Evaluation of a Tissue Culture-Based Approach for Differentiating between Virulent and Avirulent Vibrio parahaemolyticus Strains Based on Cytotoxicity

摘要

The ability of only a subset of Vibrio parahaemolyticus strains to cause human infection underscores the need for an analytical method that can effectively differentiate between pathogenic strains and those that do not cause disease. We tested the feasibility of a tissue culture-based assay to determine whether clinical isolates could be differentiated from nonclinical isolates based on relative isolate cytopathogenicity. To screen for cytotoxic capability, we measured relative extracellular lactate dehydrogenase as an indicator of host cell damage in five different mammalian cell lines in the presence of V. parahaemolyticus. Isolates originating from clinical sources exhibited 15.5 to 59.3% relative cytotoxicity, whereas those originating from food sources exhibited 4.4 to 54.9% relative cytotoxicity. In the presence of ～1.2 X 10~6 cells, cytotoxicity was 1.6- to 3.5-fold higher (P < 0.05) for clinical isolates than for nonclinical isolates in L2, Henle 407, and Caco-2 cell lines. V. parahaemolyticus serotype O3:K6 clinical isolates had 1.6- to 2.1-fold higher cytotoxicity than did the non-O3:K6 clinical isolates, with significantly higher cytotoxicity in HeLa, J774A.1, and Henle 407 cells than in L2 and Caco-2 cells. Because V. parahaemolyticus often is found in oysters, the effect of the presence of an oyster matrix on assay efficacy was also tested with L2 cells. The cytotoxicity elicited by a highly cytotoxic V. parahaemolyticus isolate was not affected by the presence of oyster tissue, suggesting that an oyster matrix will not interfere with assay sensitivity. In the present format, this assay can detect the presence of >10~5 cells of a virulent V. parahaemolyticus strain in an oyster matrix.