McGee William M, Faron Matthew L, Neil Jason R, Kronewitter Scott R, Buchan Blake W, Ledeboer Nathan A, Stephenson James L
Thermo Fisher Scientific, Cambridge, MA, United States.
Medical College of Wisconsin, Milwaukee, WI, United States.
Clin Mass Spectrom. 2020 Jul 18;17:12-21. doi: 10.1016/j.clinms.2020.07.001. eCollection 2020 Aug.
Carbapenemase-producing organisms (CPOs) are a growing threat to human health. Among the enzymes conferring antibiotic resistance produced by these organisms, carbapenemase (KPC) is considered to be a growing global health threat. Reliable and specific detection of this antibiotic resistance-causing enzyme is critical both for effective therapy and to mitigate further spread.
The objective of this study is to develop an intact protein mass spectrometry-based method for detection and differentiation of clinically-relevant KPC variants directly from bacterial cell lysates. The method should be specific for any variant expressed in multiple bacterial species, limit false positive results and be rapid in nature to directly influence clinical outcomes.
Lysates obtained directly from bacterial colonies were used for intact protein detection using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Bottom-up and top-down proteomic methods were used to characterize the KPC protein targets of interest. Comparisons between KPC-producing and KPC-non-producing isolates from a wide variety of species were also performed.
Characterization of the mature KPC protein revealed an unexpected signal peptide cleavage site preceding an AXA signal peptide motif, modifying the molecular weight (MW) of the mature protein. Taking the additional AXA residues into account allowed for direct detection of the intact protein using top-down proteomic methods. Further validation was performed by transforming a KPC-harboring plasmid into a negative control strain, followed by MS detection of the KPC variant from the transformed cell line. Application of this approach to clearly identify clinically-relevant variants among several species is presented for KPC-2, KPC-3, KPC-4 and KPC-5.
Direct detection of these enzymes contributes to the understanding of occurrence and spread of these antibiotic-resistant organisms. The ability to detect intact KPC variants via a simple LC-MS/MS approach could have a direct and positive impact on clinical therapy, by providing both direction for epidemiological tracking and appropriate therapy.
产碳青霉烯酶的微生物（CPOs）对人类健康构成的威胁日益增大。在这些微生物产生的赋予抗生素抗性的酶中，碳青霉烯酶（KPC）被认为对全球健康构成的威胁越来越大。可靠且特异性地检测这种导致抗生素抗性的酶对于有效治疗和减轻其进一步传播至关重要。
本研究的目的是开发一种基于完整蛋白质质谱分析的方法，用于直接从细菌细胞裂解物中检测和区分临床相关的KPC变体。该方法应针对多种细菌物种中表达的任何变体具有特异性，限制假阳性结果，并且本质上要快速，以便直接影响临床结果。
直接从细菌菌落获得的裂解物用于通过液相色谱与串联质谱联用（LC-MS/MS）进行完整蛋白质检测。采用自下而上和自上而下的蛋白质组学方法来表征感兴趣的KPC蛋白质靶点。还对来自多种物种的产KPC和不产KPC的分离株进行了比较。
成熟KPC蛋白的表征揭示了在AXA信号肽基序之前存在一个意外的信号肽切割位点，这改变了成熟蛋白的分子量（MW）。考虑到额外的AXA残基，使用自上而下的蛋白质组学方法可以直接检测完整蛋白。通过将携带KPC的质粒转化到阴性对照菌株中进行进一步验证，随后通过质谱检测来自转化细胞系的KPC变体。展示了将该方法应用于明确鉴定几种物种中临床相关变体的情况，包括KPC-2、KPC-3、KPC-4和KPC-5。
直接检测这些酶有助于了解这些抗生素抗性微生物的发生和传播。通过简单的LC-MS/MS方法检测完整KPC变体的能力可为临床治疗带来直接且积极的影响，为流行病学追踪和适当治疗提供指导。
