Development of a Quantitative PCR Method to Differentiate Between Viable and Non-Viable Bacteria in Environmental Water Samples

Ethidium monoazide bromide (EMA) treatment of Escherichia coli O157:H7 pure cultures, primary clarifier, secondary activated, secondary trickling filter effluent, and creek water samples successfully inhibited qPCR amplification of the uidA and fliC gene targets from injured or dead E. coli. In viable pure cultures, it was determined that 7.5 μg/ml of EMA reduced qPCR detection of the 18 hr and 40 hr cultures by approximately 1 log, but a continuous culture showed no difference between treated and untreated qPCR copy numbers at high cell concentration. Amplification of heat killed bacteria decreased from 3 to 6 logs depending on EMA concentration and culture age, while the greatest inhibition was observed in the 5 hr culture using 7.5 μg/ml EMA. Treatment exposure times had little effect on culturable numbers or qPCR copy numbers regardless of culture age and sample type. Media type (LB or mTEC) had no effect on the recovery of treated culturable cells from all culture ages and sample types examined, however all samples, both treated and untreated, were sensitive to incubation temperature (data not shown). In environmental water samples, EMA treatment had little to no effect on the recovery of bacteria on media, however qPCR results were lower in the EMA treated samples indicating a small population of non-viable bacteria that were being amplified in the untreated controls. Viability of E. coli in wastewater samples using qPCR was similar to pure culture results suggesting that the addition of EMA treatment to the pre-extraction procedure could easily be incorporated into qPCR assays for bacteria. The drawbacks of this compound are the high cost, the short shelf life and the impact of culture or cell density in environmental samples; however the benefits of rapid and accurate measurement of pathogenic bacteria as well as the potential to optimize biological treatment of wastewaters offset these disadvantages.