Thin agar layer- versus most probable number-PCR to enumerate viable and stressed

A mid-log phase broth culture of Escherichia (E.) coli O157:H7 381 (final concentration 104 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
(104 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.