Use of a 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. Va rious control strategies to reduce the disease risk associated with V. vuln ificus contamination in shellfish have been proposed. However, evaluating t he efficacy of these control strategies is complicated because of the diffi culty in distinguishing V. vulnificus from the high levels of background en vironmental Vibrio spp. The purpose of this research was to develop a model indicator V. vulnificus strain that could be readily differentiated from b ackground microflora and used to facilitate the evaluation of processing ef ficacy. A spontaneous nalidixic acid-resistant strain of V. vulnificus (Vv- NA) was prepared from a wild-type parent (Vv-WT) using selective plating te chniques. Vv-NA was very similar to Vv-WT with respect to biochemical chara cteristics, appearance on selective plating media, detection limits using m ost probable number and polymerase chain reaction, and growth rate. In comp arative freeze inactivation studies on pure cultures, Vv-WT and Vv-NA had s imilar freeze inactivation profiles at -20 degrees C (conventional freezing ), at -85 degrees C (cold blast freezing), and in liquid nitrogen (cryogeni c 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 an d 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, thes e D values were notably higher than those reported by other investigators a nd hence provided a conservative means by which to evaluate thermal inactiv ation. In oyster homogenates seeded with Vv-NA, D values of 1.3 +/- 0.09 mi n and 0.41 +/- 0.01 min were obtained at 46 degrees C and 48 degrees C, res pectively. This study demonstrated that Vv-NA is readily enumerated and cou ld be used as a surrogate for evaluating the degree of V. vulnificus inacti vation provided by freezing and thermal treatments of oyster homogenates.