Antimicrobial Resistance, Virulence Properties and Genetic Diversity of Salmonella Typhimurium Recovered from Domestic and Imported Seafood.

The quantity of seafood imported and produced by domestic aquaculture farming has increased. Recently, it has been reported that multidrug-resistant (MDR) Salmonella Typhimurium may be associated with seafood. However, information is limited to the antimicrobial resistance, virulence properties, and genetic diversity of S. Typhimurium recovered from imported and domestic seafood. This study investigated the antimicrobial resistance, virulence properties, and genetic diversity of S. Typhimurium isolated from domestic and imported catfish, shrimp, and tilapia. A total of 127 isolates were tested for the presence of multidrug-resistance (MDR), virulence genes (invA, pagC, spvC, spvR), and genetic diversity using the Sensititre micro-broth dilution method, PCR, and pulsed-field gel electrophoresis (PFGE), respectively. All isolates were uniformly susceptible to six (amoxicillin/clavulanic acid, ceftiofur, ceftriaxone, imipenem, nitrofurantoin, and trimethoprim/sulfamethoxazole) of the 17 tested antimicrobials and genetically diverse. Fifty-three percent of the Salmonella isolates were resistant to at least one antimicrobial and 49% were multidrug resistant. Ninety-five percent of the isolates possessed the invA gene, 67% pagC, and 43% for both spvC, and spvR. The results suggest that S. Typhimurium recovered from seafood is frequently MDR, virulent, and have the ability to cause salmonellosis.

The mobile gene cassette carrying tetracycline resistance genes in Aeromonas veronii strain Ah5S-24 isolated from catfish pond sediments shows similarity with a cassette found in other environmental and foodborne bacteria.

Aeromonas veronii is a Gram-negative bacterium ubiquitously found in aquatic environments. It is a foodborne pathogen that causes diarrhea in humans and hemorrhagic septicemia in fish. In the present study, we used whole-genome sequencing (WGS) to evaluate the presence of antimicrobial resistance (AMR) and virulence genes found in A. veronii Ah5S-24 isolated from catfish pond sediments in South-East, United States. We found cphA4, dfrA3, mcr-7.1, valF, bla FOX-7, and bla OXA-12 resistance genes encoded in the chromosome of A. veronii Ah5S-24. We also found the tetracycline tet(E) and tetR genes placed next to the IS5/IS1182 transposase, integrase, and hypothetical proteins that formed as a genetic structure or transposon designated as IS5/IS1182/hp/tet(E)/tetR/hp. BLAST analysis showed that a similar mobile gene cassette (MGC) existed in chromosomes of other bacteria species such as Vibrio parahaemolyticus isolated from retail fish at markets, Aeromonas caviae from human stool and Aeromonas media from a sewage bioreactor. In addition, the IS5/IS1182/hp/tet(E)/tetR/hp cassette was also found in the plasmid of Vibrio alginolyticus isolated from shrimp. As for virulence genes, we found the tap type IV pili (tapA and tapY), polar flagellae (flgA and flgN), lateral flagellae (ifgA and IfgL), and fimbriae (pefC and pefD) genes responsible for motility and adherence. We also found the hemolysin genes (hylII, hylA, and TSH), aerA toxin, biofilm formation, and quorum sensing (LuxS, mshA, and mshQ) genes. However, there were no MGCs encoding virulence genes found in A. veronii AhS5-24. Thus, our findings show that MGCs could play a vital role in the spread of AMR genes between chromosomes and plasmids among bacteria in aquatic environments. Overall, our findings are suggesting that MGCs encoding AMR genes could play a vital role in the spread of resistance acquired from high usage of antimicrobials in aquaculture to animals and humans.

High-throughput amplicon sequencing datasets of coastal sediments from three locations of the Gulf of Mexico, USA.

We present high-throughput amplicon sequence (HTS) datasets of the purified microbial metacommunity DNA of coastal surface sediments from Portersville Bay (PVB) (n = 3), Bayou La Batre (BLB) (n = 3), and Mobile Bay (MOB) (n = 3) of the U.S. Gulf of Mexico (U.S. Gulf Coast). The PVB samples were collected from the oyster aquaculture Shellevator™ system; the BLB samples were from locations on the shoreline adjacent to wild oysters attached to rocks and likely polluted from sewage and possibly chemical contamination from boats, shipyards, and seafood processing facilities; and MOB samples were adjacent to aquaculture oysters in bottom cages. The amplicons of the V4 hypervariable segment of the 16S rRNA gene from each sample were sequenced on an Illumina MiSeq to generate these HTS datasets. The raw sequences were quality-checked, demultiplexed into FASTQ files, denoised using DADA2, and subsampled. Then, the FASTA formatted sequences were assigned the taxonomic ids to amplicon sequence variants (ASVs) against the silva-138-99-nb-classifier using the Quantitative Insights Into Microbial Ecology (QIIME2 v2022.2). The applicability of the HTS datasets was confirmed by microbial taxa analysis at the phylum level using the "qiime taxa collapse" command. All HTS datasets are available through the BioSample Submission Portal under the BioProject ID PRJNA876773 (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA876773).

Prevalence and Abundance of Bacterial Pathogens of Concern in Shrimp, Catfish and Tilapia Obtained at Retail Stores in Maryland, USA.

Outbreaks of human gastroenteritis have been linked to the consumption of contaminated domestic and imported seafood. This study investigated the microbiological quality of seafood obtained from retail stores on the Eastern Shore of Maryland. A total of 440 samples of domestic and imported frozen shrimp, catfish and tilapia samples were analyzed for aerobic plate count (APC), total coliforms, Escherichia coli and seafood-borne-pathogens (Vibrio parahaemolyticus, Vibrio vulnificus, Salmonella, Campylobacter jejuni). The prevalence of APC, coliforms and E. coli positive samples was 100%, 43% and 9.3%, respectively. Approximately 3.2%, 1.4%, 28.9% and 3.6% of the samples were positive for V. parahaemolyticus, V. vulnificus, Salmonella and Campylobacter jejuni, respectively. The MPN/g ranges were 150-1100 MPN/g for vibrios, 10-1100 MPN/g for Salmonella and 93-460 MPN/g for C. jejuni in seafood, respectively. Comparing bacterial prevalence by type or source of seafood, the only significant difference identified was Salmonella-positive imported tilapia (33.3%) versus domestic tilapia (19.4%). The quantitative data on pathogen levels in the present study provide additional information for quantitative risk assessment not available in previous surveys. The findings of this study suggest the association of potential food safety hazards with domestic and imported seafood and warrant further large-scale studies and risk assessment.

Nested Spatial and Temporal Modeling of Environmental Conditions Associated With Genetic Markers of Vibrio parahaemolyticus in Washington State Pacific Oysters.

The Pacific Northwest (PNW) is one of the largest commercial harvesting areas for Pacific oysters (Crassostrea gigas) in the United States. Vibrio parahaemolyticus, a bacterium naturally present in estuarine waters accumulates in shellfish and is a major cause of seafood-borne illness. Growers, consumers, and public-health officials have raised concerns about rising vibriosis cases in the region. Vibrio parahaemolyticus genetic markers (tlh, tdh, and trh) were estimated using an most-probable-number (MPN)-PCR technique in Washington State Pacific oysters regularly sampled between May and October from 2005 to 2019 (N = 2,836); environmental conditions were also measured at each sampling event. Multilevel mixed-effects regression models were used to assess relationships between environmental measures and genetic markers as well as genetic marker ratios (trh:tlh, tdh:tlh, and tdh:trh), accounting for variation across space and time. Spatial and temporal dependence were also accounted for in the model structure. Model fit improved when including environmental measures from previous weeks (1-week lag for air temperature, 3-week lag for salinity). Positive associations were found between tlh and surface water temp, specifically between 15 and 26°C, and between trh and surface water temperature up to 26°C. tlh and trh were negatively associated with 3-week lagged salinity in the most saline waters (> 27 ppt). There was also a positive relationship between tissue temperature and tdh, but only above 20°C. The tdh:tlh ratio displayed analogous inverted non-linear relationships as tlh. The non-linear associations found between the genetic targets and environmental measures demonstrate the complex habitat suitability of V. parahaemolyticus. Additional associations with both spatial and temporal variables also suggest there are influential unmeasured environmental conditions that could further explain bacterium variability. Overall, these findings confirm previous ecological risk factors for vibriosis in Washington State, while also identifying new associations between lagged temporal effects and pathogenic markers of V. parahaemolyticus.

Risk Assessment of Norovirus Illness from Consumption of Raw Oysters in the United States and in Canada.

Human norovirus (NoV) is the leading cause of foodborne illness in the United States and Canada. Bivalve molluscan shellfish is one commodity commonly identified as being a vector of NoV. Bivalve molluscan shellfish are grown in waters that may be affected by contamination events, tend to bioaccumulate viruses, and are frequently eaten raw. In an effort to better assess the elements that contribute to potential risk of NoV infection and illness from consumption of bivalve molluscan shellfish, the U.S. Department of Health and Human Services/Food and Drug Administration (FDA), Health Canada (HC), the Canadian Food Inspection Agency (CFIA), and Environment and Climate Change Canada (ECCC) collaborated to conduct a quantitative risk assessment for NoV in bivalve molluscan shellfish, notably oysters. This study describes the model and scenarios developed and results obtained to assess the risk of NoV infection and illness from consumption of raw oysters harvested from a quasi-steady-state situation. Among the many factors that influence the risk of NoV illness for raw oyster consumers, the concentrations of NoV in the influent (raw, untreated) and effluent (treated) of wastewater treatment plants (WWTP) were identified to be the most important. Thus, mitigation and control strategies that limit the influence from human waste (WWTP outfalls) in oyster growing areas have a major influence on the risk of illness from consumption of those oysters.

A case-control analysis of traceback investigations for Vibrio parahaemolyticus infections (vibriosis) and pre-harvest environmental conditions in Washington State, 2013-2018.

BACKGROUND: Vibrio parahaemolyticus is a major cause of seafood-borne illness. It is naturally prevalent in brackish waters and accumulates in shellfish. Vibriosis cases are rising globally, likely due to rising temperatures. OBJECTIVES: To identify associations between vibriosis in Washington State and pre-harvest environmental and V. parahaemolyticus genetic measurements sampled from shellfish. METHODS: Successful vibriosis traceback investigations were spatiotemporally matched to routine intertidal oyster (Crassostrea gigas) sampling events, which included measurements of temperature, salinity, and V. parahaemolyticus genetic targets (thermolabile hemolysin: tlh; thermostable direct hemolysin: tdh; thermostable direct-related hemolysin: trh). Unmatched sampling events were treated as controls. Associations were evaluated using logistic regression models. RESULTS: Systematic differences were observed across Washington harvesting zones. These included positive associations between the odds of vibriosis and all three genetic targets in South Puget Sound, with a large odds ratio (OR) = 13.0 (95% CI: 1.5, 115.0) for a 1-log10 increase in tdh when total bacterium abundance was low (tlh < 1 log10 MPN/g). A positive association also occurred for a 1 °C increase in tissue temperature OR = 1.20 (95% CI: 1.10, 1.30) while a negative association occurred for a similar increase in water temperature OR = 0.70 (95% CI: 0.59, 0.81). In contrast, the coastal bays displayed positive associations for water temperature OR = 2.16 (95% CI, 1.15, 4.05), and for a 1-log10 increase in the tdh:trh ratio OR = 5.85 (95% CI, 1.06, 32.26). DISCUSSION: The zonal variation in associations indicates unique pathogenic strain prominence, suggesting tdh+/trh+ strains in South Puget Sound, such as the O4:K12 serotype, and tdh+/trh- strains in the coastal bays. The temperature discrepancy between water and oyster tissue suggests that South Puget Sound pathogenic strains flourish with exposure to relatively warm air during low tide. These findings identify new ecological risk factors for vibriosis in Washington State that can be used in future prevention efforts.

A systematic review of post-harvest interventions for Vibrio parahaemolyticus in raw oysters.

BACKGROUND: Non-cholera Vibrio bacteria are a major cause of foodborne illness in the United States. Raw oysters are commonly implicated in gastroenteritis caused by pathogenic Vibrio parahaemolyticus. In response to outbreaks in 1997-1998, the US Food and Drug Administration developed a nation-wide quantitative microbial risk assessment (QMRA) of V. parahaemolyticus in raw oysters in 2005. The QMRA identified information gaps that new research may address. Incidence of sporadic V. parahaemolyticus illness has recently increased and, as oyster consumption increases and sea temperatures rise, V. parahaemolyticus outbreaks may become more frequent, posing health concerns. Updated and region-specific QMRAs will improve the accuracy and precision of risk of infection estimates. OBJECTIVES: We identify research to support an updated QMRA of V. parahaemolyticus from oysters harvested in Chesapeake Bay and Puget Sound, focusing on observational and experimental research on post-harvest practices (PHPs) published from 2004 to 2019. METHODS: A predefined search strategy was applied to PubMed, Embase, Scopus, Science.gov, NAL Agricola, and Google Scholar. Study eligibility criteria were defined using a population, intervention, comparator, and outcome statement. Reviewers independently coded abstracts for inclusion/exclusion using predefined criteria. Data were extracted and study quality and relevance evaluated based on published guidance for food safety risk assessments. Findings were synthesized using a weight of evidence approach. RESULTS: Of 12,174 articles retrieved, 93 were included for full-text review. Twenty-seven studies were found to be high quality and high relevance, including studies on cold storage, high hydrostatic pressure, depuration, and disinfectant, and other PHPs. High hydrostatic pressure consistently emerged as the most effective PHP in reducing abundance of V. parahaemolyticus. DISCUSSION: Limitations of the knowledge base and review approach involve the type and quantity of data reported. Future research should focus on PHPs for which few or no high quality and high relevance studies exist, such as irradiation and relaying.

Associations of Environmental Conditions and Vibrio parahaemolyticus Genetic Markers in Washington State Pacific Oysters.

Vibrio parahaemolyticus is a naturally occurring bacterium in estuarine waters and is a major cause of seafood-borne illness. The bacterium has been consistently identified in Pacific Northwest waters and elevated illness rates of vibriosis in Washington State have raised concerns among growers, risk managers, and consumers of Pacific oysters (Crassostrea gigas). In order to better understand pre-harvest variation of V. parahaemolyticus in the region, abundance of total and potentially pathogenic strains of the bacterium in a large number of Washington State Pacific oyster samples were compared with environmental conditions at the time of sampling. The Washington Department of Health regularly sampled oysters between June and September at over 21 locations from 2014 to 2018, resulting in over 946 samples. V. parahaemolyticus strains carrying three genetic markers, tlh, trh, and tdh, were enumerated in oyster tissue using a most probable number-PCR analysis. Tobit regressions and seemingly unrelated estimations were used to formally assess relationships between environmental measures and genetic markers. All genetic markers were found to be positively associated with temperature, independent of the abundance of other genetic markers. Surface water temperature displayed a non-linear relationship, with no association observed between any genetic marker in the warmest waters. There were also stark differences between surface and shore water temperature models. Salinity was not found to be substantially associated with any of the genetic variables. The relative abundance of tdh+ strains given total V. parahaemolyticus abundance (pathogenic ratio tdh:tlh) was negatively associated with water temperature in colder waters and decreased exponentially as total V. parahaemolyticus abundance increased. Strains carrying the trh gene had a pronounced positive association with strains carrying the tdh gene but was also negatively associated with the tdh:tlh pathogenic ratio. These results suggest that there are ecological relationships of competition, growth, and survival for V. parahaemolyticus strains in the oyster tissue matrix. This work also improves the overall understanding of environmental associations with V. parahaemolyticus in Washington State Pacific oysters, laying the groundwork for future risk mitigation efforts in the region.

Vibrio parahaemolyticus in the Chesapeake Bay: Operational In Situ Prediction and Forecast Models Can Benefit from Inclusion of Lagged Water Quality Measurements.

Vibrio parahaemolyticus is a leading cause of seafood-borne gastroenteritis. Given its natural presence in brackish waters, there is a need to develop operational forecast models that can sufficiently predict the bacterium's spatial and temporal variation. This work attempted to develop V. parahaemolyticus prediction models using frequently measured time-indexed and -lagged water quality measures. Models were built using a large data set (n = 1,043) of surface water samples from 2007 to 2010 previously analyzed for V. parahaemolyticus in the Chesapeake Bay. Water quality variables were classified as time indexed, 1-month lag, and 2-month lag. Tobit regression models were used to account for V. parahaemolyticus measures below the limit of quantification and to simultaneously estimate the presence and abundance of the bacterium. Models were evaluated using cross-validation and metrics that quantify prediction bias and uncertainty. Presence classification models containing only one type of water quality parameter (e.g., temperature) performed poorly, while models with additional water quality parameters (i.e., salinity, clarity, and dissolved oxygen) performed well. Lagged variable models performed similarly to time-indexed models, and lagged variables occasionally contained a predictive power that was independent of or superior to that of time-indexed variables. Abundance estimation models were less effective, primarily due to a restricted number of samples with abundances above the limit of quantification. These findings indicate that an operational in situ prediction model is attainable but will require a variety of water quality measurements and that lagged measurements will be particularly useful for forecasting. Future work will expand variable selection for prediction models and extend the spatial-temporal extent of predictions by using geostatistical interpolation techniques.IMPORTANCEVibrio parahaemolyticus is one of the leading causes of seafood-borne illness in the United States and across the globe. Exposure often occurs from the consumption of raw shellfish. Despite public health concerns, there have been only sporadic efforts to develop environmental prediction and forecast models for the bacterium preharvest. This analysis used commonly sampled water quality measurements of temperature, salinity, dissolved oxygen, and clarity to develop models for V. parahaemolyticus in surface water. Predictors also included measurements taken months before water was tested for the bacterium. Results revealed that the use of multiple water quality measurements is necessary for satisfactory prediction performance, challenging current efforts to manage the risk of infection based upon water temperature alone. The results also highlight the potential advantage of including historical water quality measurements. This analysis shows promise and lays the groundwork for future operational prediction and forecast models.

Efficacy of Vibrio parahaemolyticus depuration in oysters (Crassostrea gigas).

This study investigated the influences of seawater to oyster ratio on depuration for decontaminating V. parahaemolyticus in raw oysters with a goal of identifying the proper ratio of oyster to seawater capable of improving the efficacy of the depuration process. The water to oyster ratios tested in this study ranged from 1.0 to 2.5 L of artificial seawater (ASW) per oyster (40 oysters in 40, 60, 80 and 100 L ASW). The depuration efficacy for purging V. parahaemolyticus from oysters was highest when we applied a 2:1, followed by 1.5:1, 2.5:1, and 1:1 L of ASW/oyster. Further studies of depuration with 2:1 L of ASW/oyster found that the concentration of V. parahaemolyticus in oysters decreased in a nonlinear manner. The depuration curve was fitted to a one phase decay model with a coefficient of determination (R2) of 0.933. The time for a 3 log reduction was 1.75 days with a 95% confidence interval from 1.65 to 1.85 days, which meets the FDA's requirement of larger than a 3.0 log (MPN/g) reduction as a post-harvest process for V. parahaemolyticus control. After 4 days levels in all trials were <100 MPN/g meeting performance standards established by Japan and Canada. Furthermore, the time for a 3.52 log reduction was 3.17 days with a 95% confidence interval from 2.92 to 3.54 days but it took 5 days to reduce levels to <30 MPN/g, which satisfies FDA's requirement as a post-harvest control process (>3.52 log MPN/g reduction) for the purpose of making safety added labeling claims for V. parahaemolyticus.

Characterization of the Microbiota of Oyster Larvae (Crassostrea virginica) and Tank Water from an Aquaculture System with High and Low Larval Survival Rates.

Aquaculture plays an increasingly important role in the growing demand for seafood. Hatchery production of oyster larvae is an integral component of oyster farming, providing single seed for off-bottom farming or larvae for setting on larger substrates for on-bottom farming. Larvae from certain tanks in an established aquaculture enterprise were dying from an unknown etiologic agent. A metagenomic approach was used to examine oyster larvae and water from larval tanks with high and low survival rates to evaluate the epidemiological efficacy of this approach.

Environmental Determinants of Vibrio parahaemolyticus in the Chesapeake Bay.

Vibrio parahaemolyticus naturally occurs in brackish and marine waters and is one of the leading causes of seafood-borne illness. Previous work studying the ecology of V. parahaemolyticus has often been limited in geographic extent and lacked a full range of environmental measures. This study used a unique large data set of surface water samples in the Chesapeake Bay (n = 1,385) collected from 148 monitoring stations from 2007 to 2010. Water was analyzed for more than 20 environmental parameters, with additional meteorological and surrounding land use data. The V. parahaemolyticus-specific genetic markers thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and tdh-related hemolysin (trh) were assayed using quantitative PCR (qPCR), and interval-censored regression models with nonlinear effects were estimated to account for limits of detection and quantitation. tlh was detected in 19.6% of water samples; tdh or trh markers were not detected. The results confirmed previously reported positive associations for V. parahaemolyticus abundance with temperature and turbidity and negative associations with high salinity (>10 to 23‰). Furthermore, the salinity relationship was determined to be a function of both low temperature and turbidity, with an increase of either nullifying the high salinity effect. Associations with dissolved oxygen and phosphate also appeared stronger when samples were taken near human developments. A renewed focus on the V. parahaemolyticus ecological paradigm is warranted to protect public health.IMPORTANCE Vibrio parahaemolyticus is one of the leading causes of seafood-borne illness in the United States and across the globe. Exposure is often through consuming raw or undercooked shellfish. Given the natural presence of the bacterium in the marine environment, an improved understanding of its environmental determinants is necessary for future preventative measures. This analysis of environmental Vibrio parahaemolyticus is one of only a few that utilize a large data set measured over a wide geographic and temporal range. The analysis also includes a large number of environmental parameters for Vibrio modeling, many of which have previously only been tested sporadically, and some of which have not been considered before. The results of the analysis revealed previously unknown relationships between salinity, turbidity, and temperature that provide significant insight into the abundance and persistence of V. parahaemolyticus bacterium in the environment. This information will be essential for developing environmental forecast models for the bacterium.

Microbiomes of American Oysters (Crassostrea virginica) Harvested from Two Sites in the Chesapeake Bay.

In this study, we used 16S rRNA gene amplicons to describe the bacterial microbiota associated with oysters (Crassostrea virginica) and seawater collected from two sites in the Chesapeake Bay. The dominant bacterial groups included those belonging to the order Pelagibacteraceae, family Enterobacteriaceae, and genus Synechococcus The microbiomes varied among oysters from the same site and between the two sites and months.

Meta-Analysis of the Reduction of Norovirus and Male-Specific Coliphage Concentrations in Wastewater Treatment Plants.

Human norovirus (NoV) is the leading cause of foodborne illness in the United States and Canada. Wastewater treatment plant (WWTP) effluents impacting bivalve mollusk-growing areas are potential sources of NoV contamination. We have developed a meta-analysis that evaluates WWTP influent concentrations and log10 reductions of NoV genotype I (NoV GI; in numbers of genome copies per liter [gc/liter]), NoV genotype II (NoV GII; in gc/liter), and male-specific coliphage (MSC; in number of PFU per liter), a proposed viral surrogate for NoV. The meta-analysis included relevant data (2,943 measurements) reported in the scientific literature through September 2013 and previously unpublished surveillance data from the United States and Canada. Model results indicated that the mean WWTP influent concentration of NoV GII (3.9 log10 gc/liter; 95% credible interval [CI], 3.5, 4.3 log10 gc/liter) is larger than the value for NoV GI (1.5 log10 gc/liter; 95% CI, 0.4, 2.4 log10 gc/liter), with large variations occurring from one WWTP to another. For WWTPs with mechanical systems and chlorine disinfection, mean log10 reductions were -2.4 log10 gc/liter (95% CI, -3.9, -1.1 log10 gc/liter) for NoV GI, -2.7 log10 gc/liter (95% CI, -3.6, -1.9 log10 gc/liter) for NoV GII, and -2.9 log10 PFU per liter (95% CI, -3.4, -2.4 log10 PFU per liter) for MSCs. Comparable values for WWTPs with lagoon systems and chlorine disinfection were -1.4 log10 gc/liter (95% CI, -3.3, 0.5 log10 gc/liter) for NoV GI, -1.7 log10 gc/liter (95% CI, -3.1, -0.3 log10 gc/liter) for NoV GII, and -3.6 log10 PFU per liter (95% CI, -4.8, -2.4 PFU per liter) for MSCs. Within WWTPs, correlations exist between mean NoV GI and NoV GII influent concentrations and between the mean log10 reduction in NoV GII and the mean log10 reduction in MSCs.

Comparison of plating media for recovery of total and virulent genotypes of Vibrio vulnificus in U.S. market oysters.

Vibrio vulnificus is the leading cause of seafood associated mortality in the United States and is generally associated with consumption of raw oysters. Two genetic markers have emerged as indicators of strain virulence, 16S rDNA type B (rrnB) and virulence correlated gene type C (vcgC). While much is known about the distribution of V. vulnificus in oysters, a limited number of studies have addressed the more virulent subtypes. Therefore, the goals of this study were to (1) determine the suitability of media for recovery of total and virulent genotypes of V. vulnificus and (2) evaluate the geographical and seasonal distribution of these genotypes. Market oysters from across the United States and the strains isolated from them during a year-long study in 2007 were used. For media evaluation, VVA and CPC+ were compared using direct plating of oyster tissues while mCPC and CPC+ were compared for isolation following MPN enrichment. Representative isolates from each media/method were tested for rrn and vcg types to determine their seasonal and geographical distribution. No statistically significant difference was observed between VVA/CPC+ or mCPC/CPC+ for isolation of total or virulent (rrnB/vcgC) genotypes of V. vulnificus. Overall, 32% of recovered isolates possessed the virulent genotype. The prevalence of these genotypes was highest in oysters from the Gulf Coast during Oct-Dec, and demonstrated a statistically significant geographical and seasonal pattern. This is the first report on the distribution of virulent V. vulnificus genotypes across the United States, which provides novel insight into the occurrence of this pathogen.

Development and validation of a predictive model for the growth of Vibrio parahaemolyticus in post-harvest shellstock oysters.

Information is limited about the growth and survival of naturally-occurring Vibrio parahaemolyticus in live oysters under commercially relevant storage conditions harvested from different regions and in different oyster species. This study produced a predictive model for the growth of naturally-occurring V. parahaemolyticus in live Eastern oysters (Crassostrea virginica) harvested from the Chesapeake Bay, MD, USA and stored at 5-30 °C until oysters gapped. The model was validated with model-independent data collected from Eastern oysters harvested from the Chesapeake Bay and Mobile Bay, AL, USA and Asian (C. ariakensis) oysters from the Chesapeake Bay, VA, USA. The effect of harvest season, region and water condition on growth rate (GR) was also tested. At each time interval, two samples consisting of six oysters each were analyzed by a direct-plating method for total V. parahaemolyticus. The Baranyi D-model was fitted to the total V. parahaemolyticus growth and survival data. A secondary model was produced using the square root model. V. parahaemolyticus slowly inactivated at 5 and 10 °C with average rates of -0.002 and -0.001 log cfu/h, respectively. The average GRs at 15, 20, 25, and 30 °C were 0.038, 0.082, 0.228, and 0.219 log cfu/h, respectively. The bias and accuracy factors of the secondary model for model-independent data were 1.36 and 1.46 for Eastern oysters from Mobile Bay and the Chesapeake Bay, respectively. V. parahaemolyticus GRs were markedly lower in Asian oysters. Harvest temperature, salinity, region and season had no effect on GRs. The observed GRs were less than those predicted by the U.S. Food and Drug Administration's V. parahaemolyticus quantitative risk assessment.

Biochemical, serological, and virulence characterization of clinical and oyster Vibrio parahaemolyticus isolates.

In this study, 77 clinical and 67 oyster Vibrio parahaemolyticus isolates from North America were examined for biochemical profiles, serotype, and the presence of potential virulence factors (tdh, trh, and type III secretion system [T3SS] genes). All isolates were positive for oxidase, indole, and glucose fermentation, consistent with previous reports. The isolates represented 35 different serotypes, 9 of which were shared by clinical and oyster isolates. Serotypes associated with pandemic strains (O1:KUT, O1:K25, O3:K6, and O4:K68) were observed for clinical isolates, and 7 (9%) oyster isolates belonged to serotype O1:KUT. Of the clinical isolates, 27% were negative for tdh and trh, while 45% contained both genes. Oyster isolates were preferentially selected for the presence of tdh and/or trh; 34% contained both genes, 42% had trh but not tdh, and 3% had tdh but not trh. All but 1 isolate (143/144) had at least three of the four T3SS1 genes examined. The isolates lacking both tdh and trh contained no T3SS2α or T3SS2ß genes. All clinical isolates positive for tdh and negative for trh possessed all T3SS2α genes, and all isolates negative for tdh and positive for trh possessed all T3SS2ß genes. The two oyster isolates containing tdh but not trh possessed all but the vopB2 gene of T3SS2α, as reported previously. In contrast to the findings of previous studies, all strains examined that were positive for both tdh and trh also carried T3SS2ß genes. This report identifies the serotype as the most distinguishing feature between clinical and oyster isolates. Our findings raise concerns about the reliability of the tdh, trh, and T3SS genes as virulence markers and highlight the need for more-detailed pathogenicity investigations of V. parahaemolyticus.