Listeria monocytogenes Assessment in a Ready-to-Eat Salad Shelf-Life Study Using Conventional Culture-Based Methods, Genetic Profiling, and Propidium Monoazide Quantitative PCR

Abstract

Listeriosis is almost entirely transmitted through foods contaminated with Listeria monocytogenes. Ready-to-eat foods present a particular challenge due to their long refrigerated shelf-life, not requiring any heat treatment before consumption. In this work, a shelf-life assessment of an industrially produced ready-to-eat salad was performed using conventional culture-based and molecular methods. L. monocytogenes isolates were confirmed and serogrouped using multiplex PCR, and genetic subtyping was performed by pulsed-field gel electrophoresis (PFGE). PMAxx-qPCR was used as an alternative method for L. monocytogenes quantification in foods. Salad samples were kept at 4 °C, 12 °C, and 16 °C for eight days and analysed. At 4 °C, acceptable results were obtained considering hygiene indicators, i.e., Enterobacteriaceae (ranging from 3.55 ± 0.15 log cfu/g to 5.39 ± 0.21 log cfu/g) and aerobic mesophilic colony counts (5.91 ± 0.90 log cfu/g to 9.41 ± 0.58 log cfu/g) throughout the study, but the same did not happen at 12 °C and 16 °C. L. monocytogenes culture-based quantification exhibited low numbers (<1 log cfu/g) for all temperatures. From 30 presumptive isolates, 10 (33.3%) were confirmed as L. monocytogenes with the majority belonging to serogroup IVb. PFGE subtyping showed that 7 of the 10 L. monocytogenes isolates had 100% of pulsotype similarity, suggesting a possible common contamination source. PMAxx-qPCR revealed a statistically higher L. monocytogenes quantification (>3 log cfu/g) when compared to the conventional culture-based method, suggesting viable but non-culturable forms. Taken together, results underline the need to combine conventional methods with more sensitive, specific, and rapid ones for L. monocytogenes assessment in ready-to-eat foods shelf-life studies to reduce the potential risk for consumers.