Resuscitation rate in different enterococcal species in the viable but non-culturable state

Aims: The viable but non-culturable (VBNC) state is a survival strategy adopted by bacteria when exposed to environmental stress. When in this state bacteria are no longer culturable on conventional growth media, but cells display metabolic activity and maintain pathogenicity factors/genes and, in some cases, resuscitation from the VBNC state has been shown. This state has been described for both human pathogens and faecal pollution indicators. In this study, we present evidence for entry of different enterococcal species into the VBNC state in an oligotrophic microcosm. Methods and Results: The duration of the viability of the cells in the VBNC state was measured either by detecting the presence of pbp5 mRNA or by quantifying their resuscitation capability. Enterococci showed different behaviours. Enterococcus faecalis and Enterococcus hirae entered into the VBNC state within 2 weeks and remained in that state for 3 months. In the experiments described the resuscitation rate was 1 : 10 000 cells as soon as the cells entered the VBNC state and decreased gradually to undetectable levels over the following 3 months. Enterococcus faecium, however, remained culturable Up to 4 weeks. After this time period, when the population was totally unculturable, the cells were far less resuscitable than other enterococci and only over a narrow time interval (2 weeks). Conclusions: These results suggest that Ent. faecalis and Ent. hirae enter the VBNC state but that Ent. faecium, in an oligotrophic laboratory environment, tends to die instead of entering the VBNC state. Significance and Impact of the Study: These experiments may mimic what happens when enterococci are released by humans and animals in natural environments.