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. Signi®cance and Impact of the Study: These experiments may mimic what happens when enterococci are released by humans and animals in natural environments.

The protein expression patterns of exponentially growing, starved, and viable but nonculturable (VBNC) Enterococcus faecalis cells were analyzed to establish whether differences exist between the VBNC state and other stress responses. The results indicate that the protein profile of VBNC cells differs from that of either starved or exponentially growing bacteria. This demonstrates that the VBNC state is a distinct physiological phase within the life cycle of E. faecalis, which is activated in response to multiple environmental stresses.

When exposed to stress-provoking environmental conditions such as those of ground waters, many medically important bacteria have been shown to be capable of activating a survival strategy known as the viable but non-culturable (VBNC) state. In this state bacteria are no longer culturable on conventional growth media, but the cells maintain their viability and pathogenicity genes/factors and can start dividing again, in a part of the cell population, upon restoration of favourable environmental conditions. Little is known about the genetic mechanisms underlying the VBNC state. In this study we show evidence of involvement of the rpoS gene in persistence of Escherichia coli in the VBNC state. The kinetics of entry into the non-culturable state and duration of cell viability were measured in two E. coli mutants carrying an inactivated rpoS gene and compared with those of the parents. For these experiments, laboratory microcosms consisting of an artificial oligotrophic medium incubated at 4 degrees C were used. The E. coli parental strains reached the non-culturable state in 33 days when the plate counts were evaluated on Luria-Bertani agar containing sodium pyruvate, whereas cells of the rpoS mutants lost their culturability in only 21 days. Upon reaching unculturability the parents yielded respiring cells and cells with intact membranes for at least the next three weeks and resuscitation was allowed during this time. In contrast, the RpoS- mutant cells demonstrated intact membranes for only two weeks and a very restricted (<7 days) resuscitation capability. Guanosine 3',5'-bispyrophosphate (ppGpp) acts as a positive regulator during the production and functioning of RpoS. A mutant deficient in ppGpp production behaved like the rpoS mutants, while overproducers of ppGpp displayed a vitality at least comparable to that of RpoS+ strains. These results suggest that the E. coli parental strains enter the VBNC state which lasts for, at least, three weeks, after which apparently all the cells die. The rpoS mutants do not activate this survival strategy and early die. This implies involvement of the rpoS gene in E. coli persistence in the VBNC state.

Aims:The current standard culture methods are unable to detect nongrowing bacteria and, thus, might not be sufficient for precise monitoring of the microbiological quality of waters. The use of a molecular method such as PCR could be a valid alternative to detect bacterial faecal contamination indicators such as Escherichia coli and Enterococcus faecalis and reveal the presence of culturable and nonculturable bacterial forms. Methods and Results: The presence of E. coli and Ent. faecalis cells in 30 groundwater samples was evaluated with the standard culture method and compared with a specific PCR protocol. A substantial percentage (50%) of the samples not containing culturable cells proved positive in the search for Ent. faecalis DNA by PCR. Quantification by competitive PCR (cPCR) of the DNA detected allowed us to calculate the number of nonculturable cells present in water samples: the number varied from 2 to 120 cells ml )1 . Only four samples were positive for E. coli DNA and the corresponding nonculturable cells varied from 24 to 70 ml )1 .Conclusions: This study demonstrates that the standard culture methods in use are unable to detect a substantial proportion of the bacterial population which is nonculturable but, as previously demonstrated, potentially still viable and able to express those pathogenic factors needed for causing infections in humans.Significance and Impact of the Study: To protect human health it is necessary to develop and use methods which detect the nonculturable as well as culturable bacteria present in water.

Stressed vancomycin-resistant enterococci (VRE) can activate a survival strategy known as the viable but nonculturable (VBNC) state and are able to maintain vancomycin resistance. During restoration of division they continue to express the vancomycin resistance trait. We suggest that VBNC enterococci may constitute further reservoirs of VRE and therefore represent an additional risk for human health.