Use of propidium monoazide and quantitative PCR for differentiation of viable Escherichia coli from E. coli killed by mild or pasteurizing heat treatments

Abstract

Suspensions of Escherichia coli in peptone water were heated at temperatures between 52 and 90 °C, inclusive. Samples withdrawn at suitable times were not or were treated with propidium monoazide (PMA) or deoxycholate then PMA before extraction of DNA. DNA was quantified by real-time PCR for estimation of the numbers of E. coli from which template DNA for the PCR was obtained. Numbers of viable E. coli in suspensions at the times of sampling were determined from plate counts. For samples from suspensions heated at temperatures ≥52 ≤ 72 °C, PCR cycle threshold (Ct) values were little or no different for DNA from corresponding samples that were or were not treated with PMA. PMA treatment of samples heated to ≥80 °C largely inactivated E. coli DNA for PCR. When samples heated to ≤72 °C were treated with deoxycholate before treatment with PMA, Ct values for treated samples were greater than the Ct values for the corresponding untreated samples. Similar results were obtained with E. coli suspended in milk or fluid from ground beef pummeled with diluent. The results indicate that cells killed by heating to ≥80 °C are permeable to PMA, but most cells killed by heating to ≤72 °C are not. However, treatment with deoxycholate renders a substantial fraction of the latter cells permeable to PMA. Numbers of viable or dead E. coli can then be estimated from Ct values for samples not treated or treated with deoxycholate and PMA, provided viable cells are ≥1% of the total.