Monitoring Sublethal Injury in

Listeria monocytogenes is a foodborne pathogen capable of contaminating ready-to-eat meat products, e.g., frankfurters. Post-packaging mild heat treatment via water immersion is commonly employed; however, this may be sublethal to cells located in protected niches or beneath the product surface. The objectives of this study were to evaluate thermal injury of L. monocytogenes on frankfurters at single-cell versus population level and to comparatively estimate pathogens' physiological status. Pork frankfurter-type sausages were inoculated (ca. 7.0-7.5 log CFU/cm2) with L. monocytogenes strain EGDE-e. Heat treatment was performed at 61 °C (max. 60 min) and 64 °C (max. 12 min). To determine the injured subpopulation from the total, tryptic soy agar with 0.6% yeast extract (TSAYE), supplemented or not with 5% NaCl, was used. Plating-based quantification of injured cells was compared to CFDA/PIstained cells analysed by fluorescence microscopy and quantified with Fiji software. Injury was recorded mainly after 2 and 4 min at 64 °C, whereas no injury was detected at 61 °C, at population level. Following exposure to 61 °C for 60 min, culturable cells dropped below the enumeration limit (0.3 log CFU/cm2), while a considerable number of CFDA+/PI- and CFDA+/PI+ cells indicated viable-but-non-culturable induction and sublethal injury, respectively. These findings suggest that non-culturability may limit the accuracy of solely culture-based enumeration methods.