ObjectiveThis study aims to assess the in vitro drug susceptibility of various Carbapenemase-Producing Enterobacteriaceae (CPE) genotypes and elucidate the underlying mechanisms of amikacin resistance.MethodsA total of 72 unique CPE strains were collected from the Second Hospital of Jiaxing between 2019 and 2022, including 51 strains of Klebsiella pneumoniae, 11 strains of Escherichia coli, 6 strains of Enterobacter cloacae, 2 strains of Klebsiella aerogenes, 1 strain of Citrobacter freundii, and 1strain of Citrobacter werkmanii. Among these strains, 24 carried blaKPC gene, 20 carried blaNDM gene, 23 carried blaOXA−48−like gene, and 5 carried both blaKPC and blaNDM. We measured the in vitro activity of amikacin and other common antibiotics. Strains carrying blaOXA−48-like gene were selected for whole genome sequencing (WGS) via next-generation sequencing to identify genes related to antimicrobial resistance (AMR) and virulence factor (VF).ResultsOut of the 72 CPE strains tested, 41.7% exhibited resistance to amikacin. The drug resistance rates for K. pneumoniae, E. coli, and Enterobacter spp. were 51.0%, 27.3%, and 10.0%, respectively. The majority of the CPE strains (> 90%) displayed resistance to cephalosporins and carbapenems, while most of them were sensitive to polymyxin B and tigecycline (97.2% and 94.4%). The amikacin resistance rate was 100% for strains carrying blaOXA−48, 20.8% for those with blaKPC, 5.0% for those with blaNDM, and 20.0% for those with both blaKPC and blaNDM. These differences were statistically significant (P < 0.05). Through sequencing, we detected aminoglycoside resistance genes rmtF and aac(6’)-Ib, VF genes iucABCD and rmpA2 in OXA-48-producing multidrug resistance and highly virulent strains. These genes were located on a IncFIB- and IncHI1B-type plasmid, respectively. Both plasmids were highly homologous to the plasmid from OXA-232 strains in Zhejiang province and Shanghai province. Integration of these resistance genes into the IncFIB plasmid, facilitated by the IS6 and/or Tn3 transposons, resulted in OXA232-producing K. pneumoniae with amikacin resistance.ConclusionThis study identified significant amikacin resistance in CPE strains, particularly in those carrying the blaOXA−48 gene. Resistance genes rmtF and aac(6’)-Ib were identified on plasmids. These results highlight the need for careful monitoring of amikacin resistance.