Targeting efflux pumps prevent the multi-step evolution of high-level resistance to fluoroquinolone in Pseudomonas aeruginosa

Fluoroquinolone antibiotics, a common antibiotic for the treatment of Pseudomonas aeruginosa infection, are facing challenges due to the rapid evolution of bacterial resistance. Through designed evolutionary experiments in vitro, we find that there are significant differences in evolutionary trajectories and outcomes of resistant bacteria obtained in different induction modes, among which fitness benefit of resistant strains obtained in high-dose induction mode under high level of antibiotic is significantly higher than that of wild-type strain, and collateral sensitivity to aminoglycosides and some other antibiotics will be obtained. Resistance strains obtained in the low-dose induction mode exhibit higher heterogeneity, which is accompanied by multiple drug resistance (MDR). Through second generation resequencing and proteomic techniques, overexpression of MexCD-OprJ efflux pump induced by mutations in the nfxB gene significantly improved the fitness benefit of Pseudomonas aeruginosa PAO1 under high level of fluoroquinolones. This is the precondition of the further evolution of the fleroxacin-resistant strains in the high dose induction mode, the addition of the efflux pump inhibitor could effectively repress the evolution of bacterial resistance in the high dose induction mode. Fleroxacin use followed by gentamicin helped drive infectious P.aeruginosa to extinction, causing nfxB mutation to cause collateral sensitivity to gentamicin.