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

Abstract

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 bla_KPC-2, bla_KPC-3, and bla_KPC-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.

Keywords: cirA gene; fiu gene; ybiX gene; Cefiderocol resistance; Clinical KPC variants; KPC beta-lactamase; Mutagenesis.

Fig. 1

Results of the enrichment experiment of mutant libraries under increasing concentrations of cefiderocol (10 days of evolution).

Fig. 2

Kiviat diagram showing the distribution of mean inhibition zone diameters (mm) for the principal antibiotic molecules tested on selected variants (blue—variants enriched from KPC-2 (D179Y-D209V), orange—variants from KPC-3 (L169P), grey variants from KPC-33 (R6H, F20L + /- G291S) as well as ancestral KPC-2 variant (yellow). AMX, Amoxicillin; AMX/CLAV, Amoxicillin/clavulanate; PIP, Piperacillin; PTZ, Piperacillin/tazobactam; AZT, Aztreonam; FEP, Cefepime; FIX, Cefixime; CTX, Cefotaxime; FOX, Cefoxitin; CAZ, Ceftazidime; CZA, Ceftazidime-avibactam; IMP, Imipenem; MER, Meropenem Diameters: millimeters; Blue points: EUCAST Breakpoints.

Fig. 3

Interplay between bla_KPC mutations and siderophore transport deficiencies driving high cefiderocol MICs.