Liu Hui, Niu Yan D, Li Jinquan, Stanford Kim, McAllister Tim A
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.
Alberta Agriculture and Rural Development, Agriculture Centre, Lethbridge, AB, Canada T1J 4V6.
Biomed Res Int. 2014;2014:319351. doi: 10.1155/2014/319351. Epub 2014 Nov 2.
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.
由于需要培养细菌，传统方法测定噬菌体对大肠杆菌进行生物防治的效果需要数天时间。此外，细胞表面附着的噬菌体颗粒在实验过程中可能会裂解细菌细胞，导致对噬菌体活性的高估。基于DNA的实时定量聚合酶链反应（qPCR）是一种快速、灵敏且高度特异的病原体计数方法。然而，由于qPCR无法区分活细胞和死细胞，可能会低估噬菌体活性。在本研究中，我们评估了单叠氮化丙锭（PMA）的适用性，PMA是一种微生物膜不可渗透的染料，可抑制细胞外DNA以及死亡或膜受损细胞内DNA的扩增，作为一种利用qPCR仅鉴定在噬菌体暴露后存活的完整大肠杆菌细胞的方法。我们研究了大肠杆菌O157:H7菌株R508N和4种噬菌体（T5样、T1样、T4样和O1样）。结果比较了PMA-qPCR和直接平板计数法，证实PMA可以成功抑制受损/破坏的大肠杆菌O157:H7细胞DNA的扩增。与PMA-qPCR相比，直接平板计数法高估了（P < 0.01）噬菌体的功效，因为细胞表面附着的噬菌体颗粒在平板计数过程中裂解了大肠杆菌O157:H7。因此，在评估噬菌体对大肠杆菌O157:H7进行生物防治的效果时，将PMA与qPCR结合处理样品可能是有益的。
