Abstract
Ethidium monoazide (EMA) quantitative polymerase chain reaction (qPCR), propidium monoazide (PMA)-qPCR and DNase treatment in combination with qPCR were compared for the determination of microbial cell viability. Additionally, varying EMA and PMA concentrations were analysed to determine which dye and concentration allowed for the optimal identification of viable cells. Viable, heat treated (70 °C for 15 min) and autoclaved cultures of Legionella pneumophila, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus and Enterococcus faecalis were utilised in the respective viability assays. Analysis of the viable and heat-treated samples indicated that variable log reductions were recorded for both EMA [log reductions ranging from 0.01 to 2.71 (viable) and 0.27 to 2.85 (heat treated)], PMA [log reductions ranging from 0.06 to 1.02 (viable) and 0.62 to 2.46 (heat treated)] and DNase treatment [log reductions ranging from 0.06 to 0.82 (viable) and 0.70 to 2.91 (heat treated)], in comparison to the no viability treatment controls. Based on the results obtained, 6 μM EMA and 50 μM PMA were identified as the optimal dye concentrations as low log reductions were recorded (viable and heat-treated samples) in comparison to the no viability treatment control. In addition, the results recorded for the 6 μM EMA concentration were comparable to the results obtained for both the 50 μM PMA and the DNase treatment. The use of EMA-qPCR (6 μM) may therefore allow for the rapid identification and quantification of multiple intact opportunistic pathogens in water sources, which would benefit routine water quality monitoring following disinfection treatment.
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This study was funded by the Water Research Commission under Grant K5/2368//3 (WRC project) and the National Research Foundation of South Africa under Grant 90320–opinions expressed and conclusions arrived at, are those of the authors and not necessarily to be attributed to the National Research Foundation (South Africa).
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Conceived and designed the experiments: B.R. and W.K. Performed the experiments: B.R. Analysed the data: B.R. and W.K. Contributed reagents/materials/analysis tools: S.K. and W.K. Compiled the manuscript: B.R., T.N., S.K. and W.K.
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Reyneke, B., Ndlovu, T., Khan, S. et al. Comparison of EMA-, PMA- and DNase qPCR for the determination of microbial cell viability. Appl Microbiol Biotechnol 101, 7371–7383 (2017). https://doi.org/10.1007/s00253-017-8471-6
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DOI: https://doi.org/10.1007/s00253-017-8471-6