Discovery of clinical isolation of drug-resistant
Klebsiella pneumoniae
with overexpression of OqxB efflux pump as the decisive drug resistance factor
Authors: Tian Ping , Guo Ming-Juan , Li Qing-Qing , Li Xu-Feng , Liu Xiao-Qiang , Kong Qin-Xiang , Zhang Hui , Yang Yi , Liu Yan-Yan , Yu Liang , Li Jia-Bin , Li Ya-Sheng 
Published: August 2024

ABSTRACT

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
Klebsiella pneumoniae
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
K. pneumoniae
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
oqxB
gene was overexpressed in
K. pneumoniae
isolates. Additional analyses assessed whether the
oqxB
gene was overexpressed in
K. pneumoniae
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
K. pneumoniae
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
K. pneumoniae
and builds on the foundation for addressing the threat posed by quinolone resistance.

IMPORTANCE

The emergence of antimicrobial resistance is a growing and significant health concern, particularly in the context of
K. pneumoniae
infections. The upregulation of efflux pump systems is a key factor that contributes to this resistance. Our results indicated that the
K. pneumoniae
strain GN 172867 exhibited a higher
oqxB
gene expression compared to the reference strain ATCC 43816. Deletion of
oqxB
led a decrease in the minimum inhibitory concentration of levofloxacin. Complementation with
oqxB
rescued antibiotic resistance in the
oqxB
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
K. pneumoniae
and promotes further study of antimicrobial resistance.