Listeria sanitizer tolerance at use-level concentrations shows limited association with genetic loci

The ability of Listeria to show reduced susceptibility to sanitizers commonly used in fresh produce packing and processing environments continues to be mentioned as a concern. We assessed the survival of 501 produce-associated Listeria isolates (328 Listeria monocytogenes [LM] and 173 Listeria spp. [LS]) after 30 s of exposure to benzalkonium chloride (BC, 300 ppm) and peroxyacetic acid (PAA, 80 ppm). A subset of 108 isolates was also exposed to sodium hypochlorite (NaOCl, 500 ppm) for 30 s. Isolates showed a range of log reductions, including 2.76-5.73 log for BC, 0.15-6.16 log for PAA, and 1.34-7.02 log for NaOCl; the variance of log reductions was significantly lower for BC compared to PAA and NaOCl. Cluster analysis on log reduction data identified four clusters, including one cluster of five LM isolates that showed reduced susceptibility to all three sanitizers. Log reductions of LS were significantly lower than LM after exposure to PAA, indicating reduced PAA susceptibility among LS. Whole genome sequence (WGS)-based characterization of all isolates revealed that the presence of known BC resistance genes (i.e., bcrABC, mdrL, and sugE1/2) was not significantly associated with log reductions to BC, and the presence of stress survival islet SSI-2 was not significantly associated with log reductions to PAA and NaOCl. Genome-wide association studies did not reveal any association of pangenome genes with phenotypic sanitizer susceptibility but identified several SNPs in core genes as associated with sanitizer susceptibility.IMPORTANCEDespite frequently stated concerns about LM and LS with reduced susceptibility to sanitizers (which could facilitate persistence and increase risk of product contamination), there are limited data available on Listeria susceptibility to sanitizers used in produce packing and processing environments at their recommended use-level concentrations. Importantly, our data showed that reduced sanitizer susceptibility of Listeria is not linked to the presence of any previously reported sanitizer resistance genes. However, we identified a group of five LM isolates that showed reduced susceptibility to all three sanitizers tested; these isolates represented lineages I, II, and III. Combined, these data suggest that there are no distinct "sanitizer-resistant" clonal Listeria groups and that WGS data may not be particularly valuable for predicting sanitizer susceptibility at use-level concentrations. Moreover, the high variability of log reductions observed across all three sanitizers highlights the importance of considering log reduction variability, in addition to average log reduction, when assessing different sanitizers.