title: Rapid and accurate detection of bacteriophage activity against Escherichia coli O157:H7 by propidium monoazide real-time PCR.
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.
authors.affiliation: College of Animal Science, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia 010018, China ; Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, AB, Canada T1J 4B1.
authors.forename: Hui
authors.identifier: 0000-0002-1738-8537
authors.initials: H
authors.lastname: Liu
authors.affiliation: Alberta Agriculture and Rural Development, Agriculture Centre, Lethbridge, AB, Canada T1J 4V6.
authors.forename: Yan D
authors.initials: YD
authors.lastname: Niu
authors.affiliation: College of Animal Science, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia 010018, China.
authors.forename: Jinquan
authors.initials: J
authors.lastname: Li
authors.affiliation: Alberta Agriculture and Rural Development, Agriculture Centre, Lethbridge, AB, Canada T1J 4V6.
authors.forename: Kim
authors.identifier: 0000-0001-8784-0042
authors.initials: K
authors.lastname: Stanford
authors.affiliation: Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, AB, Canada T1J 4B1.
authors.forename: Tim A
authors.initials: TA
authors.lastname: McAllister
mesh_terms: D001386:Azides / Q000494:pharmacology*; D001435:Bacteriophages / Q000235:genetics*; D004269:DNA, Bacterial / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D019453:Escherichia coli O157 / Q000187:drug effects / Q000235:genetics / Q000472:pathogenicity; D005516:Food Microbiology; D006801:Humans; D011419:Propidium / Q000031:analogs & derivatives* / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D001386:Azides; D004269:DNA, Bacterial; C533957:propidium monoazide; D011419:Propidium