Data_Sheet_3_Parallel Reaction Monitoring Mass Spectrometry for Rapid and Accurate Identification of β-Lactamases Produced by Enterobacteriaceae.docx

The increasing spread of drug-resistant bacterial strains presents great challenges to clinical antibacterial treatment and public health, particularly with regard to β-lactamase-producing Enterobacteriaceae. A rapid and accurate detection method that can expedite precise clinical diagnostics and rational administration of antibiotics is urgently needed. Targeted proteomics, a technique involving selected reaction monitoring or multiple reaction monitoring, has been developed for detecting specific peptides. In the present study, a rapid single-colony-processing procedure combined with an improved parallel reaction monitoring (PRM) workflow based on HRAM Orbitrap MS was developed to detect carbapenemases (Klebsiella pneumoniae carbapenemase, KPC; imipenemase, IMP; Verona integron-encoded metallo-β-lactamase, VIM; New Delhi metallo-β-lactamase, NDM; and oxacillinase, OXA), extended spectrum β-lactamases (TEM and CTX-M), and AmpC (CMY-2) produced by Enterobacteriaceae. Specific peptides were selected and validated, and their coefficients of variation and stability were evaluated. In total, 188 Enterobacteriaceae strains were screened using the workflow. Fourteen out of total 19 peptides have 100% specificity; three peptides have specificity >95% and two peptides have specificity ranged from 74∼85%. On the sensitivity, only nine peptides have 95∼100% sensitivity. The other 10 peptides have sensitivity ranged from 27∼94%. Thus, a screening method based on peptide groups was developed for the first time. Taken together, this study described a rapid extraction and detection workflow for widespread β-lactamases, including KPC, IMP, VIM, NDM, OXA, CMY, CTX-M, and TEM, using single colonies of Enterobacteriaceae strains. PRM-targeted proteomics was proven to be a promising approach for the detection of drug-resistant enzymes.

耐药菌株的持续扩散给临床抗菌治疗与公共卫生带来了严峻挑战，尤以产β-内酰胺酶的肠杆菌科细菌（Enterobacteriaceae）为甚。目前亟需能够助力精准临床诊断与合理抗生素给药的快速、精准检测方法。靶向蛋白质组学是一种基于选择性反应监测（selected reaction monitoring, SRM）或多重反应监测（multiple reaction monitoring, MRM）的技术，可用于特异性肽段的检测。本研究开发了一种快速单菌落处理流程，并结合基于高分辨准确质量轨道阱质谱（HRAM Orbitrap MS）的改良平行反应监测（parallel reaction monitoring, PRM）工作流，用于检测肠杆菌科细菌产生的碳青霉烯酶、超广谱β-内酰胺酶以及AmpC酶：其中碳青霉烯酶包括肺炎克雷伯菌碳青霉烯酶（Klebsiella pneumoniae carbapenemase, KPC）、亚胺培南酶（imipenemase, IMP）、维罗纳整合子编码金属β-内酰胺酶（Verona integron-encoded metallo-β-lactamase, VIM）、新德里金属β-内酰胺酶（New Delhi metallo-β-lactamase, NDM）与苯唑西林酶（oxacillinase, OXA）；超广谱β-内酰胺酶包括TEM与CTX-M；AmpC酶则包括CMY-2。研究人员筛选并验证了特异性肽段，并对其变异系数与稳定性进行了评估。本研究共使用该工作流筛选了188株肠杆菌科菌株。19条肽段中，14条的特异性达100%；3条肽段的特异性高于95%，剩余2条肽段的特异性介于74%~85%之间。在敏感性方面，仅9条肽段的敏感性介于95%~100%之间，其余10条肽段的敏感性介于27%~94%之间。据此，本研究首次开发了基于肽组的筛查方法。综上，本研究建立了一种基于肠杆菌科细菌单菌落的、针对常见β-内酰胺酶（包括KPC、IMP、VIM、NDM、OXA、CMY、CTX-M与TEM）的快速提取与检测工作流。研究证实，基于PRM的靶向蛋白质组学是一种极具应用前景的耐药酶检测手段。