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.     

Vibrio bacteria in raw oysters: managing risks to human health

The human-pathogenic marine bacteria Vibrio vulnificus and V. parahaemolyticus are strongly correlated with water temperature, with concentrations increasing as waters warm seasonally. Both of these bacteria can be concentrated in filter-feeding shellfish, especially oysters. Because oysters are often consumed raw, this exposes people to large doses of potentially harmful bacteria. Various models are used to predict the abundance of these bacteria in oysters, which guide shellfish harvest policy meant to reduce human health risk. Vibrio abundance and behaviour varies from site to site, suggesting that location-specific studies are needed to establish targeted risk reduction strategies. Moreover, virulence potential, rather than simple abundance, should be also be included in future modeling efforts.     

Temporal and Spatial Distribution Patterns of Potentially Pathogenic Vibrio spp. at Recreational Beaches of the German North Sea

The number of reported Vibrio-related wound infections associated with recreational bathing in Northern Europe has increased within the last decades. In order to study the health risk from potentially pathogenic Vibrio spp. in the central Wadden Sea, the seasonal and spatial distribution of Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio cholerae were investigated at ten recreational beaches in this area over a 2-year period. V. alginolyticus and V. parahaemolyticus were found to be omnipresent all year round in the study area, while V. vulnificus occurrence was restricted to summer months in the estuaries of the rivers Ems and Weser. Multiple linear regression models revealed that water temperature is the most important determinant of Vibrio spp. occurrence in the area. Differentiated regression models showed a species-specific response to water temperature and revealed a particularly strong effect of even minor temperature increases on the probability of detecting V. vulnificus in summer. In sediments, Vibrio spp. concentrations were up to three orders of magnitude higher than in water. Also, V. alginolyticus and V. parahaemolyticus were found to be less susceptible towards winter temperatures in the benthic environment than in the water, indicating an important role of sediments for Vibrio ecology. While only a very small percentage of tested V. parahaemolyticus proved to be potentially pathogenic, the presence of V. vulnificus during the summer months should be regarded with care.     

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.     

Vibrio parahaemolyticus and Vibrio vulnificus in South America: water,seafood and human infections

The bacterial species, Vibrio parahaemolyticus and Vibrio vulnificus, are ubiquitous in estuaries and coastal waters throughout the world, but they also happen to be important human pathogens. They are concentrated by filter‐feeding shellfish which are often consumed raw or undercooked, providing an important potential route of entry for an infective dose of these bacteria. Vibrio parahaemolyticus can cause abdominal cramping, nausea, diarrhoea, vomiting, chills and fever. Vibrio vulnificus can cause similar gastrointestinal‐related symptoms, but can also spread to the bloodstream, resulting in primary septicaemia, and it can also cause disease via wound infections. The objective of this article is to summarize, for the first time, the incidence and importance of V. parahaemolyticus and V. vulnificus in South America, in environmental waters and seafood, especifically molluscan shellfish, as well as human infection cases and outbreaks. It appears that infections from V. parahaemolyticus have been more strongly related to shellfish ingestion and have been more frequently reported on the Pacific coast of South America. Conversely, V. vulnificus has been more frequently acquired by water contact with open wounds and its presence has been more heavily reported along the Atlantic coast of South America, and while documented to cause serious mortality, have been relatively few in number. The impacts of El Nino Southern Oscillation (ENSO) have been observed to cause an increase in V. parahaemolyticus outbreaks on the Pacific coast of South America. The implementation of a regulated monitoring approach, along with the use of faster, more accurate and virulence‐specific detection approaches, such as PCR confirmation, should be considered to detect the presence of pathogenic Vibrio strains in environmental and seafood samples for protection of public health. Furthermore, improved clinical surveillance with suspected cases should be implemented. This review highlights the need for more research and monitoring of vibrios in South America, in water, shellfish and clinical samples.     

Abundance of Vibrio cholerae,V. vulnificus,and V. parahaemolyticus in Oysters (Crassostrea virginica) and Clams (Mercenaria mercenaria) from Long Island Sound

Vibriosis is a leading cause of seafood-associated morbidity and mortality in the United States. Typically associated with consumption of raw or undercooked oysters, vibriosis associated with clam consumption is increasingly being reported. However, little is known about the prevalence of Vibrio spp. in clams. The objective of this study was to compare the levels of Vibrio cholerae, Vibrio vulnificus, and Vibrio parahaemolyticus in oysters and clams harvested concurrently from Long Island Sound (LIS). Most probable number (MPN)–real-time PCR methods were used for enumeration of total V. cholerae, V. vulnificus, V. parahaemolyticus, and pathogenic (tdh⁺ and/or trh⁺) V. parahaemolyticus. V. cholerae was detected in 8.8% and 3.3% of oyster (n = 68) and clam (n = 30) samples, with levels up to 1.48 and 0.48 log MPN/g in oysters and clams, respectively. V. vulnificus was detected in 97% and 90% of oyster and clam samples, with median levels of 0.97 and −0.08 log MPN/g, respectively. V. parahaemolyticus was detected in all samples, with median levels of 1.88 and 1.07 log MPN/g for oysters and clams, respectively. The differences between V. vulnificus and total and pathogenic V. parahaemolyticus levels in the two shellfish species were statistically significant (P < 0.001). These data indicate that V. vulnificus and total and pathogenic V. parahaemolyticus are more prevalent and are present at higher levels in oysters than in hard clams. Additionally, the data suggest differences in vibrio populations between shellfish harvested from different growing area waters within LIS. These results can be used to evaluate and refine illness mitigation strategies employed by risk managers and shellfish control authorities.     

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.     

Defining the Niche of Vibrio parahaemolyticus During Pre- and Post-Monsoon Seasons in the Coastal Arabian Sea

The bacteria Vibrio parahaemolyticus is an important component of coastal ecosystems worldwide, and in recent years, V. parahaemolyticus has caused several cases of food-borne gastroenteritis. However, research investigating which parameters are important in regulating V. parahaemolyticus abundance in tropical areas with relatively stable temperatures and salinity are largely lacking. The objective here was to investigate which environmental forces are driving elevated abundances of V. parahaemolyticus in a tropical oligotrophic coastal area in the Arabian Sea. We analysed a large number of environmental parameters in parallel with cell densities of V. parahaemolyticus and Vibrio spp. Abundance data was obtained using real-time PCR, during two different sampling periods, representative for two distinct seasons. Water temperature and salinity were stable during and between sampling periods, but V. parahaemolyticus abundances were on average six times higher during the first sampling period in December, compared to the second period in February–March. V. parahaemolyticus abundance was found to be positively correlated to inorganic phosphate concentration and copepod abundance. We thus hypothesise that these are important factors regulating V. parahaemolyticus abundance in coastal tropical areas during these periods.     

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.     

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.     

Conventional and molecular methods to detect bacterial pathogens in mussels

Aim: To detect Aeromonas spp., Salmonella spp., Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus in mussels and water samples from a farming area, conventional and molecular methods were applied to enrichment cultures. Methods and Results: The aerolysin gene (aero) of Aeromonas spp., the invasion plasmid antigen B (ipaB) gene of Salmonella spp., the enterotoxin secretion protein (epsM) gene of V. cholerae, the species‐specific region of 16S rRNA gene of V. vulnificus, the 16S–23S rDNA (IGS) gene of V. parahaemolyticus and the pR72H fragment of V. parahaemolyticus were amplified by multiplex polymerase chain reaction (PCR) assays on DNA extracted from enrichment cultures. The haemolysin gene (tdh) of pathogenic V. parahaemolyticus was also amplified. Conventional culture method allowed the isolation of V. parahaemolyticus and V. vulnificus from water and mussels. The genes aero, epsM and 16S rRNA of V. vulnificus were occasionally detected in the enrichment cultures. In mussels, the ipaB and IGS genes were detected from June to September and from April to November, respectively. All genes, except aero, were amplified from mussels collected in September, when pathogenic V. parahaemolyticus (tdh+) strains were also isolated. Conclusions: Multiplex‐PCR assays were more sensitive and faster than conventional procedures. Significance and Impact of the Study: The results emphasize the need of an accurate and rapid detection of bacterial pathogens in mussels to protect human health.     

Prey bacteria shape the community structure of their predators

Although predator–prey interactions among higher organisms have been studied extensively, only few examples are known for microbes other than protists and viruses. Among the bacteria, the most studied obligate predators are the Bdellovibrio and like organisms (BALOs) that prey on many other bacteria. In the macroscopical world, both predator and prey influence the population size of the other''s community, and may have a role in selection. However, selective pressures among prey and predatory bacteria have been rarely investigated. In this study, Bacteriovorax, a predator within the group of BALOs, in environmental waters were fed two prey bacteria, Vibrio vulnificus and Vibrio parahaemolyticus. The two prey species yielded distinct Bacteriovorax populations, evidence that selective pressures shaped the predator community and diversity. The results of laboratory experiments confirmed the differential predation of Bacteriovorax phylotypes on the two bacteria species. Not only did Bacteriovorax Cluster IX exhibit the versatility to be the exclusive efficient predator on Vibrio vulnificus, thereby, behaving as a specialist, but was also able to prey with similar efficiency on Vibrio parahaemolyticus, indicative of a generalist. Therefore, we proposed a designation of versatilist for this predator. This initiative should provide a basis for further efforts to characterize the predatory patterns of bacterial predators. The results of this study have revealed impacts of the prey on Bacteriovorax predation and in structuring the predator community, and advanced understanding of predation behavior in the microbial world.     

A highly sensitive and specific multiplex PCR assay for simultaneous detection of Vibrio cholerae,Vibrio parahaemolyticus and Vibrio vulnificus

Aims: To develop an effective multiplex PCR for simultaneous and rapid detection of Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus, the three most important Vibrio species that can cause devastating health hazards among human. Methods and Results: Species‐specific PCR primers were designed based on toxR gene for V. cholerae and V. parahaemolyticus, and vvhA gene for V. vulnificus. The multiplex PCR was validated with 488 Vibrio strains including 322 V. cholerae, 12 V. vulnificus, and 82 V. parahaemolyticus, 20 other Vibrio species and 17 other bacterial species associated with human diseases. It could detect the three target bacteria without any ambiguity even among closely related species. It showed good efficiency in detection of co‐existing target species in the same sample. The detection limit of all the target species was ten cells per PCR tube. Conclusions: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient for simultaneous detection of these potentially pathogenic Vibrio species in clinical and environmental samples. Significance and Impact of the Study: This simple, rapid and cost‐effective method can be applicable in a prediction system to prevent disease outbreak by these Vibrio species and can be considered as an effective tool for both epidemiologist and ecologist.     

Vibrio vulnificus Bacteriophage SSP002 as a Possible Biocontrol Agent

A novel Vibrio vulnificus-infecting bacteriophage, SSP002, belonging to the Siphoviridae family, was isolated from the coastal area of the Yellow Sea of South Korea. Host range analysis revealed that the growth inhibition of phage SSP002 is relatively specific to V. vulnificus strains from both clinical and environmental samples. In addition, a one-step growth curve analysis and a bacteriophage stability test revealed a latent period of 65 min, a burst size of 23 ± 2 PFU, as well as broad temperature (20°C to 60°C) and pH stability (pH 3 to 12) ranges. A Tn5 random transposon mutation of V. vulnificus and partial DNA sequencing of the inserted Tn5 regions revealed that the flhA, flhB, fliF, and fleQ mutants are resistant to SSP002 phage infection, suggesting that the flagellum may be the host receptor for infection. The subsequent construction of specific gene-inactivated mutants (flhA, flhB, fliF, and fleQ) and complementation experiments substantiated this. Previously, the genome of phage SSP002 was completely sequenced and analyzed. Comparative genomic analysis of phage SSP002 and Vibrio parahaemolyticus phage vB_VpaS_MAR10 showed differences among their tail-related genes, supporting different host ranges at the species level, even though their genome sequences are highly similar. An additional mouse survival test showed that the administration of phage SSP002 at a multiplicity of infection of 1,000 significantly protects mice from infection by V. vulnificus for up to 2 months, suggesting that this phage may be a good candidate for the development of biocontrol agents against V. vulnificus infection.     

Effects of Temperature and Salinity on Vibrio vulnificus Population Dynamics as Assessed by Quantitative PCR

The abundance of Vibrio vulnificus in coastal environments has been linked to water temperature, while its relationship to salinity is less clear. We have developed a culture-independent, most-probable-number quantitative PCR approach to examine V. vulnificus population dynamics in Barnegat Bay, N.J. Based on the combined analysis of our results from Barnegat Bay and from the literature, the present data show that (i) V. vulnificus population dynamics are strongly correlated to water temperature and (ii) although the general trend is for V. vulnificus abundance to be inversely correlated with salinity, this relationship depends on salinity levels. Irrespective of temperature, high abundances of V. vulnificus are observed at 5 to 10 ppt, which thus appears to be the optimal salinity regime for their survival. At 20 to 25 ppt, V. vulnificus abundances show a positive correlation to salinity. Unsuccessful attempts to resuscitate V. vulnificus, combined with our inability to detect cells during the winter despite an assay adapted to detect viable but nonculturable (VBNC) cells, suggest that the decline and eventual disappearance of V. vulnificus from the water column during the winter months is due primarily to a significant reduction in population size and is not only the consequence of cells entering the VBNC state. These findings are in line with the hypothesis that the sediment serves as a refuge for a subpopulation of V. vulnificus over the winter and weather-driven mixing events during the spring initiate a summer bloom in the water column.     

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.     

Occurrence of Vibrio vulnificus Biotypes in Danish Marine Environments

During the unusually warm summer in Denmark in 1994, 11 clinical cases of Vibrio vulnificus infection were reported. These reports initiated an investigation of the occurrence of V. vulnificus biotypes in Danish marine environments. Samples of coastal water, sediment, shellfish, and wild fish were analyzed by preenrichment in alkaline peptone water amended with polymyxin B (2.0 × 10⁴ U/liter) followed by streaking onto modified cellobiose-polymyxin B-colistin agar. V. vulnificus-like colonies were tested with a V. vulnificus-specific DNA probe. Low densities of V. vulnificus were detected in water (0.8 to 19 CFU/liter) from June until mid-September and in sediment (0.04 to >11 CFU/g) from July until mid-November. The presence of V. vulnificus was strongly correlated with water temperature. However, we isolated V. vulnificus from water from a mussel farm at a lower temperature than previously reported (7°C). In 1 of the 13 locations studied, V. vulnificus was found in mussels in 7 of 17 samples analyzed; this is the first report of V. vulnificus in European shellfish. V. vulnificus was also isolated from gills, intestinal contents, and mucus from wild fish. Although biotyping of 706 V. vulnificus strains isolated during our investigations revealed that the majority of the strains (99.6%) belonged to biotype 1, biotype 2 was detected in seawater at a low frequency (0.4%). Our findings provide further evidence that seawater can serve as a reservoir and might facilitate spread of V. vulnificus biotype 2 to eels, with subsequent spread to persons handling eels. In conclusion, our data demonstrate that V. vulnificus is ubiquitous in a temperate marine environment and that V. vulnificus biotype 2 is not strictly confined to eels.     

Occurrence of Vibrio parahaemolyticus, V. cholerae, and V. vulnificus in Norwegian Blue Mussels (Mytilus edulis)

Vibrio parahaemolyticus, V. cholerae, and V. vulnificus were isolated from 10.3%, 1.0%, and 0.1% of 885 blue mussel samples, respectively. Four of the samples contained trh⁺ V. parahaemolyticus, while no tdh-positive isolates were detected. The V. cholerae isolates were non-O:1/non-O:139 serotypes and were ctxA negative.     

PVv3, a New Shuttle Vector for Gene Expression in Vibrio vulnificus

An efficient electroporation procedure for Vibrio vulnificus was designed using the new cloning vector pVv3 (3,107 bp). Transformation efficiencies up to 2 × 10⁶ transformants per μg DNA were achieved. The vector stably replicated in both V. vulnificus and Escherichia coli and was also successfully introduced into Vibrio parahaemolyticus and Vibrio cholerae. To demonstrate the suitability of the vector for molecular cloning, the green fluorescent protein (GFP) gene and the vvhBA hemolysin operon were inserted into the vector and functionally expressed in Vibrio and E. coli.