Coexistence of blaNDM–1 and blaIMP–4 in an IncHI5 plasmid harbored by carbapenem- resistant Raoultella ornithinolytica

Background Raoultella ornithinolytica is an emerging pathogen. This study aimed to characterize the genomic and molecular features of a carbapenem-resistant R.ornithinolytica strain co-harboring bla NDM–1 and bla IMP–4 . Methods Strain he2023 was identified and subjected to antimicrobial susceptibility testing using the BD Phoenix-M50 fully automated system. The strain was verified using MALDI-TOF. Carbapenemase genes were confirmed by PCR. Whole-genome sequencing (WGS) was performed to identify resistance genes and plasmid types. Conjugation experiments were conducted to assess transferability, and plasmid stability was evaluated through serial subculturing. Results The BD Phoenix-M50 system misidentified strain he2023 as Klebsiella pneumoniae , whereas MALDI-TOF and WGS correctly identified it as R. ornithinolytica . The strain was resistant to most antimicrobial agents, remaining susceptible only to amikacin,Tigecycline and polymyxin B. The bla NDM–1 and bla IMP–4 genes were both located on the plasmid pNDM-IMP, which belongs to the replicon type IncHI5. The bla NDM–1 was located in the structure of ISAba125 - bla NDM–1 - ble MBL - trpF - dsbC , residing in a region within the remnant of transposon Tn125 , flanked by two copies of ISCR1 . The bla IMP–4 was located downstream of bla NDM–1 within a novel class 1 integron ( In1965 ) carrying the structure sul1 - qacE - arr-3-ltrA - bla IMP–4 - IntI1 . Conjugation and transformation assays showed that the plasmid pNDM-IMP was non-transferable but exhibited high structural stability. Conclusion This study is the first to report an IncHI5 plasmid co-harboring bla NDM–1 and bla IMP–4 in R. ornithinolytica . Non-transferable plasmids may serve as reservoirs or initial carriers for antibiotic resistance genes. Our findings highlights the importance of mobile genetic elements in the formation of multidrug resistance.