Graduate Thesis Or Dissertation
 

Characterization of Vibrio parahaemolyticus isolated from Oregon and Washington coastal water and development of improved methods for Vibrio parahaemolyticus detection

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  • Distribution of Vibrio parahaemolyticus in Oregon and Washington oystergrowing areas was studied between November 2002 and October 2003. V. parahaemolyticus was detected in 14.3% of oyster, 23.0% of seawater, and 44.4% of sediment samples with very low levels (≤7.4 MPN/g) of pathogenic strains being detected in oysters. Occurrence of V. parahaemolyticus in oysters and the environments was positively correlated to water temperatures and were more frequent in sediment than in seawater or oyster. Thirty-four pathogenic V. parahaemolyticus isolated during the study were examined for virulence factors [tdh and trh genes encoding thermostable direct hemolysin (TDH) and TDH-related hemolysis (TRH), respectively], urease production, and O-group antigenicity. The tdh and trh genes were detected in 85% and 88% of the isolates, respectively while almost all of them (97%) produced urease. Six O serotypes were identified among the isolates with 05 and 01 being the most prevalent. Pulsed-field gel electrolysis (PFGE) analysis of the surveyed isolates and five clinical reference strains using Notl and Sfil enzymes revealed 22 restriction patterns with NISI pattern being the most prevalent {25.6%) followed by N2S2 pattern (10.3%). Results of molecular, serological, and virulence analysis demonstrated that 12 isolates were identical to two clinical strains involved in 1997's outbreak in Oregon and Washington. A newly developed chromogenic medium (Bio-Chrome Vibrio medium, BCVM) was compared with widely used thiosulfate-citrate-bile salts-sucrose agar (TCBS) for their specificities and accuracies for detecting V. parahaemolyticus in seawater, sediment, and oysters with a commonly used 3-tube most probably number (MPN) method. The specificities of BCVM and TCBS for V. parahaemolyticus detection were determined to be 94% and 77%, respectively. The accuracies of detecting V. parahaemolyticus were 84% for BCVM and 54%) for TCBS. Application of BCVM in a double layer agar plate (DLAP) for direct enumeration of injured V. parahaemolyticus cells was developed by overlaying an equal volume of a non-selective medium on top of a BCVM layer. The direct-plating procedure using DLAP was found as effective as the MPN method for recovering heat- and cold-injured V. parahaemolyticus cells. The DLAP can be used as a simple one-step procedure for quick screening of V. parahaemolyticus in foods.
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