Evolution and high transferability of an IncN/FII plasmid harboring blaKPC-2/blaKPC-33 in Enterobacter intestinihominis under ceftazidime pressure

Carbapenem-resistant Enterobacteriaceae (CRE), primarily driven by plasmid-mediated KPC enzymes, pose a major clinical threat, and resistance to ceftazidime-avibactam (CAZ-AVI) is emerging. This study aimed to investigate the emergence of the blaKPC-33 variant in Enterobacter intestinihominis (E. intestinihominis) following ceftazidime (CAZ) treatment and to explore the evolution of blaKPC-2 under CAZ pressure and the mechanisms of resistance gene dissemination. Two E. intestinihominis isolates, JNQH617 and JNQH618, were obtained from the same urine sample of an ICU patient undergoing CAZ therapy. We employed a combination of antimicrobial susceptibility testing, whole-genome sequencing (WGS), pulsed-field gel electrophoresis (PFGE), conjugation assays, and CRISPR/Cas9-based plasmid curing to explore the genetic basis of CAZ-AVI resistance and the roles of conjugative plasmids in gene dissemination. Strains JNQH617 and JNQH618 belong to sequence type 78 (ST78), harbored KPC-2 and KPC-33 respectively. Both variants were located on highly transmissible IncN/FII hybrid plasmids (nearly 100% transfer efficiency). In vitro selection experiments confirmed that prolonged exposure to CAZ alone could drive the emergence of novel KPC variants, which conferred resistance to CAZ-AVI. However, this mutational resistance could not be selected in K. pneumoniae species complex (KpSC), Serratia marcescens and Citrobacter freundii strains. CRISPR/Cas9-based dual-sgRNA strategy enables complete curing of the hybrid IncN/FII plasmid. Interestingly, the presence of an additional IncFIB/FII plasmid significantly enhanced the IncN/FII plasmid transfer efficiency. This study reports the first identification of a blaKPC-33–producing E. intestinihominis strain. Its emergence occurred independently of CAZ-AVI therapy and is likely attributable to selective pressure from CAZ exposure. The high conjugative efficiency of the blaKPC-carrying IncN/FII plasmid underscores the risk of rapid dissemination of carbapenem and CAZ-AVI resistance. These findings highlight the importance of further investigating plasmid-plasmid and plasmid-host interactions, which may play crucial roles in the evolution and transmission of antimicrobial resistance determinants.