Research Center of Food Safety and Detection, College of Light Industry and Food Sciences, South China University of Technology, Wusan Road 381, Tianhe District, Guangzhou, 510640, People's Republic of China.
Can J Microbiol. 2013 Mar;59(3):157-63. doi: 10.1139/cjm-2012-0577. Epub 2012 Dec 10.
Escherichia coli O157:H7 can enter into a viable but nonculturable (VBNC) state under stress conditions. The aims of the present study were to examine the influences of environmental factors on the survivability and culturability of E. coli O157:H7 and to develop an approach for accurate detection of VBNC E. coli O157:H7. The E. coli O157:H7 strain ATCC 6589 was inoculated into 3 induction microcosm models: (i) Luria-Bertani broth, (ii) sterilized tap water, and (iii) sterilized physiological saline solution. Our results showed that low temperature and nutritional starvation significantly impacted on the survivability of E. coli O157:H7 cells and that the in-vitro-induced VBNC cells were capable of resuscitating under normal temperature and appropriate nutrients. We tested the effectiveness of an approach combining propidium monoazide (PMA) treatment with real-time polymerase chain reaction (PMA-qPCR) for accurate quantification of total, viable, dead, and VBNC cells under different induction microcosm models. Our results indicated different threshold cycle (Ct) values for PMA-treated cells and untreated cells (ΔCt = 4.97, 4.29, and 3.30 for Luria-Bertani broth, sterilized tap water, and sterilized physiological saline solution, respectively). We determined the quantification limit of this PMA-qPCR approach to be 1 × 10(2) cells·mL(-1), providing sufficient sensitivity for detection of VBNC E. coli O157:H7 cells to no less than 100 cells·mL(-1). This study clearly demonstrated the feasibility and effectiveness of using PMA-qPCR to accurately quantify E. coli O157:H7 in a VBNC state.
大肠杆菌 O157:H7 在应激条件下可进入存活但非可培养（VBNC）状态。本研究旨在研究环境因素对大肠杆菌 O157:H7 存活和可培养性的影响，并开发一种准确检测 VBNC 大肠杆菌 O157:H7 的方法。将大肠杆菌 O157:H7 菌株 ATCC 6589 接种到 3 种诱导微宇宙模型中：（i）Luria-Bertani 肉汤，（ii）消毒自来水和（iii）消毒生理盐水。我们的结果表明，低温和营养饥饿对大肠杆菌 O157:H7 细胞的存活有显著影响，体外诱导的 VBNC 细胞在正常温度和适当营养条件下能够复苏。我们测试了结合吖啶橙单加合物（PMA）处理和实时聚合酶链反应（PMA-qPCR）的方法，以在不同诱导微宇宙模型中准确定量总细胞、活细胞、死细胞和 VBNC 细胞的有效性。我们的结果表明，PMA 处理细胞和未处理细胞的阈值循环（Ct）值不同（Luria-Bertani 肉汤、消毒自来水和消毒生理盐水分别为 4.97、4.29 和 3.30）。我们确定了该 PMA-qPCR 方法的定量下限为 1×10(2)细胞·mL(-1)，为检测 VBNC 大肠杆菌 O157:H7 细胞提供了足够的灵敏度，最低可达 100 细胞·mL(-1)。本研究清楚地表明，使用 PMA-qPCR 准确定量 VBNC 状态下的大肠杆菌 O157:H7 是可行和有效的。
