Novel approach towards the quantification of viable Shiga toxin-producing Escherichia coli in beef products

REY, M.A.; CAP, M.; VAUDAGNA S.R.; MOZGOVOJ, M.

Sorrento

Workshop; 2018 IFT-EFFoST International Nonthermal Processing Workshop and Short Course; 2018

ProdAl Scarl y Universidad de Salerno, Italia

Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen associated with beef products. Several strategies for inactivation of STEC cells have been studied. PCR-based techniques that allow quantification and differentiation between viable and non-viable cells after treatments are promising alternatives to traditional culture-based methods. PMA (propidium monoazide) is a nucleic acid-intercalating dye used as real time PCR pre-treatment and can be effectively used for this purpose as it only penetrates damaged membranes of dead cells and inhibits PCR amplification.The aim of this study was to optimize PMA coupled with real time PCR (PMA-qPCR) for quantification STEC viable cells in beef products treated by applying non-thermal technologies (high pressure, sonication, cold plasma).The sample consisted of 10 g of minced beef inoculated with STEC cells as follows: viable cells (109 CFU/ml), dead cells (109 CFU/ml) and 3 different concentrations of viable cells (109, 108 and 107 CFU/ml) mixed with a single concentration of dead cells (109 CFU/ml). Homogenates were treated with 3 different concentrations of PMA: 50, 75 y 100 µM, followed by a photo activation procedure for 15 min. Finally, DNA was isolated and a stx2 amplification was performed.Samples inoculated with only dead cells showed an increase of 10 units in Ct (Cycle Threshold) value after the PMA treatment. Samples inoculated with the mixture of dead and viable cells showed an increase of 3 units in Ct value following the PMA treatment, at all of the concentrations analyzed. Samples inoculated only with viable cells, showed similar Ct values following the PMA treatment. No significant differences were observed among PMA concentrations. As the PMA treatment was not able to completely inhibit stx2 amplification from dead cells, a new set of experiments was performed with 104 and 105 CFU/ml of dead cells. At both concentrations inhibition was complete and will be used for further experiments. This novel methodology would be of great value to quantify and to differentiate viable and non-viable STEC cells in beef products treated by non-thermal technologies.