blaKPC-2 amplification and a novel mrcA mutation drive Cefiderocol heteroresistance in ST11 CRKP

Abstract

Objectives

Cefiderocol (FDC) is a promising treatment for infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP), but emerging heteroresistance (HR) may compromise its clinical efficacy. This study aimed to elucidate the molecular mechanisms underlying FDC heteroresistance arising during in vivo evolution of CRKP. in vivo-evolved.

Methods

A total of 477 clinical CRKP isolates were analyzed. FDC-heteroresistant (FDC-HR) subpopulations emerging during in vivo evolution were characterized using population analysis profiling, antimicrobial susceptibility testing, quantitative PCR, plasmid copy number analysis, transcriptomics, whole-genome sequencing, molecular modeling, and CRISPR-Cas9–based functional validation.

Results

Among the 477 CRKP isolates, 376 (78.8%) belonged to ST11-KL64, 4 (0.84%) were non-susceptible to FDC, and 12 (2.52%) exhibited an FDC-HR phenotype. Resistant subpopulations showed approximately a twofold increase in bla_KPC-2 plasmid copy number with upregulated expression compared with susceptible counterparts. A G652A point mutation in mrcA was identified and predicted to impair FDC binding. Functional validation demonstrated that the mrcA mutation alone increased the FDC minimum inhibitory concentration (MIC), while its combination with bla_KPC-2 amplification elevated the MIC from 2 to 32 mg/L, fully recapitulating the clinical heteroresistance phenotype. In vivo evolution reduced virulence without affecting growth.

Conclusions

Amplification of bla_KPC-2 plasmid copy number together with the mrcA G652A mutation drives FDC-HR in CRKP during in vivo evolution. This mechanism may lead to underestimation of resistance by standard susceptibility testing and increase the risk of treatment failure.