Regulatory role of RamA and RamR in AcrAB-TolC-mediated carbapenem resistance in carbapenem-resistant hypervirulent Klebsiella pneumoniae

OBJECTIVE: Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP), which possesses both high pathogenicity and drug resistance, has become a major clinical challenge. The role of AcrAB-TolC efflux pump in its resistance mechanism has attracted increasing attention. This study aimed to clarify the role of the AcrAB-TolC efflux pump in carbapenem resistance of CR-hvKP, confirm the role of AcrB in the function of the AcrAB-TolC efflux pump, and reveal the regulatory mechanism of RamA and RamR. This study provides a theoretical basis for the treatment and prevention of CR-hvKP infections. METHODS: Thirty-eight non-repetitive clinical CR-hvKP isolates were analyzed. Minimum inhibitory concentrations (MICs) were determined using broth microdilution. Efflux pump activity was evaluated by comparing MICs before and after addition of CCCP. The Carbapenemase Nordmann-Poirel (Carb NP) test excluded carbapenemase-mediated resistance. PCR was used to detect virulence (iucA, rmpA, rmpA2, iroB, Peg344) and efflux pump (acrA, acrB, tolC) genes. Bioinformatics tools (KaKs_Calculator and Datamonkey) and RT-qPCR identified and quantified acrB expression. Gene knockout and overexpression, combined with MIC testing and a Galleria mellonella infection model, assessed effects on resistance and virulence. RT-qPCR evaluated RamA and RamR expression. RESULTS: acrB was identified as a major contributor to efflux-mediated resistance. Its expression decreased by 65% in the ramA knockout stra...