Abstract
Plasmid-mediated colistin-resistance genes have been reported in human origin clinical samples worldwide which raises its threats to human infections. Notably, mcr-1, mcr-3, mcr-8, and mcr-10 have been reported isolated directly from clinical samples which creates more seriously threaten to human health than other mcr gene types. A multiplex polymerase chain reaction (Multi-PCR) protocol was developed to detect and genotype mobile colistin-resistance genes (mcr-1, mcr-3, mcr-8, mcr-10) in Enterobacteria for clinical laboratory purposes. We first designed four pairs of new primers for the amplification of mcr-1, mcr-3, mcr-8, and mcr-10 gene respectively to achieve stepwise separation of amplicons between 216 and 241 bp, and complete this Multi-PCR system with the assistance of another pair of universal primer. Among which the forward primers for mcr-8 and mcr-10 amplicons were identical. The protocol was validated by testing 11 clinical isolates of Escherichia coli and 3 clinical isolates of Klebsiella from human origin, each well characterized and prospectively validated. The Multi-PCR assay showed full concordance with whole-genome sequence data and displayed higher sensitivity and 100% specificity. The assay could detect all variants of the various mcr alleles described. The Multi-PCR assay successfully genotyped of mcr alleles described in one test.
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We appreciated Miss Changfeng Peng for her assistance improving the language of manuscript.
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This work was supported by funding from the National Key Research and Development Program of China (No. 2018YFD0500303 and 2019YFC1605104), the China Postdoctoral Science Foundation (No. 2018M643202), the Sanming Project of Medicine in Shenzhen (SZSM201611068), and the Shenzhen Science and Technology Planning Project (JCYJ20170413101841798 and JCYJ20190807103003724).
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HSF performed microbiological and molecular experiments, analyzed the data, and wrote the manuscript. HSF, LZQ and KYB acquired laboratory data and performed bioinformatics analysis. HSF, WY, and SJZ designed the study, wrote, reviewed, and edited the manuscript.
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Hu, S., Lv, Z., Wang, Y. et al. Rapid detection of human origin colistin-resistance genes mcr-1, mcr-3, mcr-8, mcr-10 in clinical fecal samples. Arch Microbiol 203, 4405–4417 (2021). https://doi.org/10.1007/s00203-021-02407-2
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DOI: https://doi.org/10.1007/s00203-021-02407-2