First Identification of an ST11-KL64 Hypervirulent Klebsiella pneumoniae Strain Coproducing KPC-2 and NDM-1 with an OmpK36 GD Mutation

Jiaoli Chen¹Cheng Wang¹Lingbin Wu^1,2Ziling Lan^2,3Mei Li³Jianfen Xu¹Xiaolei Hu^1*Jiansheng Huang^1*

• ¹Department of Clinical Laboratory, the Fifth Affiliated Hospital of Wenzhou Medical University, Lishui, China
• ²Department of Clinical Laboratory, Lishui Second People's Hospital Affiliated to Wenzhou Medical University, Lishui, China
• ³Department of the School of Medical Technology and Information Engineering, Zhejiang Chinese Medical University, Hangzhou, China

The convergence of hypervirulence and carbapenem resistance in Klebsiella pneumoniae represents a critical clinical threat. We characterized 27 NDM-producing K. pneumoniae isolates from a tertiary hospital in Lishui, China, between 2017 and 2023 and detected high clonal diversity across 19 sequence types. Among the isolates, we chose an ST11 strain, CRKP26, carrying blaKPC-2 and blaNDM-1, for further investigation. Whole-genome sequencing (WGS) revealed that blaKPC-2 and blaNDM-1 were located on separate plasmids and that insertion of glycine – aspartate (GD) at positions 137–138 was identified in the ompK36 gene. CRKP26 exhibited extensive drug resistance, with resistance to ceftazidime, cefepime, aztreonam, piperacillin-tazobactam, and amikacin and high-level carbapenem resistance, with MICs of 64 μg/mL for imipenem and 256 μg/mL for meropenem. This strain was susceptible only to polymyxin B and tigecycline. WGS further identified CRKP26 as capsular serotype KL64 and confirmed the presence of core virulence determinants, including the aerobactin operon (iucABCD-iutA), on an IncFIB virulence plasmid. Strain CRKP26 was defined as hypervirulent (LD50≤1×106 CFU), despite showing slightly attenuated lethality compared to the classic hypervirulent strain NTUH-K2044. Furthermore, the survival rate of CRKP26 was 93.2% in serum killing assays and 90.7% in neutrophil killing assays, comparable to that of the hypervirulent control NTUH-K2044. To our knowledge, this is the first report of a hypervirulent ST11-KL64 K. pneumoniae strain carrying both blaKPC-2 and blaNDM-1 with an OmpK36 GD mutation. This convergence of plasmid-mediated resistance, chromosomal mutation, and hypervirulence in a high-risk clone highlights an urgent threat requiring increased genomic surveillance.