Modeling the effect of light and salinity on viable but non-culturable (VBNC) Enterococcus.

Enterococci have been recommended as suitable bacteria indicators for assessing the microbial quality of recreational waters. However, recent studies have shown that bacteria, including enterococci, are able to enter a viable but non-culturable (VBNC) state under environmentally stressed conditions, where they may remain undetected if culture-based methods are employed. To appreciate the extent of transformation of these cells in surface waters, a model Enterococcus organism, E. faecalis, was examined in laboratory controlled microcosms under different light and salinity conditions. Cells were detected by both standard culture-based and PMA-qPCR (propidium monoazide quantitative PCR) methods so that the VBNC cells could be enumerated. The decay rates from the culture based method (kc) and PMA-qPCR method (kp) were established for the different conditions. In general, the kC values (ranging from 0.0088 hr(-1) to 0.9755 hr(-1)) were always higher than the kP values (0.0019 hr(-1) to 0.2373 hr(-1)), implying that cells were able to retain their viability for much longer periods than what is shown by the culture-based method. In both cases, the k values generally showed an increasing trend with an increase in light irradiation, implying greater die-off with light. For freshwater microcosms, the kp values were 3-6 times lower than the kc values for different irradiation conditions, whereas for seawater the difference was up to 12 times, showing that E. faecalis adapts well to seawater. The kinetic data were used to develop models to describe the dynamics of VBNC formation in natural waters. At low light intensities (less than about 20 Wm(-2)), the proportion of VBNC cells was found to steadily increase to as high as 50%, even after 4 days. However, at higher light levels, this proportion was achieved more quickly (less than 5 h) but also diminished more rapidly. Hence, at high light levels, the percentage of VBNC cells is expected to be significant only for a few hours, whereas at low light levels, the VBNC cells can be expected to be present for a long period of time. These results have implications on the interpretation of microbial water quality data that are based on culture based methods.