Coexistence of a novel NDM-1-encoding MDR plasmid and an IMP-4-encoding IncN-IncU hybrid plasmid in a clinical isolate of Citrobacter freundii BC73

Objectives: To investigate the genetic characteristics and transmission mechanism of the NDM-1-, IMP-4-, and SHV-12-producing multidrug-resistant (MDR) clinical isolate, Citrobacter freundii BC73.

Methods: C. freundii BC73 was isolated from a urine specimen of a urological patient diagnosed with bladder cancer at a Chinese teaching hospital. Antimicrobial susceptibility testing was carried out using DL-120E susceptibility cards and DL-96A system. Whole genome sequencing (WGS) of the isolate was performed using the Illumina and Oxford Nanopore platforms to analyze the genetic context of drug resistance genes and plasmid characteristics. The phylogenetic tree was constructed and visualized by KSNP3.0 software and iTOL5.0 online database.

Results: C. freundii isolate BC73 co-carrying bla _NDM-1, bla _IMP-4 and bla _SHV-12 were multidrug-resistant. bla _NDM-1 and bla _IMP-4 were located on a novel IncFIB-like plasmid, pCFBC1, and an IncN-IncU hybrid plasmid, pCFBC2, respectively. The transferability of bla _NDM-1 and bla _IMP-4 from C. freundii BC73 to E. coli J53 was successfully demonstrated. The genetic context of the bla _NDM-1 and bla _IMP-4 genes were ISCR27-groEL-∆groES-cutA-dsbD-trpF-ble _MBL-bla _NDM-1-∆ISAba125-IS3000 and intI1-bla _IMP-4-Kl.pn.13-mobC-IS6100, respectively. Additionally, two extensive transposition units (MGE1 in pCFBC1, MGE2 in pCFBC2) were identified and numerous antimicrobial resistance genes were discovered on it.

Conclusion: To our knowledge, our study represents the first characterization of a ST22 C. freundii isolate co-harboring bla _NDM-1, bla _IMP-4, and bla _SHV-12, obtained from a urine sample. The dissemination of this MDR isolate should be of close concern in future clinical surveillance.