Quantitative Microbial Risk Assessment of Pathogenic Vibrios in Marine Recreational Waters of Southern California

This study investigated the occurrence of three types of vibrios in Southern California recreational beach waters during the peak marine bathing season in 2007. Over 160 water samples were concentrated and enriched for the detection of vibrios. Four sets of PCR primers, specific for Vibrio cholerae, V. parahaemolyticus, and V. vulnificus species and the V. parahaemolyticus toxin gene, respectively, were used for the amplification of bacterial genomic DNA. Of 66 samples from Doheny State Beach, CA, 40.1% were positive for V. cholerae and 27.3% were positive for V. parahaemolyticus, and 1 sample (1.5%) was positive for the V. parahaemolyticus toxin gene. Of the 96 samples from Avalon Harbor, CA, 18.7% were positive for V. cholerae, 69.8% were positive for V. parahaemolyticus, and 5.2% were positive for the V. parahaemolyticus toxin gene. The detection of the V. cholerae genetic marker was significantly more frequent at Doheny State Beach, while the detection of the V. parahaemolyticus genetic marker was significantly more frequent at Avalon Harbor. A probability-of-illness model for V. parahaemolyticus was applied to the data. The risk for bathers exposed to recreational waters at two beaches was evaluated through Monte Carlo simulation techniques. The results suggest that the microbial risk from vibrios during beach recreation was below the illness benchmark set by the U.S. EPA. However, the risk varied with location and the type of water recreation activities. Surfers and children were exposed to a higher risk of vibrio diseases. Microbial risk assessment can serve as a useful tool for the management of risk related to opportunistic marine pathogens.     

Predatory Bacteria as Natural Modulators of Vibrio parahaemolyticus and Vibrio vulnificus in Seawater and Oysters

This study shows that naturally occurring Vibrio predatory bacteria (VPB) exert a major role in controlling pathogenic vibrios in seawater and shellfish. The growth and persistence of Vibrio parahaemolyticus and Vibrio vulnificus were assessed in natural seawater and in the Eastern oyster, Crassostrea virginica. The pathogens examined were V. vulnificus strain VV1003, V. parahaemolyticus O1:KUT (KUT stands for K untypeable), and V. parahaemolyticus O3:K6 and corresponding O3:K6 mutants deficient in the toxRS virulence regulatory gene or the rpoS alternative stress response sigma factor gene. Vibrios were selected for streptomycin resistance, which facilitated their enumeration. In natural seawater, oysters bioconcentrated each Vibrio strain for 24 h at 22°C; however, counts rapidly declined to near negligible levels by 72 h. In natural seawater with or without oysters, vibrios decreased more than 3 log units to near negligible levels within 72 h. Neither toxRS nor rpoS had a significant effect on Vibrio levels. In autoclaved seawater, V. parahaemolyticus O3:K6 counts increased 1,000-fold over 72 h. Failure of the vibrios to persist in natural seawater and oysters led to screening of the water samples for VPB on lawns of V. parahaemolyticus O3:K6 host cells. Many VPB, including Bdellovibrio and like organisms (BALOs; Bdellovibrio bacteriovorus and Bacteriovorax stolpii) and Micavibrio aeruginosavorus-like predators, were detected by plaque assay and electron microscopic analysis of plaque-purified isolates from Atlantic, Gulf Coast, and Hawaiian seawater. When V. parahaemolyticus O3:K6 was added to natural seawater containing trace amounts of VPB, Vibrio counts diminished 3 log units to nondetectable levels, while VPB increased 3 log units within 48 h. We propose a new paradigm that VPB are important modulators of pathogenic vibrios in seawater and oysters.     

Relationships between Environmental Factors and Pathogenic Vibrios in the Northern Gulf of Mexico

Although autochthonous vibrio densities are known to be influenced by water temperature and salinity, little is understood about other environmental factors associated with their abundance and distribution. Densities of culturable Vibrio vulnificus containing vvh (V. vulnificus hemolysin gene) and V. parahaemolyticus containing tlh (thermolabile hemolysin gene, ubiquitous in V. parahaemolyticus), tdh (thermostable direct hemolysin gene, V. parahaemolyticus pathogenicity factor), and trh (tdh-related hemolysin gene, V. parahaemolyticus pathogenicity factor) were measured in coastal waters of Mississippi and Alabama. Over a 19-month sampling period, vibrio densities in water, oysters, and sediment varied significantly with sea surface temperature (SST). On average, tdh-to-tlh ratios were significantly higher than trh-to-tlh ratios in water and oysters but not in sediment. Although tlh densities were lower than vvh densities in water and in oysters, the opposite was true in sediment. Regression analysis indicated that SST had a significant association with vvh and tlh densities in water and oysters, while salinity was significantly related to vibrio densities in the water column. Chlorophyll a levels in the water were correlated significantly with vvh in sediment and oysters and with pathogenic V. parahaemolyticus (tdh and trh) in the water column. Furthermore, turbidity was a significant predictor of V. parahaemolyticus density in all sample types (water, oyster, and sediment), and its role in predicting the risk of V. parahaemolyticus illness may be more important than previously realized. This study identified (i) culturable vibrios in winter sediment samples, (ii) niche-based differences in the abundance of vibrios, and (iii) predictive signatures resulting from correlations between environmental parameters and vibrio densities.Vibrio spp. occur naturally in estuarine and marine environments, and two species of this genus, V. vulnificus and V. parahaemolyticus, are responsible for the majority of reported vibrio illnesses in the United States (2). V. vulnificus infections are most commonly associated with the Gulf of Mexico, either via consumption of raw oysters harvested from these waters or wound infections following exposure to seawater. On average, about 50 cases of V. vulnificus septicemia are reported in the United States each year, with a case fatality rate of approximately 50% (31), the highest of any food-borne pathogen. In contrast, V. parahaemolyticus is the most common cause of seafood-associated bacterial gastroenteritis in the United States, with an estimated annual rate of 4,500 cases per year according to the Centers for Disease Control and Prevention. V. parahaemolyticus also causes wound infections, though these are less frequent and less severe compared to those caused by V. vulnificus (5). Primary septicemia can occur following V. parahaemolyticus infection, but it is relatively rare for this pathogen. In the United States, V. parahaemolyticus illness most often results from consumption of raw or undercooked seafood, particularly oysters.It is well established that vibrio densities correlate strongly with sea surface temperature (SST), with densities increasing as temperatures increase; however, with the exception of salinity, little is definitively known about the influence of other environmental parameters, such as turbidity and chlorophyll a (22, 33). Consequently, while SST has been estimated to explain approximately 50% of the annual variation of V. parahaemolyticus abundance in oysters harvested from the northern Gulf of Mexico (40), a considerable amount of variation remains unexplained. It is of interest to delineate the effects of other environmental parameters independent of SST, as these parameters may be associated with spatial and temporal variation of vibrio densities within seasonal periods when SST is relatively constant and risk of human exposure and illness is high. Moreover, the majority of what is known about V. parahaemolyticus in the environment is based on total populations; little information is available on the pathogenic subpopulations. Isolates containing genetic markers for pathogenicity factors, including the thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) typically constitute <1% of the population in marine or postharvest oyster samples, but they account for >90% of clinical isolates (12). The basis for V. vulnificus pathogenicity remains unclear, as few pathogenicity factors have been described definitively (31). To address these data gaps, we monitored densities of culturable V. vulnificus containing vvh (the V. vulnificus hemolysin gene) and V. parahaemolyticus containing tlh (the thermolabile hemolysin gene, ubiquitous in V. parahaemolyticus), tdh, and trh in water, oysters, and sediment collected from coastal waters of Mississippi and Alabama. Associations between bacterial densities and environmental parameters were analyzed by regressing observations against sea surface temperature, chlorophyll a, turbidity, and salinity.     

Improved Method for Detection of Vibrio parahaemolyticus in Seafood

We have developed a new, effective procedure for detecting Vibrio parahaemolyticus in seafoods using enrichment and plating onto a chromogenic agar medium. Samples were cultured in salt Trypticase soy broth, which is a nonselective medium, and then a portion of the culture was cultured with salt polymyxin broth, which is a selective medium for V. parahaemolyticus. This two-step enrichment was more effective than the one-step enrichment in salt polymyxin broth alone. The enrichment cultures were then plated onto a new chromogenic agar containing substrates for beta-galactosidase. The V. parahaemolyticus colonies developed a purple color on this growth medium that distinguished them from other related bacterial strains. V. parahaemolyticus was isolated more frequently from naturally contaminated seafood samples using the chromogenic agar than thiosulfate citrate bile salts sucrose agar medium, which is currently used for the isolation of V. parahaemolyticus. Our findings suggest that this new enrichment and isolation scheme is more sensitive and accurate for identifying V. parahaemolyticus in seafood samples than previously used methods.     

Antibiotic resistance and plasmid profiling of Vibrio parahaemolyticus isolated from shrimp farms along the southwest coast of India

Shrimp, water, and sediment samples were collected from various shrimp farms located in and around Cochin. V. parahaemolyticus was identified by standard biochemical tests and plasmid profiling was carried out for the isolates. Susceptibility was tested against 15 antibiotics before and after the plasmid curing. Incidence of V. parahaemolyticus was found in 46% of the samples screened. Antibiogram studies showed, above 50% of the strains sensitive to chlorotetracycline, chloramphenicol and nitrofurantoin. Multiple antibiotic resistance (MAR) index was found to be 0.2. Total presumptive Vibrio parahaemolyticus count (TPVPC) and resistance to antibiotics was found to be more in sediment samples particularly in pre-monsoon season. Plasmid profiles of V. parahaemolyticus isolates revealed seven plasmids in the size range of 0.75, 1.2, 6.0, and 8.0 kb sizes and 3 plasmids above 10.0 kb. The MAR index suggests the low risk potential involved in consuming seafoods. Resistance to antibiotics did not vary even after curing of plasmids with sodium dodecyl sulphate suggesting that resistance to antibiotics in V. parahaemolyticus is chromosomal borne.     

Antimicrobial Susceptibilities of Vibrio parahaemolyticus and Vibrio vulnificus Isolates from Louisiana Gulf and Retail Raw Oysters

The antimicrobial susceptibilities of 168 Vibrio parahaemolyticus and 151 Vibrio vulnificus isolates recovered from 82 Louisiana Gulf and retail oysters in 2005 and 2006 were determined. Overall, the two vibrios remained susceptible to the majority of antimicrobials tested; reduced susceptibility was detected only in V. parahaemolyticus for ampicillin (81%; MIC ≥ 16 μg/ml). Additionally, V. parahaemolyticus displayed significantly higher MICs for cefotaxime, ciprofloxacin, and tetracycline than V. vulnificus.     

Variability of Total and Pathogenic Vibrio parahaemolyticus Densities in Northern Gulf of Mexico Water and Oysters

Vibrio parahaemolyticus is indigenous to coastal environments and a frequent cause of seafood-borne gastroenteritis in the United States, primarily due to raw-oyster consumption. Previous seasonal-cycle studies of V. parahaemolyticus have identified water temperature as the strongest environmental predictor. Salinity has also been identified, although it is evident that its effect on annual variation is not as pronounced. The effects of other environmental factors, both with respect to the seasonal cycle and intraseasonal variation, are uncertain. This study investigated intraseasonal variations of densities of total and pathogenic V. parahaemolyticus organisms in oysters and overlying waters during the summer of 2004 at two sites in the northern Gulf of Mexico. Regression analyses indicated significant associations (P < 0.001) between total V. parahaemolyticus densities and salinity, as well as turbidity in water and in oysters at the Mississippi site but not at the Alabama site. Pathogenic V. parahaemolyticus organisms in Mississippi oyster and water samples were detected in 56% (9 out of 16) and 78% (43 out of 55) of samples, respectively. In contrast, 44% (7 out of 16) of oyster samples and 30% (14 out of 47) of water samples from Alabama were positive. At both sites, there was greater sample-to-sample variability in pathogenic V. parahaemolyticus densities than in total V. parahaemolyticus densities. These data suggest that, although total V. parahaemolyticus densities may be very informative, there is greater uncertainty when total V. parahaemolyticus densities are used to predict the risk of infection by pathogenic V. parahaemolyticus than previously recognized.     

Seasonal Abundance of Total and Pathogenic Vibrio parahaemolyticus in Alabama Oysters

Recent Vibrio parahaemolyticus outbreaks associated with consumption of raw shellfish in the United States focused attention on the occurrence of this organism in shellfish. From March 1999 through September 2000, paired oyster samples were collected biweekly from two shellfish-growing areas in Mobile Bay, Ala. The presence and densities of V. parahaemolyticus were determined by using DNA probes targeting the thermolabile hemolysin (tlh) and thermostable direct hemolysin (tdh) genes for confirmation of total and pathogenic V. parahaemolyticus, respectively. V. parahaemolyticus was detected in all samples with densities ranging from <10 to 12,000 g⁻¹. Higher V. parahaemolyticus densities were associated with higher water temperatures. Pathogenic strains were detected in 34 (21.8%) of 156 samples by direct plating or enrichment. Forty-six of 6,018 and 31 of 6,992 V. parahaemolyticus isolates from enrichments and direct plates, respectively, hybridized with the tdh probe. There was an apparent inverse relationship between water temperature and the prevalence of pathogenic strains. Pathogenic strains were of diverse serotypes, and 97% produced urease and possessed a tdh-related hemolysin (trh) gene. The O3:K6 serotype associated with pandemic spread and recent outbreaks in the United States was not detected. The efficient screening of numerous isolates by colony lift and DNA probe procedures may account for the higher prevalence of samples with tdh⁺ V. parahaemolyticus than previously reported.     

Ecology of Vibrio parahaemolyticus and Vibrio vulnificus in the Coastal and Estuarine Waters of Louisiana,Maryland, Mississippi,and Washington (United States)

Vibrio parahaemolyticus and Vibrio vulnificus, which are native to estuaries globally, are agents of seafood-borne or wound infections, both potentially fatal. Like all vibrios autochthonous to coastal regions, their abundance varies with changes in environmental parameters. Sea surface temperature (SST), sea surface height (SSH), and chlorophyll have been shown to be predictors of zooplankton and thus factors linked to vibrio populations. The contribution of salinity, conductivity, turbidity, and dissolved organic carbon to the incidence and distribution of Vibrio spp. has also been reported. Here, a multicoastal, 21-month study was conducted to determine relationships between environmental parameters and V. parahaemolyticus and V. vulnificus populations in water, oysters, and sediment in three coastal areas of the United States. Because ecologically unique sites were included in the study, it was possible to analyze individual parameters over wide ranges. Molecular methods were used to detect genes for thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and tdh-related hemolysin (trh) as indicators of V. parahaemolyticus and the hemolysin gene vvhA for V. vulnificus. SST and suspended particulate matter were found to be strong predictors of total and potentially pathogenic V. parahaemolyticus and V. vulnificus. Other predictors included chlorophyll a, salinity, and dissolved organic carbon. For the ecologically unique sites included in the study, SST was confirmed as an effective predictor of annual variation in vibrio abundance, with other parameters explaining a portion of the variation not attributable to SST.     

Prevalence of Pandemic Thermostable Direct Hemolysin-Producing Vibrio parahaemolyticus O3:K6 in Seafood and the Coastal Environment in Japan

Although thermostable direct hemolysin (TDH)-producing Vibrio parahaemolyticus has caused many infections in Asian countries, the United States, and other countries, it has been difficult to detect the same pathogen in seafoods and other environmental samples. In this study, we detected and enumerated tdh gene-positive V. parahaemolyticus in Japanese seafoods with a tdh-specific PCR method, a chromogenic agar medium, and a most-probable-number method. The tdh gene was detected in 33 of 329 seafood samples (10.0%). The number of tdh-positive V. parahaemolyticus ranged from <3 to 93/10 g. The incidence of tdh-positive V. parahaemolyticus tended to be high in samples contaminated with relatively high levels of total V. parahaemolyticus. TDH-producing strains of V. parahaemolyticus were isolated from 11 of 33 tdh-positive samples (short-necked clam, hen clam, and rock oyster). TDH-producing strains of V. parahaemolyticus were also isolated from the sediments of rivers near the coast in Japan. Representative strains of the seafood and sediment isolates were examined for the O:K serovar and by the PCR method specific to the pandemic clone and arbitrarily primed PCR and pulsed-field gel electrophoresis techniques. The results indicated that most O3:K6 tdh-positive strains belonged to the pandemic O3:K6 clone and suggested that serovariation took place in the Japanese environment.     

Physiological and Biochemical Characteristics of the Halophilic Bacteria Vibrio parahaemolyticus and V. alginolyticus Isolated from Marine Invertebrates of Peter the Great Bay, Sea of Japan

One hundred strains of halophilic vibrios were isolated from 16 species of marine invertebrates of Peter the Great Bay. Based on their morphological and biochemical characteristics, the bacteria were identified as Vibrio parahaemolyticus and Vibrio alginolyticus. Bacterial isolates possessed virulence enzymes (DNAase, lecithinase, catalase) and were characterized by a high enterotoxigenicity. It was determined that 76% of the V. parahaemolyticus strains and 43% of the V. alginolyticus strains were Kanagawa-positive. The isolates showed a high adhesive capability, the average adhesion index was 18.06 cells per erythrocyte for V. parahaemolyticus and 12.55 for V. alginolyticus. The results of this study suggest a high pathogenic potential of the isolated halophilic vibrios, which are an epidemic hazard to marine invertebrates and to humans.     

Genes Similar to the Vibrio parahaemolyticus Virulence-Related Genes tdh,tlh, and vscC2 Occur in Other Vibrionaceae Species Isolated from a Pristine Estuary

Detection of the human pathogen Vibrio parahaemolyticus often relies on molecular biological analysis of species-specific virulence factor genes. These genes have been employed in determinations of V. parahaemolyticus population numbers and the prevalence of pathogenic V. parahaemolyticus strains. Strains of the Vibrionaceae species Photobacterium damselae, Vibrio diabolicus, Vibrio harveyi, and Vibrio natriegens, as well as strains similar to Vibrio tubiashii, were isolated from a pristine salt marsh estuary. These strains were examined for the V. parahaemolyticus hemolysin genes tdh, trh, and tlh and for the V. parahaemolyticus type III secretion system 2α gene vscC2 using established PCR primers and protocols. Virulence-related genes occurred at high frequencies in non-V. parahaemolyticus Vibrionaceae species. V. diabolicus was of particular interest, as several strains were recovered, and the large majority (>83%) contained virulence-related genes. It is clear that detection of these genes does not ensure correct identification of virulent V. parahaemolyticus. Further, the occurrence of V. parahaemolyticus-like virulence factors in other vibrios potentially complicates tracking of outbreaks of V. parahaemolyticus infections.     

Development of a Matrix Tool for the Prediction of Vibrio Species in Oysters Harvested from North Carolina

The United States has federal regulations in place to reduce the risk of seafood-related infection caused by the estuarine bacteria Vibrio vulnificus and Vibrio parahaemolyticus. However, data to support the development of regulations have been generated in a very few specific regions of the nation. More regionally specific data are needed to further understand the dynamics of human infection relating to shellfish-harvesting conditions in other areas. In this study, oysters and water were collected from four oyster harvest sites in North Carolina over an 11-month period. Samples were analyzed for the abundances of total Vibrio spp., V. vulnificus, and V. parahaemolyticus; environmental parameters, including salinity, water temperature, wind velocity, and precipitation, were also measured simultaneously. By utilizing these data, preliminary predictive management tools for estimating the abundance of V. vulnificus bacteria in shellfish were developed. This work highlights the need for further research to elucidate the full suite of factors that drive V. parahaemolyticus abundance.     

Seasonal Variation in Abundance of Total and Pathogenic Vibrio parahaemolyticus Bacteria in Oysters along the Southwest Coast of India

The seasonal abundance of Vibrio parahaemolyticus in oysters from two estuaries along the southwest coast of India was studied by colony hybridization using nonradioactive labeled oligonucleotide probes. The density of total V. parahaemolyticus bacteria was determined using a probe binding to the tlh (thermolabile hemolysin) gene, and the density of pathogenic V. parahaemolyticus bacteria was determined by using a probe binding to the tdh (thermostable direct hemolysin) gene. Furthermore, the prevalence of V. parahaemolyticus was studied by PCR amplification of the toxR, tdh, and trh genes. PCR was performed directly with oyster homogenates and also following enrichment in alkaline peptone water for 6 and 18 h. V. parahaemolyticus was detected in 93.87% of the samples, and the densities ranged from <10 to 10⁴ organisms per g. Pathogenic V. parahaemolyticus could be detected in 5 of 49 samples (10.2%) by colony hybridization using the tdh probe and in 3 of 49 samples (6.1%) by PCR. Isolates from one of the samples belonged to the pandemic serotype O3:K6. Twenty-nine of the 49 samples analyzed (59.3%) were positive as determined by PCR for the presence of the trh gene in the enrichment broth media. trh-positive V. parahaemolyticus was frequently found in oysters from India.     

Vibrio parahaemolyticus and Its Specific Bacteriophages as an Indicator in Cockles (Anadara granosa) for the Risk of V. parahaemolyticus Infection in Southern Thailand

Correlation between the numbers of Vibrio parahaemolyticus and its specific bacteriophages in cockles was investigated from June 2009 to May 2010 in Hat Yai, Songkhla, Thailand. Cockles obtained monthly from a local market were sampled to determine the numbers of V. parahaemolyticus and bacteriophages that could form plaques on ten strains of pandemic and nonpandemic V. parahaemolyticus. In addition, V. parahaemolyticus isolates from clinical samples from Hat Yai hospital over the same period were investigated. All 139 cockles sampled were positive for V. parahaemolyticus. However, only 76 of them were positive for bacteriophages. During the testing period, the number of bacteriophages was not significantly correlated with the incidence of V. parahaemolyticus-infected patients, but the numbers of V. parahaemolyticus isolates from the cockle samples were closely related to the number of infected patients. The bacteriophages isolated from V. parahaemolyticus also infected Vibrio alginolyticus and Vibrio mimicus, suggesting that the broad host range of phages may be a factor of providing the possibility of their participation in the processes of genetic exchange between V. parahaemolyticus and closely related Vibrio spp. In conclusion, this study indicated that the number of V. parahaemolyticus in cockles may be a useful tool for predicting the relative risk of infection by V. parahaemolyticus in this area of Thailand.     

Environmental Investigations of Vibrio parahaemolyticus in Oysters after Outbreaks in Washington, Texas, and New York (1997 and 1998)

Total Vibrio parahaemolyticus densities and the occurrence of pathogenic strains in shellfish were determined following outbreaks in Washington, Texas, and New York. Recently developed nonradioactive DNA probes were utilized for the first time for direct enumeration of V. parahaemolyticus in environmental shellfish samples. V. parahaemolyticus was prevalent in oysters from Puget Sound, Wash.; Galveston Bay, Tex.; and Long Island Sound, N.Y., in the weeks following shellfish-associated outbreaks linked to these areas. However, only two samples (one each from Washington and Texas) were found to harbor total V. parahaemolyticus densities exceeding the level of concern of 10,000 g⁻¹. Pathogenic strains, defined as those hybridizing with tdh and/or trh probes, were detected in a few samples, mostly Puget Sound oysters, and at low densities (usually <10 g⁻¹). Intensive sampling in Galveston Bay demonstrated relatively constant water temperature (27.8 to 31.7°C) and V. parahaemolyticus levels (100 to 1,000 g⁻¹) during the summer. Salinity varied from 14.9 to 29.3 ppt. A slight but significant (P < 0.05) negative correlation (−0.25) was observed between V. parahaemolyticus density and salinity. Based on our data, findings of more than 10,000 g⁻¹ total V. parahaemolyticus or >10 g⁻¹ tdh- and/or trh-positive V. parahaemolyticus in environmental oysters should be considered extraordinary.     

Long-Term Study of Vibrio parahaemolyticus Prevalence and Distribution in New Zealand Shellfish

The food-borne pathogen Vibrio parahaemolyticus has been reported as being present in New Zealand (NZ) seawaters, but there have been no reported outbreaks of food-borne infection from commercially grown NZ seafood. Our study determined the current incidence of V. parahaemolyticus in NZ oysters and Greenshell mussels and the prevalence of V. parahaemolyticus tdh and trh strains. Pacific (235) and dredge (21) oyster samples and mussel samples (55) were obtained from commercial shellfish-growing areas between December 2009 and June 2012. Total V. parahaemolyticus numbers and the presence of pathogenic genes tdh and trh were determined using the FDA most-probable-number (MPN) method and confirmed using PCR analysis. In samples from the North Island of NZ, V. parahaemolyticus was detected in 81% of Pacific oysters and 34% of mussel samples, while the numbers of V. parahaemolyticus tdh and trh strains were low, with just 3/215 Pacific oyster samples carrying the tdh gene. V. parahaemolyticus organisms carrying tdh and trh were not detected in South Island samples, and V. parahaemolyticus was detected in just 1/21 dredge oyster and 2/16 mussel samples. Numbers of V. parahaemolyticus organisms increased when seawater temperatures were high, the season when most commercial shellfish-growing areas are not harvested. The numbers of V. parahaemolyticus organisms in samples exceeded 1,000 MPN/g only when the seawater temperatures exceeded 19°C, so this environmental parameter could be used as a trigger warning of potential hazard. There is some evidence that the total V. parahaemolyticus numbers increased compared with those reported from a previous 1981 to 1984 study, but the analytical methods differed significantly.     

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 H₂S 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.     

Rapid identification and differentiation of Vibrio parahaemolyticus from Vibrio spp. in seafood samples using developed monoclonal antibodies

Monoclonal antibodies (MAbs) specific to Vibrio parahaemolyticus were successfully generated. According to the specificity of V. parahaemolyticus, MAbs can be classified into 5 groups. The MAbs VP-2D and VP-11H were specific to the O2 and O4 groups of V. parahaemolyticus, respectively. The MAb VP-11B reacted with 11 out of 30 isolates of V. parahaemolyticus used in this study. The MAb VP-516 bound to 27 out of 30 isolates of V. parahaemolyticus and cross reacted with all 10 isolates of V. alginolyticus. The MAb VP-618 demonstrated positive reactivity to 29 out of 30 isolates of V. parahaemolyticus and demonstrated slight cross reactivity to 3 out of 30 isolates of V. harveyi. The sensitivity of the MAbs ranged from 10⁸ to 10⁷ c.f.u. ml^?1 for V. parahaemolyticus obtained from pure cultures and depended on the group of MAbs. However, the detection capability could be improved to be equivalent to that of the PCR technique following pre-incubation of the samples in alkaline peptone water (APW). Using these MAbs along with MAbs specific to V. alginolyticus (VA-165), V. cholerae (VC-63), V. harveyi (VH-9B and VH-20C) and Vibrio spp. (VC-201) from previous studies, V. parahaemolyticus could be identified and differentiated from Vibrio spp. in various seafood samples including shrimp, green mussels, blood clams and oysters by a simple dot blot immunoassay without the requirement for bacterial isolation or biochemical characterization.     

PCR Detection of a Newly Emerged Pandemic Vibrio parahaemolyticus O3:K6 Pathogen in Pure Cultures and Seeded Waters from the Gulf of Mexico

This study describes the optimization of PCR parameters and testing of a wide number of microbial species to establish a highly specific and sensitive PCR-based method of detection of a newly emerged pandemic Vibrio parahaemolyticus O3:K6 strain in pure cultures and seeded waters from the Gulf of Mexico (gulf water). The selected open reading frame 8 (ORF8) DNA-specific oligonucleotide primers tested were found to specifically amplify all 35 pathogenic V. parahaemolyticus O3:K6 pandemic isolates, whereas these primers were not found to detectably amplify two strains of V. parahaemolyticus O3:K6 that were isolated prior to the 1996 outbreaks, 122 non-O3:K6 strains of V. parahaemolyticus, 198 non-V. parahaemolyticus spp., or 16 non-Vibrio bacterial spp. The minimum level of detection by the PCR method was 1 pg of purified genomic DNA or 10² ORF8-positive V. parahaemolyticus O3:K6 cells in 100 ml of water. The effectiveness of this method for the detection of ORF8-positive isolates in environmental samples was tested in gulf water seeded with 10-fold serial dilutions of this pathogen. A detection level of 10³ cells per 100 ml of gulf water was achieved. Also, the applicability of this methodology was tested by the detection of this pathogen in gulf water incubated at various temperatures for 28 days. This PCR approach can potentially be used to monitor with high specificity and well within the required range of sensitivity the occurrence and distribution of this newly emerged pathogenic V. parahaemolyticus O3:K6 strain in coastal, marine, and ship ballast waters. Early detection of V. parahaemolyticus O3:K6 will help increase seafood safety and decrease the risk of infectious outbreaks caused by this pathogen.