The viable but nonculturable state and cellular resuscitation

Aquatic microbial ecologists have long recognized that portions of bacterial populations seem to disappear from natural water bodies during certain times of the year, only to reappear at other times. In the case of V. vulnificus, numerous studies have documented the difficulty researchers experience in culturing this bacterium during cold months, presumably due to “die-off” of the population. This decrease in culturability is thought to be due to the entrance of the cells into a viable but nonculturable (VBNC) state, reported to occur in at east 30 other bacterial species. It is thought that the VBNC state may represent a survival response of bacteria exposed to stressful environmental conditions and, in the case of V. vulnificus, is induced by temperatures below 10 C. The ability of any bacterium to resuscitate from this dormant state would appear to be essential if the VBNC state were truly a survival strategy. Whether the culturable cells, which appear following stress removal, are a result of true resuscitation, or from re-growth of a few residual culturable cells, has long been debated. Our research, including dilution studies, time required for resuscitation to occur, and the effects of nutrient on recovery, indicates that, at least for V. vulnificus, true resuscitation does occur. Our studies have further suggested that nutrient is in some way inhibitory to the resuscitation of cells in the VBNC state, and that studies which add nutrient in an attempt to detect resuscitation are only able to detect culturable cells which might be present.