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

UNLABELLED: 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 blaKPC 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 blaNDM-1 and (ii) mutation of the blaKPC-2 gene to blaKPC-33 with an Ω-loop D179Y substitution. The blaNDM-1 gene resided within a truncated Tn125 transposon that formed part of an IS26-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 blaKPC as the most frequent carbapenemase gene (36.2%). Conjugation assays further confirmed self-transferability of pCYH4028_NDM. The blaKPC-33 gene was located within an IS26-truncated Tn6296 transposon on the hybrid plasmid pCYH1535_Vir_KPC, which formed via IS5075-mediated homologous recombination and XerD-driven site-specific recombination. Notably, acquisition of CZA resistance incurred varying in vitro fitness costs, whereas the blaKPC-33-harboring strain exhibited greater competitive advantage than the blaNDM-1-harboring strain. All strains exhibited attenuated virulence (reduced mucoviscosity and biofilm production) due to ISKpn26 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 blaKPC mutations, constitutes a critical threat to global health. Here, we reported the within-host evolution of CZA resistance, driven by IncN plasmid-encoded blaNDM-1 and blaKPC 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 blaNDM-1 plasmid acted as a helper plasmid, facilitating co-transfer of blaNDM-1 with blaKPC-2 and virulence genes. These direct insights highlight the urgent need for enhanced genomic surveillance against this evolving threat.