Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA.
Department of Laboratory Medicine, Clinical Center, Microbiology Service, National Institutes of Health, Bethesda, Maryland, USA.
J Clin Microbiol. 2019 Apr 26;57(5). doi: 10.1128/JCM.01240-18. Print 2019 May.
Phenotypic detection of the OXA-48-type class D β-lactamases in is challenging. We describe a rapid (less than 90 min) assay for the identification of OXA-48 family carbapenemases in subcultured bacterial isolates based on a genoproteomic approach. Following trypsin digestion to ascertain theoretical core peptides common to the OXA-48 family, liquid chromatography-tandem mass spectrometry (LC-MS/MS) data-dependent acquisition was used to identify candidate peptide markers. Two peptides were selected based on performance characteristics: ANQAFLPASTFK, a core peptide common to all 12 OXA-48 family β-lactamase members, and YSVVPVYQEFAR, a highly specific peptide common to 11 of 12 OXA-48 family proteins providing the basis for an LC-MS/MS multiple reaction monitoring assay. An accuracy assessment was performed that included 98 isolates, 26 of which were OXA-48 positive. Two additional specificity assessments were performed including a mixture of isolates positive for OXA-48, KPC, NDM, VIM, and IMP carbapenemases. A combination of expert rules and expert judgment was applied by blinded operators to identify positive isolates. All isolates containing an OXA-48 family carbapenemase across all three test sets were correctly identified with no false positives, demonstrating 100% sensitivity (95% confidence interval [CI], 91.2% to 100%) and 100% specificity (95% CI, 96.2% to 100%) for the assay. These findings provide a framework for an LC-MS/MS-based method for the direct detection of OXA-48 family carbapenemases from cultured isolates that may have utility in predicting carbapenem resistance and tracking hospital outbreaks of OXA-48-carrying organisms.
表型检测 OXA-48 型 D 类β-内酰胺酶具有挑战性。我们描述了一种快速（少于 90 分钟）的基于基因组蛋白质组学方法的亚培养细菌分离物中 OXA-48 家族碳青霉烯酶鉴定的方法。在进行胰蛋白酶消化以确定 OXA-48 家族共有的理论核心肽之后，使用液相色谱-串联质谱（LC-MS/MS）数据依赖采集来鉴定候选肽标记物。根据性能特征选择了两个肽：ANQAFLPASTFK，是所有 12 种 OXA-48 家族β-内酰胺酶成员共有的核心肽，以及 YSVVPVYQEFAR，是 12 种 OXA-48 家族蛋白中 11 种的高度特异性肽，为 LC-MS/MS 多重反应监测测定提供了基础。进行了准确性评估，其中包括 98 种分离物，其中 26 种为 OXA-48 阳性。还进行了另外两项特异性评估，包括 OXA-48、KPC、NDM、VIM 和 IMP 碳青霉烯酶阳性分离物的混合物。盲法操作人员应用专家规则和专家判断来识别阳性分离物。所有三个测试组中包含 OXA-48 家族碳青霉烯酶的分离物均被正确识别，没有假阳性，表明该测定具有 100%的敏感性（95%置信区间[CI]，91.2%至 100%）和 100%的特异性（95%CI，96.2%至 100%）。这些发现为基于 LC-MS/MS 的直接从培养分离物中检测 OXA-48 家族碳青霉烯酶的方法提供了框架，该方法可能有助于预测碳青霉烯类耐药性和追踪携带 OXA-48 的生物体的医院爆发。
