pathogenic vibrios and facultative bacterial pathogens to pursue strategies to prevent their infections and emergence.IMPORTANCEVibrio vulnificus is an emergent marine pathogen and is the cause of a deadly septicemia. However, the genetic factors that differentiate its clinical and environmental strains and its several biotypes remain mostly enigmatic. In this work, we investigated the underlying genomic properties and population dynamics of the V. vulnificus species to elucidate the traits that make these strains emerge as a human pathogen. The acquisition of different ecological determinants could have allowed the development of highly divergent clusters with different lifestyles within the same environment. However, we identified strains from both clusters in the mucosa of aquaculture species, indicating that manmade niches are bringing strains from the two clusters together, posing a potential risk of recombination and of emergence of novel variants. We propose a new evolutionary model that provides a perspective that could be broadly applicable to other pathogenic vibrios and facultative bacterial pathogens to pursue strategies to prevent their infections.

Vibrio species involved in seafood-borne outbreaks (Vibrio cholerae, V. parahaemolyticus and V. vulnificus): Review of microbiological versus recent molecular detection methods in seafood products.

Seafood products are widely consumed all around the world and play a significant role on the economic market. Bacteria of the Vibrio genus can contaminate seafood and thus pose a risk to human health. Three main Vibrio species, V. cholerae, V. parahaemolyticus and V. vulnificus, are potentially pathogenic to humans. These species are responsible for a dramatic increase of seafood-borne infections worldwide. Hence, early detection of total and pathogenic Vibrio is needed and should rely on quick and effective methods. This review aims to present the standard methods FDA-BAM, ISO/TS 21872-1:2007 and TS 21872-2:2007 and compare them to recent molecular biology methods including endpoint PCR, quantitative real-time PCR (qPCR) and PCR-derived methods with a focus on LAMP (loop-mediated isothermal amplification). The available methods presented here are dedicated to the detection and identification of the Vibrio species of interest in seafood.

Virulence properties of Vibrio vulnificus isolated from diseased zoea of freshness shrimp Macrobrachium rosenbergii.

Macrobrachium rosenbergii is one of the most economically important freshwater shimp, with fast growth and high nutrient content in the agricultural development of China. However, it had been suffering diseases infection, causing mass death and great economic losses. In the present study, a bacteria strain was isolated from the diseased zoea of M. rosenbergii and was identified as Vibrio vulnificus by biochemical characteristics and 16S rRNA homologous analysis. The infection test showed that the strain GXFL1-3 was pathogenic to zoea and postlarva of M. rosenbergii, and the half lethal dose (LD50) were 1.16 × 106 CFU/mL and 1.45 × 106 CFU/mL, respectively. Detection of virulence-associated genes by PCR indicated that GXFL1-3 was positive for fur, OmpU, acfA, flaA, vvhA, vvp and tcp, the detection of extracellular enzymes and hemolysin showed that GXFL1-3 was positive for protease, amylase, lecithin, urease and hemolysin activity, further supporting its pathogenicity. A duplex PCR for rapid detection of V. vulnificus was established. Only V. vulnificus could amplify two specific bands of flaA and fur, while the other six strains of Vibrio were negative. The minimum detectable amount of template was 2.4 × 103 CFU/mL through sensitivity test.

A double-quadratic model for predicting Vibrio species in water environments of Japan.

Vibrio spp. are natural inhabitants of marine and estuarine environments. Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus are the major infectious agents for humans. Their densities are affected by environmental factors such as water temperature and salinity. The detailed contribution of each factor still remains to be elucidated. Here we conducted multi-coastal study in a 21-month period to examine relationships between environmental factors and V. cholerae, V. parahaemolyticus and V. vulnificus densities in sea surface water in eight coastal sites of four prefectures in Japan. Vibrio densities were measured by a most-probable-number with PCR method which is highly sensitive and quantitative (3/100 ml of detection limit). Vibrio densities were analyzed with environmental factors including water temperature, salinity, total dissolved substance, and pH, and their quadratics. A linear regression model suited best for prediction of V. cholerae density. A novel double-quadratic model suited best for the prediction of V. parahaemolyticus and V. vulnificus densities.

Effects of Intertidal Harvest Practices on Levels of Vibrio parahaemolyticus and Vibrio vulnificus Bacteria in Oysters.

UNLABELLED: Vibrio parahaemolyticus and Vibrio vulnificus can grow rapidly in shellfish subjected to ambient air conditions, such as during intertidal exposure. In this study, levels of total and pathogenic (tdh(+) and/or trh(+)) V. parahaemolyticus and total V. vulnificus were determined in oysters collected from two study locations where intertidal harvest practices are common. Samples were collected directly off intertidal flats, after exposure (ambient air [Washington State] or refrigerated [New Jersey]), and after reimmersion by natural tidal cycles. Samples were processed using a most-probable-number (MPN) real-time PCR method for total and pathogenic V. parahaemolyticus or V. vulnificus In Washington State, the mean levels of V. parahaemolyticus increased 1.38 log MPN/g following intertidal exposure and dropped 1.41 log MPN/g after reimmersion for 1 day, but the levels were dependent upon the container type utilized. Pathogenic V. parahaemolyticus levels followed a similar trend. However, V. vulnificus levels increased 0.10 log MPN/g during intertidal exposure in Washington but decreased by >1 log MPN/g after reimmersion. In New Jersey, initial levels of all vibrios studied were not significantly altered during the refrigerated sorting and containerizing process. However, there was an increase in levels after the first day of reimmersion by 0.79, 0.72, 0.92, and 0.71 log MPN/g for total, tdh(+) and trh(+) V. parahaemolyticus, and V. vulnificus, respectively. The levels of all targets decreased to those similar to background after a second day of reimmersion. These data indicate that the intertidal harvest and handling practices for oysters that were studied in Washington and New Jersey do not increase the risk of illness from V. parahaemolyticus or V. vulnificus IMPORTANCE: Vibrio parahaemolyticus and Vibrio vulnificus are the leading causes of seafood-associated infectious morbidity and mortality in the United States. Vibrio spp. can grow rapidly in shellfish subjected to ambient air conditions, such as during periods of intertidal exposure. When oysters are submersed with the incoming tide, the vibrios can be purged. However, data on the rates of increase and purging during intertidal harvest are scarce, which limits the accuracy of risk assessments. The objective of this study was to help fill these data gaps by determining the levels of total and pathogenic (tdh(+) and/or trh(+)) V. parahaemolyticus and V. vulnificus in oysters from two locations where intertidal harvest practices are common, using the current industry practices. The data generated provide insight into the responses of Vibrio spp. to relevant practices of the industry and public health, which can be incorporated into risk management decisions.

Clinical and environmental genotypes of Vibrio vulnificus display distinct, quorum-sensing-mediated, chitin detachment dynamics.

The ability for bacteria to attach to and detach from various substrata is important for colonization, survival and transitioning to new environments. An opportunistic human pathogen, Vibrio vulnificus, can cause potentially fatal septicemia after ingestion of undercooked seafood. Based on genetic polymorphisms, strains of this species are subtyped into clinical (C) and environmental (E) genotypes. Vibrio vulnificus readily associates with chitin, thus we investigated chitin detachment dynamics in these disparate genotypes. We found that C-genotypes detach significantly more than E-genotypes after 24 hours in aerobic as well as anaerobic conditions. Furthermore, expression of genes involved in type IV pilin production was significantly downregulated in C-genotypes compared to E-genotypes, suggesting an importance in detachment. Interestingly, gbpA, a gene that has been shown to be important in host colonization in V. cholerae, was upregulated in the C-genotypes during detachment. Additionally, we found that C-genotypes detached to a greater extent, and produced more quorum-sensing (QS) autoinducer-2 molecules relative to E-genotypes, which suggests a role for QS in detachment. These findings suggest that for V. vulnificus, QS-mediated detachment may be a potential mechanism for transitioning into a human host for C-genotypes, while facilitating E-genotype maintenance in the estuarine environment.

The Fish Pathogen Vibrio vulnificus Biotype 2: Epidemiology, Phylogeny, and Virulence Factors Involved in Warm-Water Vibriosis.

Vibrio vulnificus biotype 2 is the etiological agent of warm-water vibriosis, a disease that affects eels and other teleosts, especially in fish farms. Biotype 2 is polyphyletic and probably emerged from aquatic bacteria by acquisition of a transferable virulence plasmid that encodes resistance to innate immunity of eels and other teleosts. Interestingly, biotype 2 comprises a zoonotic clonal complex designated as serovar E that has extended worldwide. One of the most interesting virulence factors produced by serovar E is RtxA13, a multifunctional protein that acts as a lethal factor for fish, an invasion factor for mice, and a survival factor outside the host. Two practically identical copies of rtxA13 are present in all biotype 2 strains regardless of the serovar, one in the virulence plasmid and the other in chromosome II. The plasmid also contains other genes involved in survival and growth in eel blood: vep07, a gene for an outer membrane (OM) lipoprotein involved in resistance to eel serum and vep20, a gene for an OM receptor specific for eel-transferrin and, probably, other related fish transferrins. All the three genes are highly conserved within biotype 2, which suggests that they are under a strong selective pressure. Interestingly, the three genes are related with transferable plasmids, which emphasizes the role of horizontal gene transfer in the evolution of V. vulnificus in nutrient-enriched aquatic environments, such as fish farms.

The Biology of Vibrio vulnificus.

Vibrio vulnificus, carrying a 50% fatality rate, is the most deadly of the foodborne pathogens. It occurs in estuarine and coastal waters and it is found in especially high numbers in oysters and other molluscan shellfish. The biology of V. vulnificus, including its ecology, pathogenesis, and molecular genetics, has been described in numerous reviews. This article provides a brief summary of some of the key aspects of this important human pathogen, including information on biotypes and genotypes, virulence factors, risk factor requirements and the role of iron in disease, association with oysters, geographic distribution, importance of salinity and water temperature, increasing incidence associated with global warming. This article includes some of our findings as presented at the "Vibrios in the Environment 2010" conference held in Biloxi, MS.

Genetic characterization of Vibrio vulnificus strains isolated from oyster samples in Mexico.

Vibrio vulnificus strains were isolated from oysters that were collected at the main seafood market in Mexico City. Strains were characterized with regard to vvhA, vcg genotype, PFGE, multilocus sequence typing (MLST), and rtxA1. Analyses included a comparison with rtxA1 reference sequences. Environmental (vcgE) and clinical (vcgC) genotypes were isolated at nearly equal percentages. PFGE had high heterogeneity, but the strains clustered by vcgE or vcgC genotype. Select housekeeping genes for MLST and primers that were designed for rtxA1 domains divided the strains into two clusters according to the E or C genotype. Reference rtxA1 sequences and those from this study were also clustered according to genotype. These results confirm that this genetic dimorphism is not limited to vcg genotyping, as other studies have reported. Some environmental C genotype strains had high similarity to reference strains, which have been reported to be virulent, indicating a potential risk for oyster consumers in Mexico City.

Multiplex PCR for detection of virulence markers of Vibrio vulnificus.

UNLABELLED: Vibrio vulnificus is a Gram-negative pathogen found in coastal and estuarine waters worldwide that can cause life threatening diseases. Characterization of the vcg (virulence correlated gene) or 16S rRNA alleles is used to distinguish virulent (clinical (C)-type) from presumably avirulent (environmental (E)-type) strains. However, some studies reported a significant number of clinical strains belonging to the E-type. In recent years more potential virulence markers have been identified, that are useful for the identification of potentially pathogenic isolates of the E-type. In this study, we successfully combined detection of pathogenicity region XII, nanA and a mannitol fermentation operon with the virulence associated alleles of the 16S rRNA and vcg genes in one multiplex PCR. Additionally, toxR primers for species confirmation and internal amplification control were included. Validation of multiplex amplification was performed with a total of 132 bacterial strains, including V. vulnificus (n = 71), other Vibrionaceae (n = 50) and non-Vibrio isolates (n = 11). Multiplex PCR showed reliable amplification of four of the five virulence markers with a high sensitivity and specificity. Amplification of the 16S rRNA type B allele was not completely reliable with conventional PCR assays, however, the positive predictive value of this marker was 100 %. SIGNIFICANCE AND IMPACT OF THE STUDY: A multiplex PCR for simultaneous detection and characterization of potentially virulent strains of Vibrio vulnificus was developed and validated. Monitoring programs will benefit from this cost and time effective method when screening large strain collections. Application of the multiplex PCR simplifies determination of risks emanating from V. vulnificus in recreational waters or mussel primary production.

Multipurpose assessment for the quantification of Vibrio spp. and total bacteria in fish and seawater using multiplex real-time polymerase chain reaction.

BACKGROUND: This study describes the first multiplex real-time polymerase chain reaction assay developed, as a multipurpose assessment, for the simultaneous quantification of total bacteria and three Vibrio spp. (V. parahaemolyticus, V. vulnificus and V. anguillarum) in fish and seawater. The consumption of raw finfish as sushi or sashimi has been increasing the chance of Vibrio outbreaks in consumers. Freshness and quality of fishery products also depend on the total bacterial populations present. RESULTS: The detection sensitivity of the specific targets for the multiplex assay was 1 CFU mL⁻¹ in pure culture and seawater, and 10 CFU g⁻¹ in fish. While total bacterial counts by the multiplex assay were similar to those obtained by cultural methods, the levels of Vibrio detected by the multiplex assay were generally higher than by cultural methods of the same populations. Among the natural samples without Vibrio spp. inoculation, eight out of 10 seawater and three out of 20 fish samples were determined to contain Vibrio spp. CONCLUSION: Our data demonstrate that this multiplex assay could be useful for the rapid detection and quantification of Vibrio spp. and total bacteria as a multipurpose tool for surveillance of fish and water quality as well as diagnostic method.

Multiplex PCR assays for the detection of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae with an internal amplification control.

A multiplex polymerase chain reaction (PCR) assay that can simultaneously detect 4 major Vibrio spp., Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae, in the presence of an internal amplification control (IAC) was developed. Species-specific PCR primers were designed based on the gyrB gene for V. alginolyticus, the collagenase gene for V. parahaemolyticus, the vvhA gene for V. vulnificus, and the ompW gene for V. cholerae. Additionally, an IAC primer pair was designed in conserved regions of the bacterial 16S rRNA gene that is used to indicate false-negative results. A multiplex PCR method was developed after optimization of the reaction conditions. The specificity of the PCR was validated by using 83 Vibrio strains and 10 other non-Vibrio bacterial species. The detection limit of the PCR was 10 CFU per tube for V. alginolyticus, V. parahaemolyticus, V. vulnificus, and 10(5) CFU per tube for V. cholerae in mixed conditions. This method was used to identify 69 suspicious Vibrio isolates, and the results were consistent with physiological and biochemical tests. This multiplex PCR method proved to be rapid, sensitive, and specific. The existence of IAC could successfully eliminate false-negative results for the detection of V. alginolyticus, V. parahaemolyticus, V. vulnificus, and V. cholerae.