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.