Rapid and Accurate Detection of Bacteriophage Activity againstEscherichia coliO157:H7 by Propidium Monoazide Real-Time PCR

Abstract

Conventional methods to determine the efficacy of bacteriophage (phage) for biocontrol ofE. colirequire several days, due to the need to culture bacteria. Furthermore, cell surface-attached phage particles may lyse bacterial cells during experiments, leading to an overestimation of phage activity. DNA-based real-time quantitative polymerase chain reaction (qPCR) is a fast, sensitive, and highly specific means of enumerating pathogens. However, qPCR may underestimate phage activity due to its inability to distinguish viable from nonviable cells. In this study, we evaluated the suitability of propidium monoazide (PMA), a microbial membrane-impermeable dye that inhibits amplification of extracellular DNA and DNA within dead or membrane-compromised cells as a means of using qPCR to identify only intactE. colicells that survive phage exposure.Escherichia coliO157:H7 strain R508N and 4 phages (T5-like, T1-like, T4-like, and O1-like) were studied. Results compared PMA-qPCR and direct plating and confirmed that PMA could successfully inhibit amplification of DNA from compromised/damaged cellsE. coliO157:H7. Compared to PMA-qPCR, direct plating overestimated (P< 0.01) phage efficacy as cell surface-attached phage particles lysedE. coliO157:H7 during the plating process. Treatment of samples with PMA in combination with qPCR can therefore be considered beneficial when assessing the efficacy of bacteriophage for biocontrol ofE. coliO157:H7.