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=14

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=<sec><title>Purpose</title><p>To explore the genetic characteristics of the IMP-4, NDM-1, OXA-1, and KPC-2 co-producing multidrug-resistant (MDR) clinical isolate, <italic>Citrobacter freundii</italic> wang9.</p></sec><sec><title>Methods</title><p>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). We performed whole genome sequencing (WGS) of the strains and analyzed the resulting data for drug resistance genes and plasmids. Phylogenetic trees were constructed with maximum likelihood, plotted using MAGA X, and decorated by iTOL.</p></sec><sec><title>Results</title><p><italic>Citrobacter freundii</italic> carrying <italic>bla</italic><sub>KPC-2</sub>, <italic>bla</italic><sub>IMP-4</sub>, <italic>bla</italic><sub>OXA-1</sub>, and <italic>bla</italic><sub>NDM-1</sub> are resistant to most antibiotics, intermediate to tigecycline, and only sensitive to polymyxin B, amikacin, and fosfomycin. The <italic>bla</italic><sub>IMP-4</sub> coexists with the <italic>bla</italic><sub>NDM-1</sub> and the <italic>bla</italic><sub>OXA-1</sub> on a novel transferable plasmid variant pwang9-1, located on the integron In<italic>1337</italic>, transposon Tn<italic>AS3</italic>, and integron In<italic>2054</italic>, respectively. The gene cassette sequence of integron In<italic>1337</italic> is <italic>IntI1-bla</italic><sub>IMP-4</sub><italic>-qacG2-aacA4′-catB3Δ</italic>, while the gene cassette sequence of In<italic>2054</italic> is <italic>IntI1-aacA4cr-bla</italic><sub>OXA-1</sub><italic>-catB3-arr3-qacEΔ1-sul1.</italic> The <italic>bla</italic><sub>NDM-1</sub> is located on the transposon Tn<italic>AS3</italic>, and its sequence is IS<italic>91-sul-</italic>IS<italic>Aba14-aph (3′)-VI-</italic>IS<italic>30-bla</italic><sub>NDM-1</sub><italic>-ble-trpF-dsbD-</italic>IS<italic>91.</italic> The <italic>bla</italic><sub>KPC-2</sub> is located on the transposon Tn<italic>2</italic> of plasmid pwang9-1, and its sequence is <italic>klcA-korC-</italic>IS<italic>kpn6-bla</italic><sub>KPC-2</sub><italic>-</italic>IS<italic>kpn27-tnpR-tnpA.</italic> Phylogenetic analysis showed that most of the 34\u00B0<italic>C. freundii</italic> isolates from China were divided into three clusters. Among them, wang1 and wang9 belong to the same cluster as two strains of <italic>C. freundii</italic> from environmental samples from Zhejiang.</p></sec><sec><title>Conclusion</title><p>We found <italic>C. freundii</italic> carrying <italic>bla</italic><sub>IMP–4</sub>, <italic>bla</italic><sub>NDM–1</sub>, <italic>bla</italic><sub>OXA-1</sub>, and <italic>bla</italic><sub>KPC-2</sub> for the first time, and conducted in-depth research on its drug resistance mechanism, molecular transfer mechanism and epidemiology. In particular, we found that <italic>bla</italic><sub>IMP-4</sub>, <italic>bla</italic><sub>OXA-1</sub>, and <italic>bla</italic><sub>NDM-1</sub> 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.</p></sec>