Detection of Vibrio cholerae in oyster (Crassostrea virginica) homogenate based on centrifugal removal of antimicrobial agents

Detection of Vibrio cholerae in oysters requires homogenization, enrichment in alkaline peptone water (APW) and isolation on selective medium. An antimicrobial substance(s) indigenous to oysters apparently inhibits V. cholerae recovery, thus, requiring extensive dilution of the oyster homogenate in APW (1:100, v/v) during enrichment. Centrifugation of oyster homogenates partitions antimicrobial agent(s) into the supernatant fraction. The antimicrobial agent(s) was not removed by filtration (0.2 mu m) of the supernatant, thus eliminating bacterial competition as the source of inhibition. Growth curves conducted in filtered sterilized oyster supernatant-APW (1:10) at 42 degrees C demonstrated logarithmic V. cholerae growth until cell density reached 10(8) ml(-1) (6-12 h) followed by logarithmic death. Antimicrobial agents were inactivated by pretreatment of the supernatant at 80 degrees C, or with pronase, but were stable at 75 degrees C and with trypsin or lipase treatment. While a rapid pH drop was noted prior to cell death, low pH alone was not sufficient for V. cholerae lethality. Vibrio vulnificus was also susceptible to inhibitory agent(s) in oysters, but no inhibition was observed with other Vibrio and non-vibrio species tested. Removal of the supernatant followed by enrichment of the pellet formed the basis of a novel procedure, which was as sensitive as the dilution procedure for V. cholerae detection (<1 cfu g(-1)). Published by Elsevier Science B.V.