Vibrio parahaemolyticus and V. vulnificus are ubiquitous marine-dwelling bacteria that cause human illness and are of particular concern when raw shellfish are consumed.These organisms can multiply in oysters during storage at ambient temperatures during harvest but prior to refrigeration.Oysters and seawater were collected from 2-3 Louisiana Gulf Coast sites seasonally over a two year period (2006-2007).Subsamples of oysters were placed on ice immediately following harvest (time 0) and then after 2.5, 5, 7.5 and 10 hours of on-deck storage at ambient air temperature.The purpose of the first study was to look at the changes in total and pathogenic V. parahaemolyticus that occur over ten hours of exposure to ambient temperature during harvest.Total V. parahaemolyticus levels were enumerated using the FDA-Bacteriological Analytical Manual (BAM) method for colony lift hybridization targeting the species-specific thermolabile hemolysin (tlh).Results show the highest increase in counts occurred during the summer season; observed levels of total V. parahaemolyticus in oysters averaged 1.7 x 102, 2.2 x 103, 7.5 x 102 and 2.5 x 102 CFU/g at harvest for winter, spring, summer, and fall seasons, respectively.After 10 hours of on-deck storage, these levels changed to 3.3 x 102, 1.9 x 103, 1.1 x 104, and 1.2 x 103 CFU/g for the same seasons.Pathogenic V. parahaemolyticus were enumerated using a multiplexed MPN-PCR targeting the thermostable direct hemolysin (tdh) and TDH-related (trh) genes.The average level of pathogenic V. parahaemolyticus at harvest were 0.027, 1.7, 0.21 and 0.040 MPN/g winter, spring, summer, and fall seasons, respectively; after 10 hours, these levels changed to 0.17, 0.21, 2.0 and 0.38 MPN/g for the same seasons. The percent of total V. parahaemolyticus which harbored pathogenic markers was 0.10% averaged over all seasons.Our data gives weight to the recent decision to reduce the time to refrigeration for Louisiana Gulf Coast oysters.The second study presented aimed to characterize V. parahaemolyticus strains isolated in the first study using pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST).PFGE of the pathogenic (tdh+ and/or trh+) isolates revealed that many isolates were the same strain.Seventeen unique pathogenic strains and 16 non-pathogenic strains were chosen for MLST and further PFGE analysis.PFGE using the NotI restriction enzyme allowed for some grouping with respect to possession of the tdh gene.Twenty-one new sequence types were added to the online MLST database for future use.Both MLST and PFGE proved useful in this study in their own right.In a similar fashion, the third study was designed to characterize V. vulnificus stains isolated during the first study utilizing 16S – 23S rRNA intergenic spacer region (ISR1) typing and MLST.Overall, V. vulnificus showed a high degree of heterogeneity; no associations could be made using ISR1 typing with respect to geographical, temporal or isolation sources.However, grouping based on virulence factors (16S rRNA and vcg genotypes) could be made, although no significant exclusive groups were formed.MLST analysis revealed similar heterogeneity, with 46 new sequence types identified of the 51 isolates tested.ISR1 proved useful in grouping the otherwise heterogeneous V. vulnificus strains, while our additions to the MLST database are valuable for future use.We hope that our studies prove useful in the future: we would like to see our enumeration data used in future regulatory documents, while our characterization data adds to the growing knowledge of the ecology of pathogenic Vibrios.
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Investigating the Ecology of Naturally-Occurring Pathogenic Vibrio Species in Gulf Coast Oysters Through the use of Molecular Sub-Typing Techniques
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