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.

Assessment of the Microbiological Quality of Ready-to-Eat Salads—Are There Any Reasons for Concern about Public Health?

“…Ready-to-eat salads could show the presence of various microbial pathogens including Escherichia coli, Staphylococcus aureus, Salmonella spp., Listeria monocytogenes, Campylobacter jejuni, Clostridium perfringens, total aerobic and spoilage bacteria, yeasts, and molds, which are concerned with serious threats [14,15]. In addition, a high number of microorganisms are also becoming a reservoir of antibiotic resistance genes, which has now become a global problem [16,17].…”

Microbiological Assessment of Various Ready-To-Eat Foods in Cairo, Egypt, and Studying the Possible Antibacterial Effects of Garlic and Cumin Oils as Food Additives

“…In the current study, it is reported that the prevalence rates of Enterobacteriaceae, Campylobacter, Aeromonas, Pseudomonas, S. aureus, coagulasenegative staphylococci (CNS), and Listeria in RTE foods were 67.26%, 0.00%, 5.36%, 1.19%, 9.82%, 11.90%, and 9.52%, respectively. In addition, RTE foods showed the existence of multiple foodborne pathogens including S. aureus, Campylobacter jejuni, Listeria monocytogenes, Clostridium perfringens, Salmonella species, Escherichia coli, spoilage bacteria, yeasts, molds, and total aerobic where all are associated with serious public health FBDs [32,33]. A high prevalence of S. aureus (12.5%) and Listeria monocytogenes (20%) were detected in desserts containing dairy cream.…”

The Viable But Non-Culturable State of Listeria monocytogenes in the One-Health Continuum

“…Exposure to light crosslinks the dye to the DNA and blocks the PCR reaction (generally in qPCR). Desneux et al, 2016;Overney et al, 2016;Overney et al, 2017;Brauge et al, 2020;Gu et al, 2020;Truchado et al, 2020;Bernardo et al, 2021 Metataxonomics 16S-rRNA 16S rRNA gene sequencing This method is based on the sequencing of the 16S rRNA gene, which is found in all bacteria and archaea. 16S rRNA gene sequencing is commonly used for identification, classification and quantification of microbes within microbial communities.…”

“…With a DVC method coupled with FISH using a 16S rRNA probe (Table 1), Moreno et al (2012) suggested the existence of VBNC Lm in RTE vegetable samples (Moreno et al, 2012). Recently, a study using PMA-qPCR provided strong arguments for the existence of VBNC Lm in RTE salads (Bernardo et al, 2021). Sources of product contamination can come directly from surfaces in the production environment.…”