Background Colistin (polymyxin E) and polymixin B are important bactericidal antibiotics used in the treatment of serious infections caused by multi-drug resistant Gram-negative organisms. Transferrable plasmid-mediated colistin resistance, conferred by the product of the mcr-1 gene, has emerged as a global healthcare threat. Consequently, the rapid detection of the MCR-1 protein in clinical bacterial isolates has become increasingly important. We used a genoproteomic approach to identify unique peptides of the MCR-1 protein that could be detected rapidly by liquid chromatography tandem mass spectrometry (LC-MS/MS). Methods MCR-1 tryptic peptides that were efficiently ionized and readily detectable were characterized in a set of mcr-1-containing isolates with triple quadrupole LC-MS. Three optimal peptides were selected for the development of a rapid multiple reaction monitoring LC-MS/MS assay for the MCR-1 protein. To investigate the feasibility of rapid detection of the MCR-1 protein in bacterial isolates using this assay, a blinded 99-sample test set was built that included three additional mcr-1-containing clinical isolates tested in triplicate (9 samples) and 90 negative control isolates. Results All of the mcr-1-containing isolates in the test set were accurately identified with no false positive detections by three independent, blinded operators, yielding an overall performance of 100% sensitivity and specificity for multiple operators. Among the three peptides tested in this study, the best performing was DTFPQLAK. The isolate-to-result time for the assay as implemented is less than 90 min. Conclusions This work demonstrates the feasibility of rapid detection of the MCR-1 protein in bacterial isolates by LC-MS/MS.

中文翻译：

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LC-MS/MS快速检测粘菌素耐药蛋白MCR-1。

背景粘菌素（多粘菌素 E）和多粘菌素 B 是重要的杀菌抗生素，用于治疗由多重耐药革兰氏阴性菌引起的严重感染。由 mcr-1 基因产物赋予的可转移质粒介导的粘菌素耐药性已成为全球医疗保健威胁。因此，快速检测临床细菌分离物中的 MCR-1 蛋白变得越来越重要。我们使用基因蛋白质组学方法来鉴定可以通过液相色谱串联质谱 (LC-MS/MS) 快速检测的 MCR-1 蛋白的独特肽。方法 使用三重四极杆 LC-MS 在一组含有 mcr-1 的分离物中表征高效电离且易于检测的 MCR-1 胰蛋白酶肽。选择了三种最佳肽用于开发 MCR-1 蛋白的快速多反应监测 LC-MS/MS 测定。为了研究使用该测定法快速检测细菌分离物中 MCR-1 蛋白的可行性，构建了一个盲法 99 样本测试集，其中包括三个额外的含有 mcr-1 的临床分离株，一式三份（9 个样本）和 90 个阴性样本进行测试控制隔离物。结果 测试集中所有含有 mcr-1 的分离株都被三个独立的、不知情的操作员准确识别，没有假阳性检测，对多个操作员产生 100% 的灵敏度和特异性的整体性能。在本研究中测试的三种肽中，表现最好的是 DTFPQLAK。所实施的分析的分离到结果时间少于 90 分钟。