Inhibition of the resuscitation from the viable but non-culturable state in Enterococcus faecalis.

The viable but non-culturable (VBNC) state is a survival strategy adopted by bacteria when exposed to environmental stresses capable of inducing cell growth inhibition and cell death. This state can be summarized as a quiescent form of life waiting for suitable conditions. This strategy has been shown to be activated by medically important bacteria either when present in natural environments or in the human body during the infection process. In this study we have evaluated the effects of antibiotics acting on peptidoglycan or protein synthesis of Enterococcus faecalis in the VBNC state. The activity of the antibiotics was determined by their ability both to inhibit resuscitation (i.e. recovery of cell division) and to bind the molecular target of action. Benzylpenicillin, piperacillin and gentamicin block cell resuscitation at the minimal inhibitory concentrations (MICs) of growing cells, while vancomycin acts only at doses 500 times higher than the MIC. This different behaviour is discussed taking into consideration the mode of action of the antibiotics.

Survival of enterococcal species in aquatic environments.

Analysis of the survival ability of faecal streptococci/enterococci in the environment has almost invariably been conducted using the standard culture method (CFU counts) despite the demonstration that these microorganisms are capable of entering a viable but nonculturable (VBNC) state. In this study we evaluated the fate, in terms of culturability and viability, of different enterococcal species under laboratory stress conditions mimicking those of the aquatic environment. The results indicate that enterococcal species may activate two different survival strategies, namely starvation and the VBNC state, depending on the specific environmental condition. Moreover, the different enterococcal species can be divided into three groups on the basis of the time needed to activate the VBNC state and the resuscitation capability. The differences in activation of the two survival strategies and the different kinetics observed among the enterococcal species reaching the VBNC state should be taken into consideration when the microbiological quality of waters has to be evaluated and because of their role as faecal contamination indicators.

Adhesion of Enterococcus faecalis in the nonculturable state to plankton is the main mechanism responsible for persistence of this bacterium in both lake and seawater.

The presence of enterococci in lake and seawater in an 18-month survey comparing molecular (PCR and quantitative PCR) and culture methods was evaluated, as well as the possibility that zooplankton could act as reservoirs for enterococci. Samples of both water and zooplankton were collected monthly from a Lake Garda site and an Adriatic Sea site. In lake water, the positive samples numbered 13 of 54 (24%) by culture and 32 of 54 (59%) when PCR was applied. In seawater, they numbered 0 of 51 by culture and 18 of 51 (35%) by PCR. Enterococci were found either totally bound to plankton or totally in water, depending on the presence or absence of plankton, respectively. These results clearly indicate that the PCR assay is a powerful tool for detecting fecal indicators and pathogens in the environment, thus providing a much more sensitive method than culture.