Direct plating procedure for enumerating Vibrio vulnificus in oysters (Crassostrea virginica)

Abstract

A procedure for enumerating and identifying Vibrio vulnificus in oysters was developed and evaluated. This method consists of growth on a direct plating medium (VVE medium) for isolating the organism from shellfish tissues, followed by biochemical tests for differentiating and identifying presumptively positive isolates. Densities of V. vulnificus are reliably obtained in 2 to 4 days, and as few as 10 culturable cells per 100 g can be identified. The procedure was evaluated by using a DNA probe technique specific for the cytotoxin-hemolysin gene of V. vulnificus and gas chromatographic analysis of the fatty acid contents of positive isolates. Only 3.2 and 0.4% of the isolates gave false-positive and false-negative results, respectively. The average level of recovery on VVE medium for 33 strains, including both clinical and environmental isolates, was 92% of the level of recovery obtained with brain heart infusion agar supplemented with 1% NaCl. The densities of V. vulnificus in oyster homogenates and individual oysters harvested from gulf and Atlantic coastal waters revealed that seasonally high levels occurred. The VVE medium procedure facilitated enumeration of this pathogen in molluscan shellfish and had a distinct advantage over the widely used most-probable-number procedure for V. vulnijus enumeration, which requlires 5 to 7 days and often gives improbable and imprecise results.

Comparison of a commercial biochemical kit and an oligonucleotide probe for identification of environmental isolates of Vibrio vulnificus

“…In the present study the VVAP probe identified a wider range of isolates of V. oulnificus than the API 20E assay. This is in agreement with previous studies that have reported a higher specificity and sensitivity for the VVAP probe for identification of both biotypes of V. vulnlfcus (Micelli et al 1993 ;Wright et al 1993 ;Amaro et ul. 1994).…”

An Overview of Vibrio vulnificus and Vibrio parahaemolyticus

“…Direct plating remains difficult because of the large amount of natural microflora that may also grow on selective media. Micelli and others (1993) developed an alternative method for direct plaing of V. vulnificus from oyster homogenates. Using their so‐called V. vulnificus enumeration (VVE) medium that contained Oxgall, sodium cholate, sodium taurocholate, and potassium tellurite, they reported reduction of 61% to 99% of marine‐associated background microflora without adversely affecting the recovery of V. vulnificus .…”

Two‐stage Nested PCR Effectiveness for Direct Detection of Vibrio vulnificus in Natural Samples

“…. effective in identifying V. vulnificus (Oliver et al, 1992;Miceli et al, 1993), these techniques still cannot provide optimal differentiation of the pathogen from other halophilic Vibrios (Choi, 1997). From this result, it was apparent that the two-stage nested PCR procedure provided a very specific means to differentiate all V. vulnificus from other Vibrios in the natural samples where the pathogen exists together with a large background of other procaryotic and eucaryotic cells.…”