Nonsynonymous Mutations in Are Associated with Adaptation of Listeria monocytogenes and Other spp. to Low Concentrations of Benzalkonium Chloride but Do Not Increase Survival of L. monocytogenes and Other spp. after Exposure to Benzalkonium Chloride Concentrations Recommended for Use in Food Processing Environments.

Selection for Listeria monocytogenes strains that are tolerant to quaternary ammonium compounds (such as benzalkonium chloride [BC]) is a concern across the food industry, including in fresh produce processing environments. This study evaluated the ability of 67 strains of produce-associated L. monocytogenes and other spp. ("parent strains") to show enhanced BC tolerance after serial passaging in increasing BC concentrations and to maintain this tolerance after substreaking in the absence of BC. After serial passaging in BC, 62/67 "BC passaged cultures" showed higher MICs (4 to 20 mg/L) than parent strains (2 to 6 mg/L). After the substreaking of two isolates from BC passaged cultures for each parent strain, 105/134 "adapted isolates" maintained MICs (4 to 6 mg/L) higher than parent strain MICs. These results suggested that adapted isolates acquired heritable adaptations that confer BC tolerance. Whole-genome sequencing and Sanger sequencing of a local repressor of the MATE family efflux pump FepA, identified nonsynonymous mutations in 48/67 adapted isolates. The mean inactivation of adapted isolates after exposure to use-level concentrations of BC (300 mg/L) was 4.48 log, which was not significantly different from inactivation observed in parent strains. Serial passaging of cocultures of L. monocytogenes strains containing or did not yield adapted isolates that showed enhanced BC tolerance in comparison to that of monocultures. These results suggest that horizontal gene transfer either did not occur or did not yield isolates with enhanced BC tolerance. Overall, this study provides new insights into selection of BC tolerance among L. monocytogenes and other spp. Listeria monocytogenes tolerance to quaternary ammonium compounds has been raised as a concern with regard to L. monocytogenes persistence in food processing environments, including in fresh produce packing and processing environments. Persistence of L. monocytogenes can increase the risk of product contamination, food recalls, and foodborne illness outbreaks. Our study shows that strains of L. monocytogenes and other spp. can acquire heritable adaptations that confer enhanced tolerance to low concentrations of benzalkonium chloride, but these adaptations do not increase survival of L. monocytogenes and other spp. when exposed to concentrations of benzalkonium chloride used for food contact surface sanitation (300 mg/L). Overall, these findings suggest that the emergence of benzalkonium chloride-tolerant strains in food processing environments is of limited concern, as even strains adapted to gain higher MICs maintain full sensitivity to the concentrations of benzalkonium chloride used for food contact surface sanitation.