NONCULTURABLE ENTEROCOCCUS-FAECALIS CELLS ARE METABOLICALLY ACTIVE AND CAPABLE OF RESUMING ACTIVE GROWTH

Entry into the viable but nonculturable (VNC) state is a survival mech anism that bacteria can adopt when they find themselves in an adverse environment. When in this state, bacteria are still viable but are una ble to form colonies on growth medium. The possibility of Gram-positiv e species entering the VNC state when environmental conditions are adv erse and remaining viable and capable of resuming active growth is dem onstrated for the first time in this study by using exponential-phase cultures of Enterococcus faecalis inoculated in filtered, sterilized w ater from Lake Garda (Italy). Over the 60-day study, the number of tot al cells stained with a fluorescent stain or counted with a Coulter Co unter remained constant, while the number of cells capable of forming colonies on Tryptic Soy Agar (TSA) declined rapidly from 10(6) CFU/ml on day zero to 10(3) CFU/ml on day 4. On day 14 no colonies could be o bserved when 50 ml of inoculated lake water were plated. E. faecalis c ells conserved their viability while in the VNC state, as can be demon strated by active uptake of amino acids, which are also incorporated i nto proteins, and by continuous detection of E. faecalis specific DNA by PCR throughout the experiment. The possibility of revival of the E. faecalis cells in the VNC state when returned to conditions supportin g its cell growth has also been demonstrated. The data obtained in thi s study lend further support to recent criticisms of the traditional m ethods used to evaluate water quality based on plate counts, assessing fecal contamination indicators such as Escherichia coli and fecal str eptococci.