Evolved populations of Listeria monocytogenes related to biofilm formation and biocide stress in the context of food production environment niches

Cleaning and disinfection of food production environments (FPE) are fundamental components of food safety programmes designed to control microbial pathogens and prevent food contamination. Yet, FPE can still harbour foodborne pathogens, including <i>Listeria monocytogenes,</i> a significant concern to food manufacturers and health authorities due to the high mortality rate associated with invasive listeriosis. Mechanisms contributing to <i>L. monocytogenes</i> persistence in FPE include biofilm formation and reduced susceptibility to biocides, such as benzalkonium chloride (BC), for which several mechanisms are known. We hypothesized that prolonged exposure to disinfectants and other FPE-associated stressors would drive <i>L. monocytogenes</i> adaptation, resulting in the accumulation of genetic mutations linked to biofilm formation and reduced biocide susceptibility. To test this, we developed a biofilm persistence model, which studied 30 consecutive passages of biofilm-associated cells grown on stainless steel under sub-inhibitory BC concentrations. Whole-genome sequencing of evolved populations identified mutations that were associated with biofilm lineages and/or BC exposure. Non-synonymous mutations were identified in genes and pathways involved in metal homeostasis, stress response and pyrimidine biosynthesis. In addition, reduced susceptibility to BC arose through multiple independent mutations within the <i>fepRA</i> operon, encoding FepR transcriptional repressor and FepA MATE efflux pump. These mutations were observed across both planktonic and biofilm lifestyles, resulting in a comparable level of reduced susceptibility to BC in both states. Several loci with fixed mutations associated with biofilm lineages were identified, including the <i>ykoK</i> riboswitch leader, the pyrimidine synthesis operon and the stress response-related gene <i>rsbU</i>. Collectively, these findings provide new insights into the genetic mechanisms underlying <i>L. monocytogenes</i> biofilm persistence and reduced biocide susceptibility in the context of FPE and reveal novel targets potentially exploited by <i>L. monocytogenes</i> to establish and maintain niches in unfavourable environments.