Isolation and molecular characterization of toxigenic Vibrio parahaemolyticus from the Kii Channel, Japan

Studies were conducted on the ecology of potentially pathogenic Vibrio parahaemolyticus in three coastal areas of Kii Channel, Tokushima, Japan. Seawater and seaweed samples were collected seasonally between June 2003 and May 2004. Total and toxigenic strains of V. parahaemolyticus were isolated using most probable number culture and colony blot hybridization. Toxigenic strains were serotyped and further characterized by random amplified polymorphic DNA (RAPD) and ribotyping. Six thousand strains of V. parahaemolyticus were isolated and 18 were found positive for tdh. V. parahaemolyticus were detected in all samples during summer and autumn, and from some samples during winter and spring. Among the toxigenic strains seven serotypes, five ribotypes and RAPD patterns were observed. Seven strains belonged to O3:K6 clone with identical ribotypes and RAPD patterns to that of a pandemic reference strain. The presence of toxigenic V. parahaemolyticus with pandemic potential might indicate a human health risk due to consumption of marine food sources.     

A persistent, productive, and seasonally dynamic vibriophage population within Pacific oysters (Crassostrea gigas)

In an effort to understand the relationship between Vibrio and vibriophage populations, abundances of Vibrio spp. and viruses infecting Vibrio parahaemolyticus (VpVs) were monitored for a year in Pacific oysters and water collected from Ladysmith Harbor, British Columbia, Canada. Bacterial abundances were highly seasonal, whereas high titers of VpVs (0.5 x 10(4) to 11 x 10(4) viruses cm(-3)) occurred year round in oysters, even when V. parahaemolyticus was undetectable (< 3 cells cm(-3)). Viruses were not detected (<10 ml(-1)) in the water column. Host-range studies demonstrated that 13 VpV strains could infect 62% of the V. parahaemolyticus strains from oysters (91 pairings) and 74% of the strains from sediments (65 pairings) but only 30% of the water-column strains (91 pairings). Ten viruses also infected more than one species among V. alginolyticus, V. natriegens, and V. vulnificus. As winter approached and potential hosts disappeared, the proportion of host strains that the viruses could infect decreased by approximately 50% and, in the middle of winter, only 14% of the VpV community could be plated on summer host strains. Estimates of virus-induced mortality on V. parahaemolyticus indicated that other host species were required to sustain viral production during winter when the putative host species was undetectable. The present study shows that oysters are likely one of the major sources of viruses infecting V. parahaemolyticus in oysters and in the water column. Furthermore, seasonal shifts in patterns of host range provide strong evidence that the composition of the virus community changes during winter.     

Development of a quantitative real-time polymerase chain reaction targeted to the toxR for detection of Vibrio vulnificus

The TaqMan assay, a quantitative real-time polymerase chain reaction (PCR), was developed to target the ToxR gene (toxR) of Vibrio vulnificus. The toxR of V. vulnificus was cloned and sequenced. Based on these results, we designed specific primers and a probe for use in the quantitative PCR assay. Twenty-nine strains of V. vulnificus that were obtained from various sources produced a single PCR product. The amount of final amplification product and threshold cycle number were the same among the strains. We used the method to detect V. vulnificus in seawater and oyster samples. We developed standard curves to quantitate V. vulnificus numbers using the PCR from seawater and oyster samples. The standard curves were not different from that of the pure culture of V. vulnificus. We found the assay was very sensitive detecting as few as 10 microbes per milliliter of seawater and oyster homogenate. Moreover, we evaluated the TaqMan assay to detect V. vulnificus in seawater samples. The numbers of V. vulnificus counted by the TaqMan assay were similar to those by a culture method in almost samples. The TaqMan assay was performed within 2 h compared to days using the culture method. The results indicate the TaqMan assay method used in this study was rapid, effective and quantitative for monitoring V. vulnificus contamination in seawater and seafoods such as oysters.     

Factors influencing in vitro killing of bacteria by hemocytes of the eastern oyster (Crassostrea virginica).

A tetrazolium dye reduction assay was used to study factors governing the killing of bacteria by oyster hemocytes. In vitro tests were performed on bacterial strains by using hemocytes from oysters collected from the same location in winter and summer. Vibrio parahaemolyticus strains, altered in motility or colonial morphology (opaque and translucent), and Listeria monocytogenes mutants lacking catalase, superoxide dismutase, hemolysin, and phospholipase activities were examined in winter and summer. Vibrio vulnificus strains, opaque and translucent (with and without capsules), were examined only in summer. Among V. parahaemolyticus and L. monocytogenes, significantly (P < 0.05) higher levels of killing by hemocytes were observed in summer than in winter. L. monocytogenes was more resistant than V. parahaemolyticus or V. vulnificus to the bactericidal activity of hemocytes. In winter, both translucent strains of V. parahaemolyticus showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. In summer, only one of the V. parahaemolyticus translucent strains showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. No significant differences (P > 0.05) in killing by hemocytes were observed between opaque (encapsulated) and translucent (nonencapsulated) pairs of V. vulnificus. Activities of 19 hydrolytic enzymes were measured in oyster hemolymph collected in winter and summer. Only one enzyme, esterase (C4), showed a seasonal difference in activity (higher in winter than in summer). These results suggest that differences existed between bacterial genera in their ability to evade killing by oyster hemocytes, that a trait(s) associated with the opaque phenotype may have enabled V. parahaemolyticus to evade killing by the oyster's cellular defense, and that bactericidal activity of hemocytes was greater in summer than in winter.     

Studies on pathogenic Vibrio parahaemolyticus during a warm weather season in the Seto Inland Sea,Japan

Vibrio parahaemolyticus is a potentially pathogenic bacterium, occurring naturally in estuarine and marine environments throughout the world. The incidence of this organism in an aquatic environment depends upon many ecofactors. Sea water and organic material were collected during the warm weather season from a coast of the Seto Inland Sea, Japan, and analysed to determine V. parahaemolyticus densities and the occurrence of pathogenic strains, defined as those possessing tdh and/or trh genes by polymerase chain reaction (PCR), using isolated DNA from enrichment culture of the samples. About 99% of samples were positive for V. parahaemolyticus with densities of 3 to >1400 cells per 100 ml of water or 10 g of organic samples by the most-probable-number (MPN)-PCR technique, but only 76.6% were positive by the conventional MPN culture technique, with densities ranging from 3 to >1400 cells per 100 ml of water or 10 g of organics. Furthermore, the tdh and trh genes were positive in 41.5% and 8.5% of samples, respectively, by the MPN-PCR technique. No tdh and trh gene-positive strains were isolated by the conventional MPN culture procedure. The difference in detection between the MPN culture and the MPN-PCR techniques appeared to be significant and may be attributed to different detection sensitivities and other factors.     

Use of sodium dodecyl sulfate-polymyxin B-sucrose medium for isolation of Vibrio vulnificus from shellfish.

The differential and selective sodium dodecyl sulfate-polymyxin B-sucrose medium (SPS) of Kitaura et al. (T. Kitaura, S. Doke, I. Azuma, M. Imaida, K. Miyano, K. Harada, and E. Yabuuchii, FEMS Microbiol. Lett. 17:205-209, 1983), which highlights alkylsulfatase activity, was evaluated for its potential use in the direct isolation and enumeration of Vibrio vulnificus from shellfish. V. vulnificus was detected by this method in six of nine shellfish samples collected from diverse geographic locales during the summer of 1986. Direct enumeration of V. vulnificus at 7.0 X 10(2) to 2.2 X 10(4) CFU/g of shellfish was achieved on SPS agar. All sample results were confirmed in parallel examinations by using conventional glucose-salt-Teepol (Shell Oil Co.) broth and alkaline peptone water enrichment with plating onto thiosulfate-citrate-bile salts-sucrose agar. Additionally, alkylsulfatase activity was evaluated in vitro for 97 strains representing 14 Vibrio spp. V. vulnificus and Vibrio cholerae-01 were the only species consistently found to possess this activity. The range of plating efficiencies for random V. vulnificus strains analyzed on SPS was 11 to 74% (mean, 39%). The use of SPS shows great promise for the study of shellfish and other environmental sources for V. vulnificus.     

Use of sodium dodecyl sulfate-polymyxin B-sucrose medium for isolation of Vibrio vulnificus from shellfish

The differential and selective sodium dodecyl sulfate-polymyxin B-sucrose medium (SPS) of Kitaura et al. (T. Kitaura, S. Doke, I. Azuma, M. Imaida, K. Miyano, K. Harada, and E. Yabuuchii, FEMS Microbiol. Lett. 17:205-209, 1983), which highlights alkylsulfatase activity, was evaluated for its potential use in the direct isolation and enumeration of Vibrio vulnificus from shellfish. V. vulnificus was detected by this method in six of nine shellfish samples collected from diverse geographic locales during the summer of 1986. Direct enumeration of V. vulnificus at 7.0 X 10(2) to 2.2 X 10(4) CFU/g of shellfish was achieved on SPS agar. All sample results were confirmed in parallel examinations by using conventional glucose-salt-Teepol (Shell Oil Co.) broth and alkaline peptone water enrichment with plating onto thiosulfate-citrate-bile salts-sucrose agar. Additionally, alkylsulfatase activity was evaluated in vitro for 97 strains representing 14 Vibrio spp. V. vulnificus and Vibrio cholerae-01 were the only species consistently found to possess this activity. The range of plating efficiencies for random V. vulnificus strains analyzed on SPS was 11 to 74% (mean, 39%). The use of SPS shows great promise for the study of shellfish and other environmental sources for V. vulnificus.     

Persistence of Vibrio vulnificus in tissues of Gulf Coast oysters, Crassostrea virginica, exposed to seawater disinfected with UV light.

Vibrio vulnificus is an estuarine bacterium which can cause opportunistic infections in humans consuming raw Gulf Coast oysters, Crassostrea virginica. Although V. vulnificus is known as a ubiquitous organism in the Gulf of Mexico, its ecological relationship with C. virginica has not been adequately defined. The objective of the present study was to test the hypothesis that V. vulnificus is a persistent microbial flora of oysters and unamenable to traditional methods of controlled purification, such as UV light depuration. Experimental depuration systems consisted of aquaria containing temperature-controlled seawater treated with UV light and 0.2-microns-pore-size filtration. V. vulnificus was enumerated in seawater, oyster shell biofilms, homogenates of whole oyster meats, and tissues including the hemolymph, digestive region, gills, mantle, and adductor muscle. Results showed that depuration systems conducted at temperatures greater than 23 degrees C caused V. vulnificus counts to increase in oysters, especially in the hemolymph, adductor muscle, and mantle. Throughout the process, depuration water contained high concentrations of V. vulnificus, indicating that the disinfection properties of UV radiation and 0.2-microns-pore-size filtration were less than the rate at which V. vulnificus was released into seawater. Approximately 10(5) to 10(6) V. vulnificus organisms were released from each oyster per hour, with 0.05 to 35% originating from shell surfaces. These surfaces contained greater than 10(3) V. vulnificus organisms per cm2. In contrast, when depuration seawater was maintained at 15 degrees C, V. vulnificus was not detected in seawater and multiplication in oyster tissues was inhibited.     

Persistence of Vibrio vulnificus in tissues of Gulf Coast oysters, Crassostrea virginica, exposed to seawater disinfected with UV light.

Vibrio vulnificus is an estuarine bacterium which can cause opportunistic infections in humans consuming raw Gulf Coast oysters, Crassostrea virginica. Although V. vulnificus is known as a ubiquitous organism in the Gulf of Mexico, its ecological relationship with C. virginica has not been adequately defined. The objective of the present study was to test the hypothesis that V. vulnificus is a persistent microbial flora of oysters and unamenable to traditional methods of controlled purification, such as UV light depuration. Experimental depuration systems consisted of aquaria containing temperature-controlled seawater treated with UV light and 0.2-microns-pore-size filtration. V. vulnificus was enumerated in seawater, oyster shell biofilms, homogenates of whole oyster meats, and tissues including the hemolymph, digestive region, gills, mantle, and adductor muscle. Results showed that depuration systems conducted at temperatures greater than 23 degrees C caused V. vulnificus counts to increase in oysters, especially in the hemolymph, adductor muscle, and mantle. Throughout the process, depuration water contained high concentrations of V. vulnificus, indicating that the disinfection properties of UV radiation and 0.2-microns-pore-size filtration were less than the rate at which V. vulnificus was released into seawater. Approximately 10(5) to 10(6) V. vulnificus organisms were released from each oyster per hour, with 0.05 to 35% originating from shell surfaces. These surfaces contained greater than 10(3) V. vulnificus organisms per cm2. In contrast, when depuration seawater was maintained at 15 degrees C, V. vulnificus was not detected in seawater and multiplication in oyster tissues was inhibited.     

Offshore suspension relaying to reduce levels of Vibrio vulnificus in oysters (Crassostrea virginica).

Oysters naturally contaminated with 10(3) to 10(4) most probable numbers (MPN) of Vibrio vulnificus per g were relayed to offshore waters (salinity, 30 to 34 ppt), where they were suspended in racks at a depth of 7.6 m. V. vulnificus counts in oysters were reduced to < 10 MPN/g within 7 to 17 days in five of the six studies. At the end of the studies (17 to 49 days), V. vulnificus levels were reduced further and ranged from a mean of 0.23 to 2.6 MPN/g. Oyster mortalities during relaying were < 6%. The reduction of V. vulnificus in relayed oysters is associated with exposure to high-salinity environments essentially devoid of V. vulnificus. Offshore suspension relaying may be a method that industry can employ to reduce V. vulnificus levels in raw Gulf Coast oysters.     

Survival of Vibrio vulnificus in shellstock and shucked oysters (Crassostrea gigas and Crassostrea virginica) and effects of isolation medium on recovery.

When two species of shellstock oysters were artificially contaminated with Vibrio vulnificus, the bacterium survived when the oysters were stored at 10 degrees C and below. Large numbers of endogenous V. vulnificus cells were found after 7 days at both 0.5 and 10 degrees C in uninoculated control oysters (Crassostrea virginica). Oysters allowed to take up V. vulnificus from seawater retained the bacterium for 14 days at 2 degrees C. The presence of V. vulnificus in the drip exuded from the shellstock presented a possibility of contamination of other shellstock in storage. V. vulnificus injected into shucked Pacific (Crassostrea gigas) and Eastern (C. virginica) oysters survived at 4 degrees C for at least 6 days. An 18-h most-probable-number enrichment step in alkaline peptone water gave higher recovery levels of V. vulnificus than did direct plating to selective agars. The survival of this pathogen in both shellstock and shucked oysters suggests a potential for human illness, even though the product is refrigerated.     

Survival of Vibrio vulnificus in shellstock and shucked oysters (Crassostrea gigas and Crassostrea virginica) and effects of isolation medium on recovery

When two species of shellstock oysters were artificially contaminated with Vibrio vulnificus, the bacterium survived when the oysters were stored at 10 degrees C and below. Large numbers of endogenous V. vulnificus cells were found after 7 days at both 0.5 and 10 degrees C in uninoculated control oysters (Crassostrea virginica). Oysters allowed to take up V. vulnificus from seawater retained the bacterium for 14 days at 2 degrees C. The presence of V. vulnificus in the drip exuded from the shellstock presented a possibility of contamination of other shellstock in storage. V. vulnificus injected into shucked Pacific (Crassostrea gigas) and Eastern (C. virginica) oysters survived at 4 degrees C for at least 6 days. An 18-h most-probable-number enrichment step in alkaline peptone water gave higher recovery levels of V. vulnificus than did direct plating to selective agars. The survival of this pathogen in both shellstock and shucked oysters suggests a potential for human illness, even though the product is refrigerated.     

Distribution of Vibrio vulnificus and other lactose-fermenting vibrios in the marine environment

During the summer of 1981, 3,887 sucrose-negative vibrios were isolated from seawater, sediment, plankton, and animal samples taken from 80 sites from Miami, Fla., to Portland, Maine. Of these, 4.2% were able to ferment lactose. The lactose-positive strains isolated from the various samples correlated positively with pH and turbidity of the water, vibrios in the sediment and oysters, and total bacterial counts in oysters. Negative correlations were obtained for water salinity. Numerical taxonomy was performed on 95 of the lactose-fermenting environmental isolates and 23 reference strains. Five clusters resulted, with the major cluster containing 33 of the environmental isolates and all of the Vibrio vulnificus reference strains. The 33 isolates, which produced an acid reaction in lactose broth within hours of initial inoculation, represented 20% of all lactose-fermenting vibrios studied. These isolates were nearly identical phenotypically to clinical strains of V. vulnificus studied by the Centers for Disease Control, Atlanta, Ga., and by our laboratory, and their identification was confirmed by DNA-DNA hybridization studies. V. vulnificus was isolated from all sample types and from Miami to Cape Cod, Mass., and comparison of the environmental parameters of the eight subsites yielding this species with those of all 80 subsites revealed no significant differences. The majority of the isolates were obtained from animals, with clams providing most (84%) of these. On injection into mice, 82% of the V. vulnificus isolates resulted in death. Members of the remaining four clusters contained strains which differed from V. vulnificus in such phenotypic traits as luminescence and in urease or H(2)S production. None of the other reference cultures, including nine other Vibrio species, were contained in the remaining clusters, and these isolates could not be identified. Most of these were also lethal for mice. Phenotypic differences, potential pathogenicity, and geographic distribution of the five clusters were examined. It is concluded that V. vulnificus is a ubiquitous organism, both geographically and in a variety of environmental sources, although it occurs in relatively low numbers. The public health significance of this organism and of the other unidentified lactose-fermenting Vibrio species is discussed.     

Occurrence and potential pathogenesis of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus on the South Coast of Sweden

During the summer of 2006, several wound infections - of which three were fatal - caused by Vibrio cholerae were reported from patients who had been exposed to water from the Baltic Sea. Before these reports, we initiated a sampling project investigating the occurrence of potential human pathogenic V. cholerae, Vibrio vulnificus and Vibrio parahaemolyticus in The Sound between Sweden and Denmark. The Blue mussel (Mytilus edulis) was used as an indicator to follow the occurrence of vibrios over time. Molecular analyses showed high frequencies of the most potent human pathogenic Vibrio spp.; 53% of mussel samples were positive for V. cholerae (although none were positive for the cholera toxin gene), 63% for V. vulnificus and 79% for V. parahaemolyticus (of which 47% were tdh(+) and/or trh(+)). Viable vibrios were also isolated from the mussel meat and screened for virulence by PCR. The mortality of eukaryotic cells when exposed to bacteria was tested in vivo, with results showing that the Vibrio strains, independent of species and origin, were harmful to the cells. Despite severe infections and several deaths, no report on potential human pathogenic vibrios in this area had been published before this study.     

Sandwich enzyme-linked immunosorbent assay for Vibrio vulnificus hemolysin to detect V. vulnificus in environmental specimens.

Vibrio vulnificus hemolysin, purified by quantitative isoelectric focusing, was used to prepare rabbit and goat anti-hemolysin. The resulting antibodies were used as capture and detector antibody reagents in a sandwich enzyme-linked immunosorbent assay (ELISA) to detect V. vulnificus in environmental samples. By this technique, 4 laboratory-maintained V. vulnificus strains and 33 environmental V. vulnificus isolates were detected. Also, the technique distinguished five other Vibrio species from V. vulnificus, and when it was used in combination with colistin-polymyxin-cellobiose agar, 31 non-V. vulnificus isolated were excluded. This sandwich ELISA compared favorably with the current Food and Drug Administration standard immunoassay in confirming presumptive V. vulnificus colonies from environmental specimens: oysters, sediment, and seawater. Among 340 presumptive V. vulnificus colonies, the sandwich ELISA detected 95% of the confirmed V. vulnificus colonies. Equally important, the technique correctly distinguished 99% of the non-V. vulnificus colonies. The sandwich ELISA offers time-saving and labor-saving advantages over the currently accepted immunoassay.     

Influence of aquatic microbiota on the survival in water of the human and eel pathogen Vibrio vulnificus serovar E

The eel and human pathogen Vibrio vulnificus serovar E (biotype 2) is seldom isolated from natural waters, although it can survive in sterilized artificial seawater microcosms for years. The main objective of the present study was to investigate whether aquatic microbiota can limit its survival and recovery from water samples. A set of preliminary experiments of survival in microcosms containing natural seawater and water from eel farms showed that the persistence of this pathogen was mainly controlled by grazing, and secondarily by bacterial competition. The bacterial competition was further analysed in artificial seawater microcosms co-inoculated with selected virulent serovar E (VSE) strains and potential competitors. Competitors included V. vulnificus biotype 1 isolates and strains of selected species that can grow on the selective media designed for V. vulnificus isolation from water samples. Evidences of bacterial competition that was detrimental for VSE recovery were recorded. Thus, some species produced a deleterious effect on VSE strains under starvation, and others were able to use the resources more efficiently under nutrient input. These results suggest that an overgrowth of more efficient competitor bacteria in conventional media used for isolation of V. vulnificus could mask the recovery of VSE strains and explain the scarcity of reports on the isolation of this human and eel pathogen from natural waters.     

Identification of Vibrio spp. (other than V. vulnificus) recovered on CPC agar from marine natural samples.

Two hundred and eighty four presumptive but not confirmed Vibrio vulnificus isolates grown on cellobiose-polymixin B-colistin agar (CPC) at 40 degrees C, recovered from sea water samples from Valencia, Spain, during a microbiological survey for V. vulnificus, were phenotypically identified. Most of the isolates (91%) corresponded to Vibrio species. V. harveyi (24%) and V. splendidus(19%) were the most abundant species identified, followed by V. navarrensis (13%), V. alginolyticus (8%) and V. parahaemolyticus (5%). The ability to grow on CPC agar and ferment cellobiose of several V. vulnificus strains from different origins and serovars, including reference strains, was tested. Most serovar E isolates and 25% of non-serovar E isolates could not grow on CPC agar.     

Intraspecific diversity of Vibrio vulnificus in Galveston Bay water and oysters as determined by randomly amplified polymorphic DNA PCR

Randomly amplified polymorphic DNA (RAPD) PCR was used to analyze the temporal and spatial intraspecific diversity of 208 Vibrio vulnificus strains isolated from Galveston Bay water and oysters at five different sites between June 2000 and June 2001. V. vulnificus was not detected during the winter months (December through February). The densities of V. vulnificus in water and oysters were positively correlated with water temperature. Cluster analysis of RAPD PCR profiles of the 208 V. vulnificus isolates revealed a high level of intraspecific diversity among the strains. No correlation was found between the intraspecific diversity among the isolates and sampling site or source of isolation. After not being detected during the winter months, the genetic diversity of V. vulnificus strains first isolated in March was 0.9167. Beginning in April, a higher level of intraspecific diversity (0.9933) and a major shift in population structure were observed among V. vulnificus isolates. These results suggest that a great genetic diversity of V. vulnificus strains exists in Galveston Bay water and oysters and that the population structure of this species is linked to changes in environmental conditions, especially temperature.     

Phase variation, capsular polysaccharide, pilus and flagella contribute to uptake of Vibrio vulnificus by the Eastern oyster (Crassostrea virginica)

Vibrio vulnificus infections are associated with raw oyster consumption, and disease reservoirs are determined by the ability of this bacterium to infect and persist in oysters. Surface structures, such as capsular polysaccharide (CPS), pili and flagella, function as virulence factors in mouse infection models. Furthermore, virulence is related to phase variation in colony morphology, which reflects CPS expression and includes opaque (encapsulated, virulent), translucent (reduced encapsulation, avirulent) and rugose (wrinkled, biofilm-enhanced) colony types. The role of these factors in environmental survival is unknown; therefore, mutational analysis and phase variation of V. vulnificus were examined in an oyster infection model. Oysters ( Crassostrea virginica ) were pre-treated with tetracycline to reduce background bacteria and subsequently inoculated via filter feeding with 10⁶ colony-forming units (cfu) ml⁻¹ of V. vulnificus wild-type strains and phase variants, as well as strains with deletion mutations in genes related to CPS (Δ wza ), pili (Δ pilA ), flagella (Δ flaCDE/ Δ flaFBA ) and motility (Δ motAB ). All mutants were significantly reduced in their dissemination to oyster haemolymph as compared with wild type; however, recovery of mutants from gills and intestinal tissue was generally similar to wild type. Translucent and rugose inocula showed induction of high-frequency phase variation to the opaque encapsulated phenotype (100% and 72% respectively) during oyster infections that did not occur in strains recovered from seawater. Thus, multiple bacterial factors determine uptake of V. vulnificus in oysters, and phase variation during oyster infection is a likely mechanism for environmental survival and for induction of the more virulent phenotype.     

Randomly amplified polymorphic DNA analysis of clinical and environmental isolates of Vibrio vulnificus and other vibrio species

Vibrio vulnificus is an estuarine bacterium that is capable of causing a rapidly fatal infection in humans. A randomly amplified polymorphic DNA (RAPD) PCR protocol was developed for use in detecting V. vulnificus, as well as other members of the genus Vibrio. The resulting RAPD profiles were analyzed by using RFLPScan software. This RAPD method clearly differentiated between members of the genus Vibrio and between isolates of V. vulnificus. Each V. vulnificus strain produced a unique band pattern, indicating that the members of this species are genetically quite heterogeneous. All of the vibrios were found to have amplification products whose sizes were within four common molecular weight ranges, while the V. vulnificus strains had an additional two molecular weight range bands in common. All of the V. vulnificus strains isolated from clinical specimens produced an additional band that was only occasionally found in environmental strains; this suggests that, as is the case with the Kanagawa hemolysin of Vibrio parahaemolyticus, the presence of this band may be correlated with the ability of a strain to produce an infection in humans. In addition, band pattern differences were observed between encapsulated and nonencapsulated isogenic morphotypes of the same strain of V. vulnificus.