Rapid replacement of blaKPC variant in ST11 carbapenem-resistant and hypervirulent Klebsiella pneumoniae contributed to ceftazidime/avibactam resistance during severe in vivo infection.

Objectives: Hypervirulent ceftazidime/avibactam (CAZ/AVI)-resistant Klebsiella pneumoniae (Kp) has emerged; however, its dynamic within-host evolution and competitive features are uncharacterized. This study aimed to clarify the systematic microevolution characteristics of the rapid transformation of blaKPC variants during long-term infection.

Methods: Thirty-nine Kp strains were isolated from a single patient with severe recurrent osteomyelitis during a 2-year period. Whole-genome sequencing and in vitro evolution assay was performed. Microbiological characteristics were examined through antimicrobial susceptibility testing, plasmid stability, growth curve, in vitro competition and Galleria mellonella larvae lethality assays.

Results: Among all the clinical Kp isolates, 37 were carbapenem-resistant Kp (CRKP), including 25 CAZ-/AVI-resistant Kp. All isolates belonged to the ST11-K47. During in vivo evolution, the blaKPC variant and its amplification emerged. Twenty-four isolates (24/39, 61.5%) harboured a novel blaKPC variant, blaKPC-144. All five Kp isolates carried blaKPC-2 in 2021. Surprisingly, 24 blaKPC-144-harbouring isolates (70.6%, 24/34) and 10 blaKPC-2-harboring isolates were identified in 2023, indicating rapid changing of blaKPC. Kp4 carried two copies of blaKPC-2, and Kp10-1 exhibited a 1.94-fold increase in the blaKPC-144 copy number. Similarly, in vitro, the blaKPC copy number increased upon exposure to low CAZ/AVI concentrations. However, at higher concentrations (4/1 mg/L), the blaKPC copy number increased significantly, and blaKPC mutations emerged simultaneously. The competition assay indicated that the blaKPC-144-harboring isolates exhibited a superior competitive capacity.

Conclusions: The blaKPC amplification and mutation emerged simultaneously or sequentially during in vivo and in vitro evolution. Kp isolates harbouring blaKPC-144, conferring resistance to CAZ/AVI, exhibited a competitive advantage, promoting the rapid replacement of blaKPC-2.