Mutations That Enhance the Ciprofloxacin Resistance of Escherichia coli with qnrA1

Plasmid-mediated qnr genes provide only a modest decrease in quinolone susceptibility but facilitate the selection of higher-level resistance. In Escherichia coli strain J53 without qnr, ciprofloxacin resistance often involves mutations in the GyrA subunit of DNA gyrase. Mutations in gyrA were absent, however, when 43 mutants with decreased ciprofloxacin susceptibility were selected from J53(pMG252) with qnrA1. Instead, in 13 mutants, individual and whole-genome sequencing identified mutations in marR and soxR associated with increased expression of marA and soxS and, through them, increased expression of the AcrAB pump, which effluxes quinolones. Nine mutants had increased expression of the MdtE efflux pump, and six demonstrated increased expression of the ydhE pump gene. Many efflux mutants also had increased resistance to novobiocin, another pump substrate, but other mutants were novobiocin hypersusceptible. Mutations in rfaD and rfaE in the pathway for inner core lipopolysaccharide (LPS) biosynthesis were identified in five such strains. Many of the pump and LPS mutants had decreased expression of OmpF, the major porin channel for ciprofloxacin entry. Three mutants had increased expression of qnrA that persisted when pMG252 from these strains was outcrossed. gyrA mutations were also rare when mutants with decreased ciprofloxacin susceptibility were selected from E. coli J53 with aac(6')-Ib-cr or qepA. We suggest that multiple genes conferring low-level resistance contribute to enhanced ciprofloxacin resistance selected from an E. coli strain carrying qnrA1, aac(6')-Ib-cr, or qepA because these determinants decrease the effective ciprofloxacin concentration and allow more common but lower-resistance mutations than those in gyrA to predominate.