Development of a quantitative PCR method to differentiate between viable and nonviable bacteria in environmental water samples

Ethidium monoazide bromide (EMA) treatment of pure culture and environmental waters at low concentrations (1.0-7.5 A mu g/ml) indicated effective enumeration of viable and viable but nonculturable Escherichia coli in pure cultures, creek waters, and secondary activated sludge effluent samples by quantitative polymerase chain reaction (qPCR) amplification of the uidA and fliC gene targets at turbidity values < 10 NTU. However, EMA treatment was not effective in primary clarifier and secondary trickling filter effluents where turbidities were a parts per thousand yen10 NTU. In viable pure cultures, rapidly dividing and senescent cells were most affected by increasing EMA concentrations. Amplification of heat-killed pure bacterial cultures decreased 4 to 6 logs depending on EMA concentration and culture age. The greatest difference was observed in 5-h cultures using 7.5 mu g/ml EMA. Turbidity (a parts per thousand yen100 NTU) in environmental samples inhibited EMA effectiveness on viability discrimination. Enumeration of E. coli in certain wastewaters using EMA-qPCR was similar to culture suggesting that EMA treatment could be incorporated into qPCR assays for the quantification of viable bacteria increasing assay time no more than 30 min. Our results indicate that EMA can be used in routine qPCR assays, but optimum conditions for exposure must be identified for each sample type due to sample matrix effects such as turbidity.