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Complete-Genome Sequencing and Comparative Genomic Characterization of an IMP-4 Producing Citrobacter freundii
Xiaohui Chi1,2, Jing Guo1, Yanzi Zhou1
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.
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.
Insights
This study investigated carbapenem-resistant Citrobacter freundii, revealing the blaIMP-4 gene on a transferable IncN1 plasmid. Enhanced surveillance is crucial to prevent the spread of this resistant pathogen in clinical settings.
Area of Science:
- Clinical Microbiology
- Molecular Biology
- Genomics
Background:
- Citrobacter freundii is a significant opportunistic pathogen, frequently associated with underlying health conditions.
- Carbapenem resistance in C. freundii poses a growing threat in healthcare settings.
- Understanding resistance mechanisms is vital for effective treatment and control strategies.
Purpose of the Study:
- To elucidate the carbapenem resistance mechanism in a clinical C. freundii isolate.
- To characterize the genetic environment and transferability of the blaIMP-4 gene.
- To analyze the IncN1 plasmid carrying the blaIMP-4 gene.
Main Methods:
- Whole-genome sequencing (Illumina and PacBio) for phylogenetic analysis.
- Antimicrobial susceptibility testing using the VITEK 2 system.
- Plasmid analysis via S1-pulsed-field gel electrophoresis (PFGE), Southern blotting, and conjugation experiments.
Main Results:
- The blaIMP-4 gene was confirmed on a conjugative IncN1 plasmid in C. freundii L91.
- The isolate and transconjugants exhibited resistance to carbapenems.
- The blaIMP-4 gene was part of a complex cassette array within a class 1 integron, flanked by mobile genetic elements.
Conclusions:
- The blaIMP-4 gene on the IncN1 plasmid confers carbapenem resistance and possesses horizontal transfer capabilities.
- The genetic environment suggests potential for spread within clinical environments.
- Increased surveillance is recommended to mitigate the dissemination of carbapenem-resistant C. freundii.
