AUTHOR=Qiao Jie , Chen Yingying , Ge Haoyu , Xu Hao , Guo Xiaobing , Liu Ruishan , Li Chenyu , Chen Ruyan , Gou Jianjun , Chen Mantao , Zheng Beiwen 

TITLE=Coexistence of blaIMP-4, blaNDM-1 and blaOXA-1 in blaKPC-2-producing Citrobacter freundii of clinical origin in China

JOURNAL=Frontiers in Microbiology

VOLUME=Volume 14 - 2023

YEAR=2023

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2023.1074612

DOI=10.3389/fmicb.2023.1074612

ISSN=1664-302X

ABSTRACT=Purpose: To explore the genetic characteristics of the IMP-4, NDM-1, OXA-1 and KPC-2 co-producing multidrug-resistant (MDR) clinical isolate, Citrobacter freundii wang9.
Methods: MALDI-TOF MS was used for species identification. PCR and Sanger sequencing analysis were used to identify resistance genes. In addition to agar dilution, broth microdilution was used for antimicrobial susceptibility testing (AST). Whole-genome sequencing (WGS) analysis was conducted using the Illumina NovaSeq 6000 and the Oxford Nanopore platforms. Annotation was performed by RAST and Prokka. Discovery of insertion sequences, transposons and integrons was performed by ISfinder and INTEGRALL. Molecular characterization and transferability of plasmids were studied by S1-PFGE, conjugation assays, and oriTfinder analysis. Plasmid characterization and structural analysis were conducted using the BLAST Ring Image Generator (BRIG) and Easyfig 2.25. Phylogenetic trees were constructed with maximum likelihood, plotted using MAGA X, and decorated by iTOL.
Results: C. freundii carrying blaKPC-2, blaIMP-4, blaOXA-1, and blaNDM-1 are resistant to most antibiotics, intermediate to tigecycline, and only sensitive to polymyxin B, amikacin, and fosfomycin. The blaIMP-4 coexists with the blaNDM-1 and the blaOXA-1 on a novel transferable plasmid pwang9-1, located on the integron In1337, transposon TnAS3, and integron In2054, respectively. The gene cassette sequence of integron In1337 is IntI1-blaIMP-4-qacG2-aacA4'-catB3Δ, while the gene cassette sequence of In2054 is IntI1-aacA4cr-blaOXA-1-catB3-arr3-qacEΔ1-sul1.The blaNDM-1 is located on the transposon TnAS3, and its sequence is IS91-sul-ISAba14-aph(3')-VI-IS30-blaNDM-1-ble-trpF-dsbD-IS91. The blaKPC-2 is located on the transposon Tn2 of plasmid pwang9-1, and its sequence is klcA-korC-ISkpn6-blaKPC-2-ISkpn27-tnpR-tnpA. Phylogenetic analysis showed that most of the 34 C. freundii isolates from China were divided into five clusters. Among them, wang1 and wang9 belong to the same cluster as two strains of C. freundii from environmental samples from Zhejiang.
Conclusion: We found C. freundii carrying blaIMP–4, blaNDM–1, blaOXA-1 and blaKPC-2 for the first time, and conducted in-depth research on its drug resistance mechanism, molecular transfer mechanism and epidemiology. In particular, we found that blaIMP-4, blaOXA-1, and blaNDM-1 coexisted on a new transferable hybrid plasmid that carried many drug resistance genes and insertion sequences. The plasmid may capture more resistance genes, raising our concern about the emergence of new resistance strains.