Abstract
Plasmid-mediated colistin-resistance genes have been reported worldwide in recent years. A multiplex polymerase chain reaction (Multi-PCR) protocol was developed to detect transferable colistinresistance genes (mcr-1 to mcr-6) in Enterobacteria for clinical laboratory purposes.The authors first designed six new primer pairs to amplify mcr-1 to mcr-6 gene products to achieve stepwise separation of amplicons between 87 to 216 bp,then divided these primers into two subgroups with the assistance of a pair of universal primers for the detection of currently described mcr genes and their variants in Enterobacteria. The protocol was validated by testing 29 clinical isolates of Escherichia coli of human origin, each well characterised 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. It was able to detect mcr-3 and mcr-4 as singletons or in combination. This type of test is critical for the epidemiological surveillance of plasmid-encoded resistance in limited resources conditions, and this method allows rapid identification of mcr-positive bacteria and overcomes the challenges of phenotypic detection of colistin resistance.
Highlights
Colistin has been used in veterinary medicine worldwide, but human use is restricted in terms of its nephrotoxicity and neurotoxicity [1,2,3]
We firstly designed six pairs of primers targeting on six mcr gene types, and artificially synthesized new primers by adding individual nucleotides as the universal bacteria primers to the 5′ end of the chain while do not change the targeting sites
We separately test the efficiency of each primer pairs via polymerase chain reaction (PCR) procedure described in section 2.3.for the primers without bacterial universal primers, the Tm peak of mcr1 was found at 84.8±0.01 °C, the Tm peak of mcr-2 was detected at 85.2±0.01 °C, the Tm peak of mcr-5 was observed at 84.1±0.01 °C, the Tm peak of mcr-6 was found at 83.3±0.02 °C, the Tm peak of mcr-3 was detected at 80.7±0.01 °C, and the Tm peak of mcr-4 was observed at 74.8±0.06 °C
Summary
Colistin (polymyxin E) has been used in veterinary medicine worldwide, but human use is restricted in terms of its nephrotoxicity and neurotoxicity [1,2,3]. Since the first report of mcr-1 [2], mobile colistinresistance (MCR) genes have been found in many clinical and environmental samples. The regular surveillance of these human isolated MCR genes help the appropriate use of colistin in human and animals. Since the positive growth on selective enrichment media was not equivalent to the presence of mcr genes, neither phenotypic resistance nor growth on colistin selective media are perfect indicators for the presence of mcr genes [16], molecular detection methods that targets specific genes are needed. Considering the global concerns, we sought to develop and validate a rapid, efficient and inexpensive method that enables screening of relevant isolates and identification of those clinical samples that may harbour plasmid-mediated colistin-resistance genes
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