The intracellular concentrations of fluoroquinolones determined the antibiotic resistance response of Escherichia coli.

The extensive consumption of antibiotics has been reported to significantly promote the generation of antibiotic resistance (ABR), however, a quantitative causal relationship between antibiotic exposure and ABR response is absent. This study aimed to pinpoint the accurate regulatory concentration of fluoroquinolones (FQs) and to understand the biochemical mechanism of the mutual action between FQ exposure and FQ resistance response. Highly sensitive analytical methods were developed by using UPLC-MS/MS to determine the total residual, extracellular residual, total intracellular, intracellular residual and intracellular degraded concentration of three representative FQs, including ciprofloxacin (CIP), ofloxacin (OFL) and norfloxacin (NOR), with detection limits in the range of 0.002-0.057 μg/L, and recoveries in the range of 80-93%. The MICs of Escherichia coli (E. coli) were 7.0-31.4-fold of the respective MIC0 after 40-day FQ exposure, and significant negative associations were discovered between the intracellular (residual, degraded or the sum) FQ concentrations and FQ resistance. Transcriptional expression and whole-genome sequencing results indicated that reduced membrane permeability and enhanced multi-drug efflux pumps contributed to the decreasing intracellular concentration. These results unveiled the pivotal role of intracellular concentration in triggering FQ resistance, providing important information to understand the dose-response relationship between FQ exposure and FQ resistance response, and ascertain the target dose metric of FQs for eliminating FQ resistance crisis.