Efficacy of Propidium Monoazide on Quantitative Real-Time PCR–Based Enumeration of Staphylococcus aureus Live Cells Treated with Various Sanitizers

Abstract

ABSTRACT Propidium monoazide (PMA) has been used together with quantitative real-time PCR (qPCR) to enumerate live bacteria, while discriminating against the residual DNA of dead bacterial cells. Although the effectiveness of PMA at increasing the accuracy of enumeration of live bacteria treated with heat has been investigated in a number of studies, few studies have involved bacteria treated with sanitizers. In this study, dead Staphylococcus aureus cells were prepared by treatment with six kinds of sanitizers (ethanol, isopropyl alcohol, benzalkonium chloride, sodium hypochlorite, hydrogen peroxide, and nisin) and were mixed with a culture of live bacteria in different ratios. PMA-qPCR was able to accurately enumerate live bacteria with a <0.5 CFU/500 μL difference with that of plate counts for cultures treated with ethanol, isopropyl alcohol, and nisin. For ethanol and isopropyl alcohol treatments, live cells were accurately enumerated for live/dead cell ratios of 10/1 to 0.01/1, while live cells for the nisin treatment were accurately enumerated for live/dead cell ratios of 10/1 to 0.1/1. In contrast, PMA-qPCR was not able to accurately enumerate live cells in bacterial cultures treated with benzalkonium chloride and hydrogen peroxide. In addition, qPCR without PMA was able to enumerate live cells as consistently as plate counts with a bacterial culture treated with sodium hypochlorite. The results of this study show that the use of PMA for qPCR-based enumeration of live cells is not always recommended, and its effectiveness depends on the treatment used on the cells.