Use of mutant strain for evaluating processing strategies to inactivate Vibrio vulnificus in oysters

Vibrio vulnificus is a ubiquitous marine bacterium frequently isolated from shellfish and associated with severe and often fatal disease in humans. Various control strategies to reduce the disease risk associated with V. vulnificus contamination in shellfish have been proposed. However, evaluating the efficacy of these control strategies is complicated because of the difficulty in distinguishing V. vulnificus from the high levels of background environmental Vibrio spp. The purpose of this research was to develop a model indicator V. vulnificus strain that could be readily differentiated from background microflora and used to facilitate the evaluation of processing efficacy. A spontaneous nalidixic acid-resistant strain of V. vulnificus (Vv-NA) was prepared from a wild-type parent (Vv-WT) using selective plating techniques. Vv-NA was very similar to Vv-WT with respect to biochemical characteristics, appearance on selective plating media, detection limits using most probable number and polymerase chain reaction, and growth rate. In comparative freeze inactivation studies on pure cultures, Vv-WT and Vv-NA had similar freeze inactivation profiles at -20 degrees C (conventional freezing), at -85 degrees C (cold blast freezing), and in liquid nitrogen (cryogenic freezing). In oyster homogenates artificially inoculated with Vv-NA, the organism was inactivated 95 to 99% after freezing, irrespective of freezing temperature. Thermal inactivation comparisons of pure cultures of Vv-WT and Vv-NA using the capillary tube method revealed statistically significant differences in D values at 47 degrees C (2.2 versus 3.0 min, respectively) and 50 degrees C (0.83 versus 0.56 min, respectively), but nearly identical values at 52 degrees C (0.21 versus 0.22 min, respectively). However, these D values were notably higher than those reported by other investigators and hence provided a conservative means by which to evaluate thermal inactivation. In oyster homogenates seeded with Vv-NA, D values of 1.3+/-0.09 min and 0.41+/-0.01 min were obtained at 46 degrees C and 48 degrees C, respectively. This study demonstrated that Vv-NA is readily enumerated and could be used as a surrogate for evaluating the degree of V. vulnificus inactivation provided by freezing and thermal treatments of oyster homogenates.