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

Area of Science:

• Microbiology
• Molecular Biology
• Antimicrobial Resistance

Background:

• Cefiderocol (FDC) is a key treatment for carbapenem-resistant Klebsiella pneumoniae (CRKP) infections.
• Emerging heteroresistance (HR) to FDC poses a threat to its clinical effectiveness.
• Understanding the molecular basis of FDC-HR in CRKP is crucial for effective treatment strategies.

Purpose of the Study:

• To investigate the molecular mechanisms driving FDC heteroresistance in CRKP during in vivo evolution.
• To characterize the genetic and molecular changes associated with FDC-HR emergence.
• To validate the identified mechanisms using functional assays.

Main Methods:

• Analysis of 477 clinical CRKP isolates.
• Characterization of FDC-heteroresistant (FDC-HR) subpopulations using population analysis profiling and antimicrobial susceptibility testing.
• Molecular techniques including quantitative PCR, transcriptomics, whole-genome sequencing, and CRISPR-Cas9 functional validation.
• Molecular modeling to predict FDC binding to mutated proteins.

Main Results:

• 12 out of 477 CRKP isolates (2.52%) exhibited an FDC-HR phenotype.
• FDC-resistant subpopulations showed increased blaKPC-2 plasmid copy number and upregulated expression.
• A G652A mutation in mrcA was identified, impairing FDC binding.
• The combination of mrcA mutation and blaKPC-2 amplification elevated FDC MIC from 2 to 32 mg/L, mimicking clinical heteroresistance.

Conclusions:

• Amplification of blaKPC-2 plasmid copy number and the mrcA G652A mutation are key drivers of FDC-HR in CRKP during in vivo evolution.
• This resistance mechanism can be underestimated by standard susceptibility testing, increasing the risk of treatment failure.
• The findings highlight the importance of detecting heteroresistance for optimizing FDC therapy in CRKP infections.