Competitive polymerase chain reaction for quantification of nonculturable Enterococcus faecalis cells in lake water

Among the survival strategies developed by bacteria when faced with adverse environmental conditions, the viable but nonculturable (VNC) state has bee n described. In this state, bacteria are unable to form colonies but are st ill alive and capable of metabolic activity. The VNC state has been describ ed in numerous Gram-negative species, but recently also in Enterococcus fae calis, a Cram-positive species which can be found in the environment. In th is study we describe a competitive PCR (cPCR) protocol to detect and quanti fy a specific sequence of DNA from culturable and nonculturable E. faecalis cells present in water samples. The protocol was found to be specific and capable of detecting amounts of DNA up to 0.1 pg corresponding to approxima tely 2 cells ml(-1). Moreover, it allows an internal standard to be used to quantify the amount of specific DNA present in samples from different envi ronments. The application of this cPCR method to water samples from Lake Ga rda enabled us to demonstrate the presence of nonculturable forms of E. fae calis in lake water and to quantify their DNA and the corresponding concent ration of nonculturable cells. (C) 1999 Federation of European Microbiologi cal Societies. Published by Elsevier Science B.V. All rights reserved.