Fluoroquinolone susceptibility of wild-type Listeria monocytogenes isolates and the role of FepR and ParC mutations in conferring fluoroquinolone tolerance

Fluoroquinolone response and associated genotypic variants were characterized in a set of 88 Listeria monocytogenes strains isolated from Canadian dairy facilities over a span of 10 years. All strains were susceptible to ciprofloxacin (CIP), moxifloxacin (MOX), and levofloxacin (LEV) using the VITEK GP-AST75 assay. However, CIP minimum inhibitory concentrations (MICs) varied in the broth dilution assay, with 47 isolates exhibiting MICs of 1 ppm and 41 isolates at ≤0.5 ppm (detection limit). Isolates with elevated MICs to one fluoroquinolone showed correlated increases in MICs to other fluoroquinolones (Spearman's correlation; p < 0.0001). MICs were confirmed using a broth dilution assay (0.125-8 ppm; two-fold dilution in MHB), with variable growth observed among replicates at sublethal CIP concentrations. After two passages in 0.5 ppm CIP, WRLP81 exhibited increased fitness (reduced lag phase duration) and MIC increases in CIP (4 ppm), LEV (2 ppm), and MOX (1 ppm). Whole genome sequencing of descendants (81.2B; 81.2C) revealed two unique frameshift mutations in fepR: V114fs and Glu139fs. Molecular modeling predicted conformational defects likely impairing FepA dimerization and efflux pump repression. Similarly, after two passages in 0.5 ppm CIP, a WRLP86 descendant (86.2C_2) exhibited increased MICs for CIP (2 ppm), LEV (4 ppm), and MOX (1 ppm), linked to a parC missense mutation that altered the winged helix domain. This amino acid change likely creates steric interference near the fluoroquinolone binding pocket and represents the first report of a parC mutation identified in L. monocytogenes in response to sublethal CIP exposure. This research highlights the rapid and varied adaptation of reduced fluoroquinolone susceptibility in L. monocytogenes.