Temperature effects on the viable but non-culturable state of Vibrio vulnificus

Abstract The non-culturable state of Vibrio vulnificus , strain C7184, was studied in artificial seawater microcosms held at 5, 10, 15, 20, and 30°C. Plate counts were made on a non-selective medium, total cell counts were monitored by acridine orange epifluorescence, and direct viable counts (DVSs) by the method of Kogure et al. (Can J. Microbiol. 25, 415–420; 1986) and by the INT method. From an initial inoculum of 10⁷ cells/ml, V. vulnificus became non-culturable within 40 days at 5°C, although both indicators of viability revealed a viable population exceeding 10⁶ cells/ml. Cells at all higher temperatures remained culturable (at least 10⁴/ml) throughout the study. The non-culturable states of the opaque and translucent colony variants of V. vulnificus , as well as those of six other clinical and environmental strains of V. vulnificus , were examined at 5°C; all but one strain and both colony variants also became non-culturable within 40 days. In contrast, six other Vibrio spp. ( V. cholerae, V. mimicus, V. parahaemolyticus, V. natriegens, V. proteolyticus , and V. campbelli ) remained culturable at 5°C. Thus, entrance of V. vulnificus into the non-culturable state appears to be highly temperature dependent and, among the vibrios, this species may be especially sensitive to low temperature. The public health aspects of these findings are discussed.