Unravelling tandem repeat-mediated mutagenesis drive rapid diversification of KPC enzymes: emergence of blaKPC-263 and enhanced resistance to ceftazidime-avibactam.

BACKGROUND: The emergence of ceftazidime-avibactam (CZA)-resistant carbapenem-resistant Klebsiella pneumoniae (CRKP) producing K. pneumoniae carbapenemase (KPC) severely challenges clinical management. Understanding the genetic mechanisms underpinning their evolution, particularly the emergence of new KPC variants conferring CZA resistance, is critical. METHODS: Fifteen CRKP isolates were prospectively collected in 2018 from a single patient at a tertiary-care hospital in Zhejiang, China, and were subjected to antimicrobial susceptibility testing and whole-genome sequencing. Bioinformatics analyses, including resistance gene identification, phylogenetic analysis, and comparative genomics of KPC variants and their plasmid contexts, were performed. Public NCBI databases were analysed to assess the prevalence of tandem repeat (TR)-mediated mutagenesis in KPC variants. FINDINGS: We identified a previously uncharacterised KPC variant, blaKPC-263, resulting from a TR-mediated 3-bp insertion in the blaKPC-2 gene, which conferred enhanced resistance to CZA. A previously reported variant, blaKPC-90, with a distinct 6-bp insertion, was also isolated from the same patient. Broader genomic analysis revealed that TR-mediated mutagenesis is prevalent across 42 distinct KPC variants. Comparative plasmid analysis suggested the genetic context of blaKPC-263 may facilitate its stability. INTERPRETATION: TR-mediated mutagenesis represents a significant, previously underappreciated genetic mechanism driving the rapid diversification and adaptive evolution of KPC enzymes under antibiotic selective pressures. These findings have broad implications for antimicrobial resistance surveillance, emphasising the need for genomic monitoring programs and targeted interventions to combat the spread of KPC variants driven by this mechanism. FUNDING: This work was funded by the National Key R&D Programme of China (2023YFC2308400); National Natural Science Foundation of China (82072314); Shandong Provincial Laboratory Project (SYS202202); Zhejiang Province Leading Geese Plan (2025C04013); Zhejiang Provincial Traditional Chinese Medicine Science and Technology Program (Grant No. 2017ZZ012); Zhejiang Provincial Medical and Health Science and Technology Project (2022KY355 &2024KY402) and Fundamental Research Funds for the Central Universities (2022ZFJH003).