Complete-Genome Sequencing and Comparative Genomic Characterization of an IMP-4 Producing Citrobacter freundii Isolate from Patient with Diarrhea

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-IS6100) 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.