Thin agar layer- versus most probable number-PCR to enumerate viable and stressed Escherichia coli O157:H7 and application in a traditional raw milk pasta filata cheese.

A mid-log phase broth culture of Escherichia (E.) coli O157:H7 381 (final concentration 10(4) cfu/mL) was monitored by conventional liquid- and solid-based enumeration techniques combined with PCR while it was subjected to thermal stress in gradually more complex systems (i.e., Tryptone Soya Broth, pasteurized milk and during lab-scale productions of a pasta filata fior di latte cheese obtained from raw or pasteurized milk). Our results highlighted: i) the incapability of the selective medium, ii) the effectiveness of the thin agar layer-PCR method, and iii) the effectiveness of the most probable number (MPN)-PCR method (in comparison with both plating-based methods) in recovering and selectively counting viable and stressed or injured E. coli O157:H7. Moreover, MPN-PCR was superior to both plating-based methods in terms of speed and easiness to get results. The thermal stresses herein applied (heating at 55 °C for 5 and 8 min) were less effective on the pasteurized milk than on the Tryptone Soya Broth and the pathogen was more protected in the raw milk-based matrices than in the pasteurized ones. Moreover, given the contamination level (10(4) cfu/mL of milk) of the strain, the temperature/time of stretching and the hardening and brining conditions herein used, the complete inactivation of the pathogen is not achievable.