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

Abstract

IntroductionKlebsiella pneumoniae carbapenemase (KPC) is one of the most widespread carbapenemases, with over 242 variants identified. Some, like KPC-33 (D179Y), resist both ceftazidime-avibactam (CZA) and cefiderocol but show only a moderate cefiderocol MIC increase. KPCs success stems from its genetic adaptability, enabling mutation accumulation and necessitating proactive resistance monitoring. This study investigates the mutational possibilities for three KPC variants to develop high-level cefiderocol resistance. MethodUsing random mutagenesis and enrichment selection over a 10-day cefiderocol exposure at increasing concentration, we investigated the mutational potential of KPC-2, KPC-3, and KPC-33 to develop high-level cefiderocol resistance. Resistance mechanisms were then analyzed through phenotypic testing and sequencing. ResultsRandom mutagenesis generated 10, 10, and 105 mutants for blaKPC-2, blaKPC- 3, and blaKPC-33, respectively. After ten days of increasing cefiderocol exposure, MICs rose significantly, with KPC-2 mutants reaching >32 mg/L. Phenotypic analysis revealed a common resistance profile across all tested mutants, with resistance to ceftazidime, ceftazidime-avibactam, cefixime, and piperacillin, but restored susceptibility to carbapenems and most other {beta}-lactams. Sequencing identified key mutations (D179Y- D209V in KPC-2, L169P in KPC-3), while chromosomal changes, particularly cirA and ybiX disruptions, played a crucial role in resistance evolution. DiscussionOur results show that the enriched mutations in KPC genes are not sufficient to confer high cefiderocol MICs, suggesting that, in the studied variants, no simple mutational pathway allows the KPC enzyme to efficiently hydrolyze cefiderocol. These findings underscore the interplay between enzymatic and iron transport mutations in cefiderocol resistance, highlighting the importance of surveillance to anticipate emerging resistance in KPC-producing pathogens.