Development of a real-time PCR procedure for quantification of viable Escherichia coli in populations of E. coli exposed to lactic acid, and the acid tolerance of verotoxigenic E. coli (VTEC) from cattle hides

Abstract

The aims of this study were to develop a real-time PCR procedure for determining the effects on Escherichia coli of treatment for decontaminating beef carcasses with lactic acid solution, and determining if there were differences in the acid tolerance of E. coli generally and verotoxigenic E. coli (VTEC). Suspensions of E. coli were incubated with 4% lactic acid at pH 3.6. The numbers of surviving E. coli at different incubation times were determined from plate counts and from quantification by real-time PCR of the uidA gene in DNA preparations. The numbers of viable E. coli progressively declined, by about 4 log units during incubation for 6 h. The mean cycle threshold (Ct) values for uidA in DNA from samples collected at different times and treated or not treated with propidium monoazide (PMA) before DNA extraction were similar. Treatment with 1% sodium deoxycholate (SD) before PMA treatment resulted in an increase of >6 Ct when the reduction in viable cell number was around 1 log. When E. coli incubated with 4% lactic acid solutions of pH 2.4, 2.8, 3.2 or 3.6 were resuscitated in half strength brain heart infusion (BHI) for 2 h before treatments with SD and PMA, the slope of the plot relating Ct values to the numbers of viable E. coli was 1.85 Ct log cfu−1 with the correlation coefficient (R2) being 0.80. The findings indicate that while the membranes of E. coli inactivated by 4% lactic acid were largely impermeable to PMA, the membranes of both dead and injured cells were rendered permeable to PMA by treatment with 1% SD. Resuscitation in BHI restored the membrane barrier properties of the injured cells. Treatment with lactic acid resulted in increases in Ct values of 4.1, 3.7, 2.5 and 1.5 for the uidA, stx1, stx2 and eae genes, respectively; and the increases in Ct values for the latter two genes were significantly different (p < 0.05) from that for the uidA gene. This indicates that VTEC carrying stx2 and/or eae were more acid resistant than other E. coli. Thus, caution should be exercised when using generic E. coli as an indicator for VTEC for assessment of the antimicrobial efficacy of organic acid decontaminating treatments at abattoirs.