Work

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

Year: 2025

Type: article

Abstract: • FDC heteroresistance was in 2.52% of 477 CRKP strains, mostly of ST11-KL64 type. • bla KPC-2 plasmid copy number doubled in HR strains, with higher expression. • A G652A point mutation in mrcA weakened FDC binding and reproduced the HR phenotype. • Combined plasmid amplification and mrcA mutation increased MIC from 2 to 32 mg/L. • Routine testing may miss HR; gene expression and mutation checks can guide FDC use. Cefiderocol (FDC) is a promising treatment for infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP), but emerging heteroresistance (HR) may compromise its clinical efficacy. Of 477 CRKP isolates, 376 (78.8%) were ST11-KL64, 4 (0.84%) were non-susceptible to FDC, and 12 (2.52%) exhibited the FDC-HR phenotype. Using population analysis profiling, antimicrobial susceptibility testing, qPCR, gene copy number analysis, transcriptomics, and whole-genome sequencing, we characterized FDC-HR subpopulations that emerged during in vivo evolution. Within in vivo -evolved heterogeneous populations, resistant versus susceptible strains showed approximately doubled bla KPC-2 plasmid copy number and upregulated expression. Whole-genome sequencing further identified a G652A point mutation in mrcA , which molecular docking and molecular dynamics (MD) simulations predicted to impair FDC binding. CRISPR-Cas9-based functional validation confirmed that the mrcA mutation alone elevated FDC minimum inhibitory concentration (MIC), while its combination with bla KPC-2 amplification increased MIC from 2 to 32 mg/L, fully replicating the clinical HR phenotype. In vivo evolution reduced virulence without affecting growth. This study is the first to reveal that bla KPC-2 plasmid copy number amplification and mrcA G652A mutation drive FDC-HR in CRKP during in vivo evolution. This mechanism may lead to underestimated resistance in standard testing and an increased risk of treatment failure. Monitoring resistance gene expression and mutations could improve FDC applications and identify HR phenotypes.

Source: International Journal of Antimicrobial Agents

DOI: 10.1016/j.ijantimicag.2025.107682