Characterization of multidrug-resistant ST363-KL151 Klebsiella variicola clinical isolates harboring blaNDM−9 from Eastern China

Abstract Background Klebsiella variicola is an emerging multidrug‑resistant opportunistic pathogen that is often misidentified as K. pneumoniae in clinical laboratories. Here, we investigated four NDM‑9-producing K. variicola ST363‑KL151 isolates from patients treated at a tertiary-care centre in China. Methods Whole-genome sequencing and genomic analyses were performed to confirm species identity, define molecular characteristics, and identify resistance determinants. Core-genome SNP analysis was used to assess phylogenetic relatedness to international strains. Carbapenem-resistance transferability was evaluated by conjugation to E. coli . Biofilm formation, epithelial-cell invasion/cytotoxicity, serum bactericidal resistance, and virulence in a Galleria mellonella infection model were assessed. Results Antimicrobial susceptibility testing revealed a broad resistance profile encompassing β‑lactams (including carbapenems), aminoglycosides, fluoroquinolones, and fosfomycin, while retaining susceptibility to tigecycline. One isolate demonstrated elevated polymyxin resistance with an MIC of 64 mg/L, mediated by mgrB truncation (p.Gln30*). Whole‑genome sequencing identified a IncHI2A plasmid carrying a unique IS 26 ‑flanked bla NDM‑9 composite transposon adjacent to a mercury resistance operon, and conjugation assays confirmed transferable carbapenem resistance. Phylogenetic analysis placed the ST363-KL151 isolates in a monophyletic clade with a Norwegian strain collected in 2015. Although hypervirulence plasmids and classical virulence loci were absent, the isolates showed substantial biofilm formation and serum resistance, but only moderate cytotoxicity in epithelial cells and limited lethality in Galleria mellonella compared with the hypervirulent control NTUH-K2044. Conclusions These findings identify NDM‑9‑producing K. variicola ST363‑KL151 as a multidrug‑resistant lineage, emphasizing the need for accurate species identification and genome‑based surveillance to prevent its further clinical spread.