Monitoring Sublethal Injury in Listeria monocytogenes During Heat Treatment of Pork Frankfurter-Type Sausages: A Single-Cell vs. Population Level Approach

<i>Listeria monocytogenes</i> 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 <i>L. monocytogenes</i> on frankfurters at single-cell versus population level and to comparatively estimate pathogens' physiological status. Pork frankfurter-type sausages were inoculated (<i>ca</i>. 7.0-7.5 log CFU/cm²) with <i>L. monocytogenes</i> strain EGDE-e. Heat treatment was performed at 61 °C (<i>max</i>. 60 min) and 64 °C (<i>max</i>. 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.