Complex evolutionary trajectories in vivo of two novel KPC variants conferring ceftazidime-avibactam resistance

More and more ceftazidime-avibactam resistant KPC-producing K. pneumoniae have been reported with its widespread use, and the detection rate of KPC variants has increased dramatically. However, the evolutionary mechanism and fitness effects during KPC mutation remained unknown. Here, we report the complex in vivo evolutionary trajectories of two novel KPC variants, KPC-155 (L169P/GT242A) and KPC-185 (D179Y/GT242A), from Klebsiella pneumoniae in the same patient. The novel variants were shown to confer ceftazidime-avibactam resistance but restore carbapenem susceptibility based on the results of plasmid transformation assays, cloning experiments, and enzyme kinetic measurements. In vitro competition experiments highlighted the adaptive advantage conferred by strains carrying these KPC variants, which could lead to the rapid spread of these ceftazidime-avibactam resistant strains. The growth curve indicated that blaKPC-185 had better growth conditions at lower avibactam concentration compared to blaKPC-155, which was consistent with ceftazidime-avibactam use in vivo. In addition, replicative transposition of the IS26-flanked translocatable unit (IS26-ISKpn6-blaKPC-ISKpn27-IS26) also contributes to the blaKPC amplification and formation of two copies (blaKPC-2 and blaKPC-185), conferring both carbapenem and ceftazidime-avibactam resistance. However, strains with double copies showed reduced competitive advantage and configuration stability. The comparative plasmid analysis of IS26 group (IS26-blaKPC-IS26) and Tn1721 group (Tn1721-blaKPC-IS26) revealed that IS26-insertion could influence the distribution of resistance genes and ability of self-conjugation. The dynamic changes in blaKPC configuration highlight the need for consistent monitoring including antimicrobial susceptibility testing and determination of blaKPC subtypes—during clinical treatment, especially when ceftazidime-avibactam is administered.