Nonsynonymous mutations in fepR are associated with Listeria adaptation to low concentrations of benzalkonium chloride, but not increased survival in use level concentrations of benzalkonium chloride.

Presence of and selection for Listeria spp. and Listeria monocytogenes that are less effectively inactivated by quaternary ammonium compounds (such as benzalkonium chloride [BC]) is a food safety concern, including in fresh produce environments. An initial MIC assay on 67 produce-associated Listeria strains showed that strains carrying BC resistance genes bcrABC (n=10) and qacH (n=1) showed higher MIC (4-6 mg/L BC) compared to strains lacking these genes (MIC of 1-2 mg/L BC). Serial passaging experiments that exposed the 67 strains to increasing BC concentrations revealed that 62/67 strains showed growth in BC concentrations above their parent MIC (range of 4-20 mg/L). Two serially passaged isolates were obtained for each parent strain and substreaked onto BHI agar in the absence of BC for seven rounds; 105/134 substreaked isolates showed higher substreaked MIC (range of 4 - 6 mg/L) compared to parent MIC. These results suggested isolates acquired genetic adaptations that confer BC resistance. Substreaked isolates were characterized by a combination of whole genome sequencing and Sanger sequencing of fepR, a local repressor of the MATE family efflux pump FepA. These data identified nonsynonymous fepR mutations in 48/67 isolates including 24 missense, 16 nonsense, and 8 frameshift mutations. The mean inactivation of substreaked 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 passage experiments performed on cocultures of Listeria strains containing bcrABC or qacH did not yield growth at higher BC concentrations than monoculture experiments. IMPORTANCEListeria resistance to quaternary ammonium compounds has been raised as a concern with regards to Listeria persistence in food environments, which can increase risk of product contamination, food recalls and foodborne illness outbreaks. Findings from our study show that individual Listeria strains can acquire genetic adaptations that confer resistance to low concentrations of benzalkonium chloride, but these genetic adaptations dont increase Listeria survival when exposed to concentrations of benzalkonium chloride used for food contact surface sanitation (300 mg/L). Our study also suggests that there is limited risk of benzalkonium chloride resistance genes bcrABC and qacH spreading through horizontal gene transfer and conferring enhanced resistance of L. monocytogenes to benzalkonium chloride. Overall, our study suggests that emergence of benzalkonium chloride resistant Listeria strains in food processing environments is of limited concern, as even strains adapted to gain higher MIC in vitro maintain full sensitivity to use level concentrations of benzalkonium chloride.