Discovery of clinical isolation of drug-resistant <i>Klebsiella pneumoniae</i> 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 <i>Klebsiella pneumoniae</i> 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 <i>K. pneumoniae</i> 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 <i>oqxB</i> gene was overexpressed in <i>K. pneumoniae</i> isolates. Additional analyses assessed whether the <i>oqxB</i> gene was overexpressed in <i>K. pneumoniae</i> 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 <i>K. pneumoniae</i> 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 <i>K. pneumoniae</i> 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 <i>K. pneumoniae</i> infections. The upregulation of efflux pump systems is a key factor that contributes to this resistance. Our results indicated that the <i>K. pneumoniae</i> strain GN 172867 exhibited a higher <i>oqxB</i> gene expression compared to the reference strain ATCC 43816. Deletion of <i>oqxB</i> led a decrease in the minimum inhibitory concentration of levofloxacin. Complementation with <i>oqxB</i> rescued antibiotic resistance in the <i>oqxB</i> 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 <i>K. pneumoniae</i> and promotes further study of antimicrobial resistance.