Enhanced Detection of Vibrio Cholerae in Oyster Homogenate Based on Centrifugal Removal of Inhibitory Agents

The disease cholera, caused by Vibrio cholerae, has been associated with consumption of contaminated seafood, including raw oysters. Detection of V. cholerae in foods typically involves blending the oysters, diluting the homogenate in alkaline peptone water (APW), overnight enrichment, and isolation on selective agar. Unfortunately, the oyster homogenate must be diluted to large volumes because lower dilutions inhibit the growth of V. cholerae. The goals of this study were to develop an alternative to large dilutions and to evaluate the basis for the inhibition observed in lower dilutions of oyster homogenates. Centrifugation of oyster homogenates at 10,000 x g for 15 min, followed by enrichment of the resulting pellet in APW, was found to eliminate the inhibition of V. cholerae growth. Inhibition appears not to be due to competing microflora but to a component(s) released when V. cholerae grows in the presence of oyster homogenate. The inhibitory component(s) kills the V. cholerae after the cell concentration reaches > 10(exp 8) cells/mL, rather than initially preventing their growth. The pH also declines from 8.0 to 5.5 during this period; however, the pH decline by itself appears not to cause V. cholerae death. Seven strains of V. cholerae (01 and non-01) and two strains of V. vulnificus were susceptible to the inhibitory agent(s). However, other Vibrio and non-Vibrio species tested were not inhibited by the oyster homogenates. Based on digestion of oyster homogenates with pronase, trypsin and lipase, the inhibitory reaction involves a protein(s). In a preliminary trial with oyster homogenate seeded with 1 cfu/g of V. cholerae, the modified centrifugation technique detected a slightly higher percentage of samples at a 1:10 dilution than the standard FDA Bacteriological Analytical Method (BAM) detected in uncentrifuged oyster homogenate at a 1:100 dilution. V. cholerae in seeded samples could also be detected more frequently by the modified centrifugation method than by PCR at a 1:10 dilution.