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

Objective: 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 bla_KPC-33 variant in Enterobacter intestinihominis (E. intestinihominis) following ceftazidime (CAZ) treatment and to explore the evolution of bla_KPC-2 under CAZ pressure and the mechanisms of resistance gene dissemination. Methods: 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. Results: 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. Conclusion: This study reports the first identification of a bla_KPC-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 bla_KPC-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.