[Source: Eurosurveillance, full page: (LINK). Abstract, edited.]
Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes
Ana Rita Rebelo1, Valeria Bortolaia1, Jette S Kjeldgaard1, Susanne K Pedersen1, Pimlapas Leekitcharoenphon1, Inge M Hansen1, Beatriz Guerra2, Burkhard Malorny3, Maria Borowiak3, Jens Andre Hammerl3, Antonio Battisti4, Alessia Franco4, Patricia Alba4, Agnes Perrin-Guyomard5, Sophie A Granier6, Cristina De Frutos1,2,3,4,5,6,7,8,9, Escobar7, Surbhi Malhotra-Kumar8, Laura Villa9, Alessandra Carattoli9, Rene S Hendriksen1
Affiliations: 1 National Food Institute, Technical University of Denmark, WHO Collaborating Center for Antimicrobial Resistance in Food borne Pathogens and European Union Reference Laboratory for Antimicrobial Resistance, Kongens Lyngby, Denmark; 2 European Food Safety Authority, Parma, Italy; 3 German Federal Institute for Risk Assessment, Berlin, Germany; 4 National Reference Laboratory for antimicrobial resistance, Istituto Zooprofilattico Sperimentale del Lazio e della Toscana, Rome, Italy; 5 Anses, Fougères Laboratory, Fougères, France; 6 Université Paris-Est, Anses, Laboratory for Food Safety, Maisons-Alfort, France; 7 Laboratorio Central de Veterinaria, (LCV Algete), Madrid, Spain; 8 Laboratory of Medical Microbiology, Vaccine & Infectious Disease Institute, University of Antwerp, Wilrijk, Belgium; 9 Department of Infectious Diseases, Istituto Superiore di Sanità, Rome, Italy
Correspondence: Rene S. Hendriksenrshe food.dtu.dk
Citation style for this article: Rebelo Ana Rita, Bortolaia Valeria, Kjeldgaard Jette S, Pedersen Susanne K, Leekitcharoenphon Pimlapas, Hansen Inge M, Guerra Beatriz, Malorny Burkhard, Borowiak Maria, Hammerl Jens Andre, Battisti Antonio, Franco Alessia, Alba Patricia, Perrin-Guyomard Agnes, Granier Sophie A, De Frutos Cristina , Escobar, Malhotra-Kumar Surbhi, Villa Laura, Carattoli Alessandra, Hendriksen Rene S. Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes. Euro Surveill. 2018;23(6):pii=17-00672. https://doi.org/10.2807/1560-7917.ES.2018.23.6.17-00672
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.
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 amplicons. 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.
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.
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.
© This work is licensed under a Creative Commons Attribution 4.0 International License.
Keywords: Antibiotics; Drugs Resistance; Colistin; MCR1; MCR2; MCR3; MCR4; Diagnostic Tests.