Historically, methods used to identify Vibrio vulnificus in environmental samples have been inadequate because isolation and identification procedures are time-consuming and fail to separate V. vulnificus from other bacterial species. We describe an enzyme immunoassay (EIA) and culture techniques which identified V. vulnificus in seawater, sediment, and oysters. The EIA used monoclonal antibody FRBT37 to a species-specific epitope of V. vulnificus. No cross-reactions were observed among 72 non-V. vulnificus strains comprising 34 species and 15 genera. In field trials, the EIA identified correctly 99.7% of 348 biochemically confirmed V. vulnificus isolates. The epitope corresponding to FRBT37 was found in cells lysed by Triton X-100, deionized H20, and ultrasonication but was not found in culture supernatants, indicating that its location was intracellular. In addition, electron micrographs of V. vulnificus labeled with FRBT37-biotin-avidin-gold showed that epitope FRBT37 reacted with fragments of lysed cells but not whole cells. FRBT37 was expressed when V. vulnificus was cultured in different growth media. The minimum level of detection of the EIA was
Pure cultures of Vibrio vulnificus held at temperatures of 4 and 0°C underwent a time-dependent decrease in number of recoverable cells. A similar pattern of decreasing numbers was observed with naturally occurring V. vulnificus in cold stored shellstock oysters and shucked oyster meats. The time required for the bacterium to reach undetectable levels (MPN <3/g) may exceed the usual storage life of 14 d for shucked oyster meats and 21 d for shellstock oysters. Freezing and storage of pure cultures of V. vulnificus at −20°C reduced the number of culturable cells more quickly than did holding the cultures at 0°C. However, the organism was cultured from oysters frozen at −20°C for 12 weeks. While cold storage reduced the numbers of V. vulnificus in oysters, such treatment cannot be relied upon to eliminate the organism. Exposure to temperatures above 45°C causes death of V. vulnificus. Decimal reduction times at 47°C for 52 strains averaged 78 s (SD ± 30 s), and D50 values for 18 of the hardiest strains averaged 39.8 s (SD ± 12.2 s). Heating oysters for 10 min in water at 50°C proved adequate to reduce V. vulnificus to a nondetectable level. This treatment does not impart a noticeable cooked appearance or taste to the oysters and may be employed as a strategy to improve the safety of raw oysters.
Changes in the levels of indicator bacteria and Vibrionaceae were monitored in post-harvest shellstock oysters during commercial transport and during storage at temperatures of 10°C, 22°C, and 30°C. Aerobic plate counts, fecal coliforms including Escherichia coli, and Vibrionaceae including Vibrio cholerae, V. parahaemolyticus, V. vulnificus, and Aeromonas hydrophila increased in numbers in shellstock oysters during transport and storage at 22°C and 30°C. Increases in the levels of indicator bacteria were generally accompanied by increases in Vibrionaceae, but sometimes the Vibrionaceae multiplied in the absence of fecal coliform multiplication. Storage of oysters at 10°C prevented multiplication of vibrios and fecal coliforms, but not Aeromonas hydrophila.
Fifty-one interstate shipments of shellstock oysters were sampled at processing plants and examined bacteriologically for Vibrio vulnificus, fecal coliforms, Escherichia coli, and standard plate count. The occurrence of V. vulnificus in the oysters was seasonal with low numbers during the winter and levels frequently exceeding 110,000/g during the summer. The numbers of V. vulnificus correlated (p < 0.01) with fecal coliform levels in the oysters and with water temperatures in the harvest areas. Normal commercial processing did not significantly (p > 0.05) reduce the levels of V. vulnificus or indicator bacteria in the oyster meats. However, storage of the processed meats in containers packed on ice usually produced a one-log and two-log unit reduction in numbers of V. vulnificus after 3 and 7 d, respectively.
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