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

Authors Chi X, Guo J, Zhou Y, Xiao T, Xu H, Lv T, Chen C, Chen J, Zheng B

Received 3 January 2020

Accepted for publication 19 March 2020

Published 14 April 2020 Volume 2020:13 Pages 1057—1065


Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 2

Editor who approved publication: Dr Joachim Wink

Xiaohui Chi, 1, 2,* Jing Guo, 1,* Yanzi Zhou, 1 Tingting Xiao, 1 Hao Xu, 1 Tao Lv, 1 Chunlei Chen, 1 Jian Chen, 3 Beiwen Zheng 1

1Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis and Treatment of Infectious Disease, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, People’s Republic of China; 2Department of Environment and Health, School of Public Health, Shandong University, Jinan, People’s Republic of China;  3Intensive Care Unit, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, People’s Republic of China

*These authors contributed equally to this work.

Correspondence: Beiwen Zheng; Jian Chen
Tel/Fax +86-571-87236421; Tel +86-571-87236666

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 blaIMP-4 gene from C. freundii isolate.
Materials and Methods: We identified a clinical isolate of C. freundii L91 carrying blaIMP-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 blaIMP-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, blaIMP-4 gene was found encoded in the blaIMP-4-qacG2-aacA4-catB3 cassette array within a class 1 integron. A conserved structure sequence (ΔISKpn27-blaIMP-4-ΔISSen2-hp-hp-IS 6100) was found in the upstream and downstream of the blaIMP-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 blaIMP-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

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