Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA.
Microbiology Service, Department of Laboratory Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA.
Sci Rep. 2017 May 31;7(1):2531. doi: 10.1038/s41598-017-02749-2.
Carbapenemase producing organisms (CPOs) represent an urgent public health threat, and the need for new rapid methods to detect these organisms has been widely recognized. CPOs carrying the Klebsiella pneumoniae carbapenemase (bla ) gene have caused outbreaks globally with substantial attributable mortality. Here we describe the validation of a rapid MS method for the direct detection of unique tryptic peptides of the KPC protein in clinical bacterial isolates with an isolate-to-result time of less than 90 minutes. Using a genoproteomic discovery approach that combines theoretical peptidome analysis and liquid chromatography-tandem MS (LC-MS/MS), we selected three high abundance peptide markers of the KPC protein that can be robustly detected following rapid tryptic digestion. Protein BLAST analysis confirmed that the chosen peptide markers were unique to KPC. A blinded validation set containing 20 KPC-positive and 80 KPC-negative clinical isolates, performed in triplicate (300 runs) demonstrated 100% sensitivity and 100% specificity (60/60 positive identifications, 240/240 negative identifications) using defined rules for positive calls. The most robust tryptic peptide marker in the validation was LTLGSALAAPQR. The peptide discovery and detection methods validated here are general and should be broadly applicable to allow the direct and rapid detection of other resistance determinants.
产碳青霉烯酶的生物体（CPO）是一种紧迫的公共卫生威胁，因此广泛需要新的快速方法来检测这些生物体。携带肺炎克雷伯氏菌碳青霉烯酶（bla ）基因的 CPO 已在全球范围内引发了爆发，其病死率相当高。在此，我们描述了一种快速 MS 方法的验证，该方法可在 90 分钟内直接检测临床细菌分离物中 KPC 蛋白的独特胰蛋白酶肽，从而实现从分离物到结果的快速转化。使用一种结合理论肽组学分析和液相色谱-串联质谱（LC-MS/MS）的基因蛋白质组学发现方法，我们选择了三个 KPC 蛋白的高丰度肽标记物，这些标记物在快速胰蛋白酶消化后可以可靠地检测到。蛋白质 BLAST 分析证实，所选的肽标记物是 KPC 所特有的。在盲法验证中，包含 20 个 KPC 阳性和 80 个 KPC 阴性的临床分离物，在三重复（300 次运行）中，使用阳性判断的明确定义规则，灵敏度为 100%，特异性为 100%（60/60 次阳性鉴定，240/240 次阴性鉴定）。验证中最稳健的胰蛋白酶肽标记物是 LTLGSALAAPQR。这里验证的肽发现和检测方法是通用的，应该广泛适用于直接快速检测其他耐药决定因素。
