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.

Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes / 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. - In: EUROSURVEILLANCE. - ISSN 1560-7917. - 23:6(2018). [10.2807/1560-7917.ES.2018.23.6.17-00672]

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

Carattoli A;
2018

Abstract

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.
2018
Colistin; antiinfective agent; colistin; Escherichia coli protein; 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; phosphotransferase, amplicon; antibiotic resistance; Article; bacterial gene; bacterium isolate; calf (bovine); controlled study; Escherichia coli; France; gene amplification; gene identification; genetic variability; Germany; Italy; mcr 1 gene; mcr 2 gene; mcr 3 gene; mcr 4 gene; mcr 5 gene; minimum inhibitory concentration; multiplex polymerase chain reaction; nonhuman; pig; plasmid; process development; Salmonella; Salmonella enterica serovar Paratyphi B; Spain; whole genome sequencing; drug effect; Enterobacteriaceae; Enterobacteriaceae infection; genetics; human; isolation and purification; metabolism; microbial sensitivity test; microbiology; multiplex polymerase chain reaction; plasmid, Anti-Bacterial Agents; Colistin; Enterobacteriaceae; Enterobacteriaceae Infections; Escherichia coli Proteins; Humans; Membrane Proteins; Microbial Sensitivity Tests; Multiplex Polymerase Chain Reaction; Plasmids; Salmonella; Transferases (Other Substituted Phosphate Groups)
01 Pubblicazione su rivista::01a Articolo in rivista
Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes / 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. - In: EUROSURVEILLANCE. - ISSN 1560-7917. - 23:6(2018). [10.2807/1560-7917.ES.2018.23.6.17-00672]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11573/1284223
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