IN-VIVO RESUSCITATION, AND VIRULENCE TOWARDS MICE, OF VIABLE BUT NONCULTURABLE CELLS OF VIBRIO-VULNIFICUS

Vibrio vulnificus is an estuarine bacterium responsible for 95% of all seafood-related deaths in the United States. The bacterium occurs nat urally in molluscan shellfish, and ingestion of raw oysters is typical ly the source of human infection. V. vulnificus is also known to enter a viable but nonculturable (VBNC) state, wherein the cells are no lon ger culturable on routine plating media but can be shown to remain via ble. Whether or not this human pathogen remains virulent when entering the VBNC state has not been definitively demonstrated. In this study, the VBNC state was induced through a temperature downshift to 5 degre es C, with cells becoming nonculturable (<0.1 CFU/ml) within 7 days. A s they became nonculturable, virulence was determined by employing an iron overload mouse model. At the point of nonculturability (7 days), injections of the diluted microcosm population resulted in death when <0.04 CFU was inoculated, although >10(5) cells in the VBNC state were present in the inoculum. Culturable cells of V. vulnificus, with iden tification confirmed through PCR, were recovered from the blood and pe ritoneal cavities of mice which had died from injections of cells pres ent in the VBNC state for at least 3 days. Thus, our data suggest that cells of V. vulnificus remain virulent, at least for some time, when present in the VBNC state and are capable of causing fatal infections following in vivo resuscitation. Our studies also indicate, however, t hat virulence decreases significantly as cells enter the VBNC state, w hich may account, at least to some extent, for the decrease in infecti ons caused by this bacterium during winter months.