Characterization of Listeria monocytogenes enhanced cold-tolerance variants isolated during prolonged cold storage

In this study, we show that growth and prolonged storage of Listeria monocytogenes at 4 °C can promote the selection of variants with enhanced cold and heat tolerance. Enhanced cold-tolerance (ECT) variants (n = 12) were successfully isolated from a strain with impaired cold growth abilities following 84 days of storage at 4 °C in brain heart infusion broth (BHIB). Whole genome sequencing, membrane fatty acid analysis, and stress tolerance profiling were performed on the parent strain and two ECT variants: one displaying regular-sized colonies and the other displaying small colonies when grown at 37 °C on BHI agar. Under cold stress conditions, the parent strain exhibited an impaired ability to produce branched-chain fatty acids which are known to be important for cold adaptation in L. monocytogenes. The ECT variants were able to overcome this limitation, a finding which is hypothesized to be associated with the identification of two independent single-nucleotide polymorphisms in genes encoding subunits of acetyl-coA carboxylase, an enzyme critical for fatty acid biosynthesis. While the ECT phenotype was not found to be associated with improved salt (BHIB + 6% NaCl, 25 °C), acid (BHIB pH 5, 25 °C) or desiccation (33% RH, 20 °C) tolerance, the small-colony variant exhibited significantly (p < 0.05) enhanced heat tolerance at 52 °C in buffered peptone water compared to the parent strain and the other variant. The results from this study demonstrate that the continuous use of refrigeration along the food-supply chain has the potential to select for L. monocytogenes variants with enhanced cold and heat tolerance, highlighting the impact that microbial intervention strategies can have on the evolution of bacterial strains and likewise, food safety.