Rapid detection and differentiation of mobile colistin resistance (mcr-1 to mcr-10) genes by real-time PCR and melt-curve analysis

Mentasti, M., David, S., Sands, K., Khan, S., Davies, L., Turner, L. and Wootton, M. 2021. Rapid detection and differentiation of mobile colistin resistance (mcr-1 to mcr-10) genes by real-time PCR and melt-curve analysis. Journal of Hospital Infection 110 , pp. 148-155. 10.1016/j.jhin.2021.01.010

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Abstract

Background The emergence of multi-drug-resistant (MDR) micro-organisms prompted new interest in older antibiotics, such as colistin, that had been abandoned previously due to limited efficacy or high toxicity. Over the years, several chromosomal-encoded colistin resistance mechanisms have been described; more recently, 10 plasmid-mediated mobile colistin resistance (mcr) genes have been identified. Spread of these genes among MDR Gram-negative bacteria is a matter of serious concern; therefore, reliable and timely mcr detection is paramount. Aim To design and validate a multiplex real-time polymerase chain reaction (PCR) assay for detection and differentiation of mcr genes. Methods All available mcr alleles were downloaded from the National Center for Biotechnology Information Reference Gene Catalogue, aligned with Clustal Omega and primers designed using Primer-BLAST. Real-time PCR monoplexes were optimized and validated using a panel of 120 characterized Gram-negative strains carrying a wide range of resistance genes, often in combination. Melt-curve analysis was used to confirm positive results. Findings In-silico analysis enabled the design of a ‘screening’ assay for detection of mcr-1/2/6, mcr-3, mcr-4, mcr-5, mcr-7, mcr-8 and mcr-9/10, paired with an internal control assay to discount inhibition. A ‘supplementary’ assay was subsequently designed to differentiate mcr-1, mcr-2, mcr-6, mcr-9 and mcr-10. Expected results were obtained for all strains (100% sensitivity and specificity). Melt-curve analysis showed consistent melting temperature results. Inhibition was not observed. Conclusions The assay is rapid and easy to perform, enabling unequivocal mcr detection and differentiation even when more than one variant is present. Adoption by clinical and veterinary microbiology laboratories would aid the surveillance of mcr genes amongst Gram-negative bacteria.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Medicine
Publisher:	Elsevier
ISSN:	0195-6701
Date of First Compliant Deposit:	9 January 2023
Date of Acceptance:	13 January 2021
Last Modified:	10 May 2023 03:30
URI:	https://orca.cardiff.ac.uk/id/eprint/150559

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