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.
European Food Safety Authority, Parma, Italy.
Euro Surveill. 2018 Feb;23(6). doi: 10.2807/1560-7917.ES.2018.23.6.17-00672.
Background and aimPlasmid-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 ( to , and variants) in was developed for surveillance or research purposes. We designed four new primer pairs to amplify , , and gene products and used the originally described primers for 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 genes and their variants present in . The protocol was validated testing 49 European and 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 and as singletons or in different combinations as they were present in the test isolates. One new variant, , was also identified. This method allows rapid identification of -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.
背景与目的
在过去的几年中，世界各地已经发现了质粒介导的多黏菌素耐药机制。为了监测或研究目的，我们开发了一种多重聚合酶链反应（PCR）方案，用于检测中所有已知的可转移多黏菌素耐药基因（mcr-1 至 mcr-9 和变体）。我们设计了四个新的引物对来扩增 mgrB、pmrD、phoP 和 lpxM 基因产物，并使用最初描述的引物来获得大约 200 bp 之间的扩增子的逐步分离。这些引物对和扩增条件允许对所有目前描述的 mcr-1 基因及其变体进行单一或多重检测，这些基因及其变体存在于中。该方案通过测试 49 株来自欧洲动物源的 和 分离株进行了验证。来自西班牙、德国、法国和意大利的牛和猪分离株的多重 PCR 结果与全基因组序列数据完全一致。该方法能够检测到 mcr-1 作为单基因或不同组合存在于测试分离株中。还鉴定了一种新的 mcr-1 变体，。该方法允许快速鉴定阳性细菌，并克服了多黏菌素耐药表型检测的挑战。对于资源有限的环境或实验室来说，多重 PCR 特别有趣，因为它可以在不需要基因组测序的情况下提供有关多黏菌素耐药机制的信息。
