Listeria monocytogenes Assessment in a Ready-to-Eat Salad Shelf-Life Study Using Conventional Culture-Based Methods, Genetic Profiling, and Propidium Monoazide Quantitative PCR
Rita Bernardo, Ana Duarte, Luís Tavares, A.S. Barreto, Ana Rita Henriques
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.