Abstract
Conventional methods to determine the efficacy of bacteriophage (phage) for biocontrol of E. coli require 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 intact E. coli cells that survive phage exposure. Escherichia coli O157: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 cells E. coli O157:H7. Compared to PMA-qPCR, direct plating overestimated (P < 0.01) phage efficacy as cell surface-attached phage particles lysed E. coli O157: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 of E. coli O157:H7.
Highlights
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 has been recognized as a major food safety concern due to its low infectious dose and severity of disease [1]
One problem with culture-based techniques is that EHEC and some other human pathogens (e.g., Salmonella enteritidis, Vibrio cholerae, Legionella pneumophila, Listeria monocytogenes, and Campylobacter jejuni) may enter a viable but nonculturable (VBNC) physiological state in which they do not grow on media [12]
The wzx gene coding for O antigen flippase has been extensively used as a genetic target for detection or quantification of E. coli O157:H7 [36, 40, 41] as only one copy of this gene is present in the genome and standard curves for converting PCR copy number to CFU using this genetic target were available
Summary
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 has been recognized as a major food safety concern due to its low infectious dose and severity of disease [1]. Despite the best efforts of the food industry, a number of E. coli O157:H7 outbreaks associated with the consumption of contaminated food including spinach [2], ready-to-eat salad, [3] and ground beef [4] have occurred These outbreaks stress the importance of developing new, more potent strategies to mitigate this important pathogen. The VBNC state can be induced by stressful conditions such as fluctuating temperatures and oxygen levels [13] and lead to underestimation of the number of viable pathogens during plate counts Another problem for traditional plating techniques is that surviving bacteria can be killed by cell surface-attached phage particles, during the BioMed Research International incubation required for plating, leading to an overestimation of phage efficacy
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