Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes

2018

EUROSURVEILLANCE

Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes

01 Pubblicazione su rivista

Rebelo Ar, Bortolaia V, Kjeldgaard Js, Pedersen Sk, Leekitcharoenphon P, Hansen Im, Guerra B, Malorny B, Borowiak M, Hammerl Ja, Battisti A, Franco A, Alba P, Perrin-Guyomard A, Granier Sa, de Frutos C, Escobar and Malhotra-Kumar S, Villa L, Carattoli A, Hendriksen Rs

DOI: 10.2807/1560-7917.ES.2018.23.6.17-00672

ISSN: 1560-7917

Background and aim: Plasmid-mediated colistin resistance mechanisms have been identified worldwide in the past years. A multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes (mcr-1 to mcr-5, and variants) in Enterobacteriaceae was developed for surveillance or research purposes. Methods: We designed four new primer pairs to amplify mcr-1, mcr-2, mcr-3 and mcr-4 gene products and used the originally described primers for mcr-5 to obtain a stepwise separation of ca 200 bp between ampli-cons. The primer pairs and amplification conditions allow for single or multiple detection of all currently described mcr genes and their variants present in Enterobacteriaceae. The protocol was validated testing 49 European Escherichia coli and Salmonella isolates of animal origin. Results: Multiplex PCR results in bovine and porcine isolates from Spain, Germany, France and Italy showed full concordance with whole genome sequence data. The method was able to detect mcr-1, mcr-3 and mcr-4 as singletons or in different combinations as they were present in the test isolates. One new mcr-4 variant, mcr-4.3, was also identified. Conclusions: This method allows rapid identification of mcr-positive bacteria and overcomes the challenges of phenotypic detection of colistin resistance. The multiplex PCR should be particularly interesting in settings or laboratories with limited resources for performing genetic analysis as it provides information on the mechanism of colistin resistance without requiring genome sequencing. © 2018, European Centre for Disease Prevention and Control (ECDC). All rights reserved.

antibiotic resistance antiinfective agent bacterial gene bacterium isolate calf (bovine) controlled study drug effect Enterobacteriaceae Enterobacteriaceae infection Escherichia coli Escherichia coli protein Escherichia coli Proteins France gene amplification gene identification genetic variability Germany Human isolation and purification Italy mcr 1 gene mcr 2 gene mcr 3 gene mcr 4 gene mcr 5 gene MCR-1 protein E coli mcr-2 protein E coli MCR-3 protein E coli MCR-4 protein E coli MCR-5 protein E coli membrane protein Membrane Proteins metabolism microbial sensitivity test microbiology minimum inhibitory concentration multiplex polymerase chain reaction nonhuman phosphotransferase amplicon pig plasmid plasmid Anti-Bacterial Agents process development Salmonella Salmonella enterica serovar Paratyphi B Spain Transferases (Other Substituted Phosphate Groups)