Experimental evolution reveals genetic and transcriptomic mechanisms underlying benzalkonium chloride tolerance and fluoroquinolone cross-resistance in <i>Listeria monocytogenes</i>

ABSTRACT Preventing Listeria monocytogenes persistence in food processing environments is challenged by tolerance to quaternary ammonium compound sanitizers such as benzalkonium chloride (BAC). To define the genetic and molecular routes to BAC tolerance and antibiotic cross-resistance, we performed an evolve-and-resequence experiment on three L. monocytogenes strains (three replicate lineages each) passaged under sublethal BAC for ~400–500 generations. All nine BAC-exposed lineages increased BAC minimum inhibitory concentrations (MICs), and several showed elevated tolerance to a commercial sanitizer. Notably, multiple lineages displayed cross-resistance to fluoroquinolones. Whole-genome sequencing of end-point lineages revealed recurrent, parallel mutations in the fepR transcriptional regulator (often involving nonsense mutations), accompanied by missense mutations in the adjacent efflux pump fepA , implicating derepressed multidrug and toxic compound extrusion (MATE) efflux activity in BAC, and fluoroquinolone tolerance. RNA-seq under BAC stress showed consistent upregulation of phosphotransferase system (PTS) genes across evolved lineages. Complementing this, we observed convergent missense mutations in ptsH (encoding the HPr phosphocarrier), including substitutions in the HPr serine phosphorylation region. Biophysical modeling predicted that these HPr variants are strongly destabilizing, consistent with altered PTS signaling under BAC sthe presence of BAC stress tress. Efflux inhibition with reserpine reduced BAC tolerance in fepR / fepA -mutant backgrounds, whereas the effects were minimal in a bcrABC-positive strain (where bcrABC encodes a plasmid-borne BAC resistance efflux cassette), underscoring strain-dependent mechanisms. Together, our data reveal efflux-driven and PTS-linked adaptation to BAC with implications for the emergence of antibiotic cross-resistance and persistence in food production environments. IMPORTANCE Listeria monocytogenes persistence in food facilities poses major public health and economic burdens. We modeled long-term exposure to sublethal benzalkonium chloride (BAC) to approximate conditions similar to those in food processing facility drains and uncovered two complementary routes to sanitizer tolerance: (i) repeated inactivation of the fepR regulator with accompanying alterations in the fepA efflux pump, driving BAC tolerance and fluoroquinolone cross-resistance, and (ii) convergence on mutations in ptsH (HPr) within the phosphotransferase system, supported by RNA-seq upregulation of phosphotransferase system (PTS) pathways and biophysical evidence of destabilized HPr variants. Efflux inhibition reduced BAC tolerance where fepR/fepA was mutated but had limited impact in a bcrABC-positive background (where bcrABC encodes a plasmid-borne BAC resistance efflux cassette), highlighting the role of genetic context. These findings connect sanitizer adaptation to antibiotic resistance and identify PTS signaling as a systems-level contributor, informing surveillance and sanitation strategies in food production.