Within-host evolution drives the emergence of ceftazidime-avibactam resistance mediated by IncN plasmid-encoded bla NDM-1 and bla KPC-33 in ST11-KL64 hypervirulent Klebsiella pneumoniae

Ceftazidime-avibactam (CZA) is considered one of the most effective antimicrobial agents for treating infections caused by Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae . However, rising CZA resistance poses a critical clinical challenge, primarily driven by bla KPC mutations. From a single CZA-treated patient, we isolated two CZA-susceptible and three CZA-resistant K. pneumoniae strains. Whole-genome sequencing of CZA-resistant strains CYH4028 and CYH1535 revealed two key resistance pathways: (i) acquisition of a conjugative IncN plasmid harboring bla NDM-1 and (ii) mutation of the bla KPC-2 gene to bla KPC-33 with an Ω-loop D179Y substitution. The bla NDM-1 gene resided within a truncated Tn 125 transposon that formed part of an IS 26 -associated translocatable unit in the self-transferable IncN plasmid pCYH4028_NDM. Phylogenetic analysis of 257 IncN plasmids revealed the highest plasmid prevalence in China (34.6%), with K. pneumoniae as the predominant host (41.6%) and bla KPC as the most frequent carbapenemase gene (36.2%). Conjugation assays further confirmed self-transferability of pCYH4028_NDM. The bla KPC-33 gene was located within an IS 26 -truncated Tn 6296 transposon on the hybrid plasmid pCYH1535_Vir_KPC, which formed via IS 5075 -mediated homologous recombination and XerD-driven site-specific recombination. Notably, acquisition of CZA resistance incurred varying in vitro fitness costs, whereas the bla KPC-33 -harboring strain exhibited greater competitive advantage than the bla NDM-1 -harboring strain. All strains exhibited attenuated virulence (reduced mucoviscosity and biofilm production) due to IS Kpn26 disruption of rmpA , a mucoid phenotype regulator. These findings elucidate within-host adaptive evolution in ST11-KL64 hypervirulent carbapenem-resistant K. pneumoniae and highlight the necessity for continuous genomic surveillance during therapy. IMPORTANCE The alarming rise of ceftazidime-avibactam (CZA) resistance, primarily through bla KPC mutations, constitutes a critical threat to global health. Here, we reported the within-host evolution of CZA resistance, driven by IncN plasmid-encoded bla NDM-1 and bla KPC mutations, in a high-risk ST11-KL64 hypervirulent carbapenem-resistant Klebsiella pneumoniae lineage during patient therapy. Our study demonstrates the emergence of simultaneous, independent evolutionary pathways under CZA pressure. Crucially, an IncN bla NDM-1 plasmid acted as a helper plasmid, facilitating co-transfer of bla NDM-1 with bla KPC-2 and virulence genes. These direct insights highlight the urgent need for enhanced genomic surveillance against this evolving threat.