Detection of viable but nonculturable Escherichia coli O157:H7 using propidium monoazide treatments and qPCR

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 (Delta Ct = 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 x 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.