Discovery of clinical isolation of drug-resistant with overexpression of OqxB efflux pump as the decisive drug resistance factor.

Background emergence of multidrug-resistant (MDR) bacterial strains is a public health concern that threatens global and regional security. Efflux pump-overexpressing MDR strains from clinical isolates are the best subjects for studying the mechanisms of MDR caused by bacterial efflux pumps. A strain overexpressing the OqxB-only efflux pump was screened from a clinical strain library to explore reverse OqxB-mediated bacterial resistance strategies. We identified non-repetitive clinical isolated strains using a matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrometry clinical TOF-II (Clin-TOF-II) and susceptibility test screening against levofloxacin and ciprofloxacin. And the polymorphism analysis was conducted using pulsed-field gel electrophoresis. Efflux pump function of resistant strains is obtained by combined drug sensitivity test of phenylalanine-arginine beta-naphthylamide (PaβN, an efflux pump inhibitor) and detection with ethidium bromide as an indicator. The quantitative reverse transcription PCR was performed to assess whether the gene was overexpressed in isolates. Additional analyses assessed whether the gene was overexpressed in isolates and gene knockout and complementation strains were constructed. The binding mode of PaβN with OqxB was determined using molecular docking modeling. Among the clinical quinolone-resistant strains, one mediates resistance almost exclusively through the overexpression of the resistance-nodulation-division efflux pump, OqxB. Crystal structure of OqxB has been reported recently by N. Bharatham, P. Bhowmik, M. Aoki, U. Okada et al. (Nat Commun 12:5400, 2021, https://doi.org/10.1038/s41467-021-25679-0). The discovery of this strain will contribute to a better understanding of the role of the OqxB transporter in and builds on the foundation for addressing the threat posed by quinolone resistance.IMPORTANCEThe emergence of antimicrobial resistance is a growing and significant health concern, particularly in the context of infections. The upregulation of efflux pump systems is a key factor that contributes to this resistance. Our results indicated that the strain GN 172867 exhibited a higher gene expression compared to the reference strain ATCC 43816. Deletion of led a decrease in the minimum inhibitory concentration of levofloxacin. Complementation with rescued antibiotic resistance in the mutant strain. We demonstrated that the overexpression of the OqxB efflux pump plays an important role in quinolone resistance. The discovery of strain GN 172867 will contribute to a better understanding of the role of the OqxB transporter in and promotes further study of antimicrobial resistance.