Wang Honghui, Chen Yong, Strich Jeffrey R, Drake Steven K, Youn Jung-Ho, Rosenberg Avi Z, Gucek Marjan, McGann Patrick T, Suffredini Anthony F, Dekker John P
1Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, MD USA.
2Proteomics Core Facility, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD USA.
Clin Proteomics. 2019 Feb 26;16:8. doi: 10.1186/s12014-019-9228-2. eCollection 2019.
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 - 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).
MCR-1 tryptic peptides that were efficiently ionized and readily detectable were characterized in a set of --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 --containing clinical isolates tested in triplicate (9 samples) and 90 negative control isolates.
All of the --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.
This work demonstrates the feasibility of rapid detection of the MCR-1 protein in bacterial isolates by LC-MS/MS.
黏菌素（多黏菌素E）和多黏菌素B是用于治疗由多重耐药革兰氏阴性菌引起的严重感染的重要杀菌抗生素。由mcr - 1基因产物介导的可转移质粒介导的黏菌素耐药性已成为全球医疗保健的一大威胁。因此，在临床细菌分离株中快速检测MCR - 1蛋白变得越来越重要。我们采用基因蛋白质组学方法来鉴定MCR - 1蛋白的独特肽段，这些肽段可通过液相色谱串联质谱（LC - MS/MS）快速检测。
在一组含有mcr - 1的分离株中，用三重四极杆液相色谱 - 质谱对能有效电离且易于检测的MCR - 1胰蛋白酶肽段进行表征。选择了三个最佳肽段用于开发针对MCR - 1蛋白的快速多反应监测LC - MS/MS检测方法。为了研究使用该检测方法在细菌分离株中快速检测MCR - 1蛋白的可行性，构建了一个包含99个样本的盲测集，其中包括另外三个含mcr - 1的临床分离株，每个分离株重复检测三次（共9个样本）以及90个阴性对照分离株。
测试集中所有含mcr - 1的分离株均被三位独立的盲法操作人员准确鉴定，无假阳性检测结果，多位操作人员的总体灵敏度和特异性均为100%。在本研究中测试的三个肽段中，表现最佳的是DTFPQLAK。该检测方法从分离株到得出结果的时间不到90分钟。
这项工作证明了通过LC - MS/MS在细菌分离株中快速检测MCR - 1蛋白的可行性。
