A continuous biofilm evolution model identified mechanisms of persistence and niche adaptation of Listeria monocytogenes.

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 Listeria monocytogenes, a significant concern to food manufacturers and health authorities due to the high mortality rate associated with invasive listeriosis. Mechanisms contributing to L. monocytogenes persistence in FPE include biofilm formation and increased tolerance to biocides, such as benzalkonium chloride (BC), for which several tolerance mechanisms are known. We hypothesised that prolonged exposure to disinfectants and other FPE-associated stressors would drive L. monocytogenes adaptation, resulting in the accumulation of genetic mutations linked to biofilm formation and biocide tolerance. To test this, we developed a biofilm persistence model, which studied 30 consecutive passages of biofilm-associated cells grown on stainless steel under subinhibitory BC concentrations. Whole-genome sequencing of evolved populations identified mechanisms of persistence and niche adaptation. Non-synonymous mutations were identified in genes and pathways involved in metal homeostasis, stress response and pyrimidine biosynthesis. In addition, tolerance to BC arose through multiple independent mutations within the fepRA 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 tolerance to BC in both states. Fixed mutations associated with biofilm lineages identified several loci involved in adaptation - including the ykoK riboswitch leader, the pyrimidine synthesis operon, and the stress response-related gene rsbU. Collectively, these findings provide new insights into the genetic mechanisms underlying L. monocytogenes biofilm persistence and biocide tolerance in the context of FPE, and reveal novel targets potentially exploited by L. monocytogenes to establish and maintain niches in unfavourable environments.