Increased Expression of Efflux Pump norA Drives the Rapid Evolutionary Trajectory from Tolerance to Resistance against Ciprofloxacin in Staphylococcus aureus

The intensively intermittent use of antibiotics promotes the rapid evolution of tolerance, which may lead to resistance acquisition in the following evolutionary trajectory. In addition to directly exporting antibiotics as an instant resistance strategy, efflux pumps are overexpressed in tolerant strains. To investigate how efflux pumps participate in resistance development from tolerance to resistance, we performed evolutional experiments against the antibiotic ciprofloxacin in efflux pump mutants of Staphylococcus aureus. These experiments demonstrated that overexpression of rapidly facilitated the development of ciprofloxacin resistance from tolerance to resistance through elevated spontaneous mutations. The generated resistance mutations were further fixed in the population by increasing survival ability. The observed Ser80Phe and Glu84Lys mutations in the topoisomerase IV ParC (GrlA in S. aureus) may be responsible for tolerant strains to develop resistance to ciprofloxacin since it has been reported that such mutations disrupt the water-metal ion bridge between quinolones and ParC. MepA and Sav1866 are related to the same antibiotic (ciprofloxacin) susceptibility as NorA, and they also contributed to resistance development against ciprofloxacin. MgrA positively regulated NorA expression and the development of ciprofloxacin resistance. Importantly, blocking the evolutionary pathway by coadministering ciprofloxacin with the efflux pump inhibitor reserpine effectively delayed the resistance acquisition in an experiment. This study illustrated the role of efflux pumps in the evolutionary trajectory from tolerance to resistance. The delayed resistance development caused by the efflux pump inhibitor illuminates a possible strategy for postponing the resistance acquisition from tolerance to resistance by disrupting efflux pumps.