Berberine inhibits the AcrB efflux pump and synergizes with meropenem against carbapenem-resistant hypervirulent Klebsiella pneumoniae: mechanistic insights and in vivo validation.

From 285 non-duplicate clinical CRKP isolates, 170 were confirmed as carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP); after efflux pump phenotyping by the CCCP inhibition method and carbapenemase exclusion via Carba NP testing combined with multiplex carbapenemase gene PCR, 30 strains with AcrAB-TolC efflux-mediated resistance were selected for mechanistic investigation. Molecular docking revealed berberine binding to the AcrB Distal Binding Pocket (free energy: - 7.5 kcal/mol; hydrogen bond with SER-996, 2.6 Å). Checkerboard assays demonstrated synergistic berberine-meropenem activity against all 30 strains (fractional inhibitory concentration index, FICI: 0.1875-0.500; dimensionless). Nile red assays confirmed AcrB efflux inhibition comparable to PAβN, evidenced by attenuated intracellular fluorescence decay (ΔF/F₀ =  - 9.7% vs. - 52.1% in AcrB-overexpressing controls). RT-qPCR showed berberine suppressed acrB transcription via upregulation of ramR and downregulation of ramA. Combination therapy significantly improved murine survival and bacterial clearance versus monotherapy (log-rank P < 0.001).