Exploring mutational possibilities of KPC variants to reach high level resistance to cefiderocol.

Klebsiella pneumoniae carbapenemase (KPC) is a frequent and widespread carbapenemase, with over 260 variants identified. While KPC often evolves resistance to ceftazidime-avibactam, cefiderocol remains a key treatment option. Some variants, such as KPC-33 (D179Y), reduce cefiderocol susceptibility, but typically with only modest MIC increases. However, KPC's genetic adaptability raises concern that further mutations could lead to high-level resistance, compromising cefiderocol's efficacy. To anticipate this risk, we explored the mutational potential of blaKPC-2, blaKPC-3, and blaKPC-33 using random mutagenesis followed by 10-day selection under increasing cefiderocol pressure and whole genome sequencing. Libraries of 105, 104, and 105 mutants, respectively, yielded isolates with significantly elevated MICs, some exceeding 32 mg/L. All resistant clones shared a phenotype marked by cross-resistance to cefiderocol, ceftazidime, ceftazidime-avibactam, cefixime, and piperacillin, but restored susceptibility to carbapenems and most other β-lactams. Our findings highlight that no single mutation enables KPC to efficiently hydrolyze cefiderocol. Instead, high-level resistance requires a combination of enzymatic mutations and chromosomal alterations-such as disruptions in cirA and ybiX-suggesting a multifactorial and stepwise evolutionary pathway. Notably, ybiX has not previously been associated with cefiderocol resistance. These results underscore the importance of ongoing surveillance to detect emerging cefiderocol resistance in KPC-producing Enterobacterales.