Background: Citrobacter freundii  is the most common class of pathogens in the genus Citrobacter  and is an important pathogen associated with certain underlying diseases or immune dysfunction. The aim of this study was to elucidate the resistance mechanism of clinically derived carbapenem-resistant C. freundii  isolate and to characterize the genetic environment and delivery pattern of the IncN1 plasmid carrying the bla _IMP-4 gene from C. freundii  isolate.
Materials and Methods: We identified a clinical isolate of C. freundii  L91 carrying bla _IMP-4 and performed phylogenetic analysis by whole-genome sequencing. The complete genomic sequence of L91 was obtained using the Illumina HiSeq 4000-PE150 and PacBio RS  II platforms. Antimicrobial susceptibility testing was determined by the VITEK 2 system. Plasmid characteristics were presented by S1-pulsed-field gel electrophoresis (PFGE), Southern blotting and conjugation experiments.
Results: S1-PFGE, Southern blot and conjugation assay confirmed the presence of bla _IMP-4 genes on a conjugative plasmid in this isolate. C. freundii  L91 and transconjugant L91-E. coli  600 strains both showed resistance to carbapenems. In silico analysis further showed that pIMP-4-L91 is an IncN1 plasmid with a length of 51,042 bp. Furthermore, bla _IMP-4 gene was found encoded in the bla _IMP-4-qacG2-aacA4-catB3  cassette array within a class 1 integron. A conserved structure sequence (ΔISKpn27-bla _IMP-4-ΔISSen2-hp-hp -IS 6100 ) was found in the upstream and downstream of the bla _IMP-4.
Conclusion: We performed a comprehensive phylogenetic analysis of carbapenemase-resistant C. freundii  and elucidated the resistance mechanism of clinically derived C. freundii  L91. Not only that, we also found that the bla _IMP-4 gene is located on the IncN1 plasmid and has a horizontal transfer function and a certain ability to spread. To lower the risk of the dissemination of such C. freundii  isolates in clinical settings, more surveillance is needed in the future.
Keywords: Citrobacter freundii , CPE, whole-genome sequencing, SNP, IncN, integron