Department of Medical Microbiology, Leiden University Medical Center, PO Box 9600, 2300 RC, Leiden, The Netherlands.
Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center, 3015 CN, Rotterdam, The Netherlands.
Eur J Clin Microbiol Infect Dis. 2017 Sep;36(9):1621-1628. doi: 10.1007/s10096-017-2975-y. Epub 2017 Apr 10.
Rapid bacterial species identification and antibiotic susceptibility testing in positive blood cultures have an important impact on the antibiotic treatment for patients. To identify extended-spectrum beta-lactamases (ESBL) directly in positive blood culture bottles, we developed a workflow of saponin extraction followed by a bottom-up proteomics approach using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The workflow was applied to positive blood cultures with Escherichia coli and Klebsiella pneumoniae collected prospectively in two academic hospitals over a 4-month period. Of 170 positive blood cultures, 22 (12.9%) contained ESBL-positive isolates based on standard susceptibility testing. Proteomic analysis identified CTX-M ESBLs in 95% of these isolates directly in positive blood cultures, whereas no false positives were found in the non-ESBL producing positive blood cultures. The results were confirmed by molecular characterisation of beta-lactamase genes. Based on this proof-of-concept study, we conclude that LC-MS/MS-based protein analysis can directly identify extended-spectrum beta lactamases in E. coli and K. pneumoniae positive blood cultures, and could be further developed for application in routine diagnostics.
快速鉴定血培养中的细菌种类和药敏试验对患者的抗生素治疗有重要影响。为了直接在阳性血培养瓶中鉴定超广谱β-内酰胺酶（ESBL），我们开发了一种皂素提取工作流程，然后采用液相色谱-串联质谱（LC-MS/MS）进行自下而上的蛋白质组学分析。该工作流程应用于前瞻性收集的来自两个学术医院的在 4 个月期间的大肠杆菌和肺炎克雷伯菌阳性血培养物。在 170 份阳性血培养物中，根据标准药敏试验，22 份（12.9%）含有 ESBL 阳性分离株。蛋白质组学分析直接在阳性血培养物中鉴定出 95%的这些分离株中的 CTX-M ESBL，而在非 ESBL 产生的阳性血培养物中未发现假阳性。通过对β-内酰胺酶基因的分子特征分析确认了结果。基于这项概念验证研究，我们得出结论，基于 LC-MS/MS 的蛋白质分析可以直接鉴定大肠杆菌和肺炎克雷伯菌阳性血培养物中的超广谱β-内酰胺酶，并且可以进一步开发用于常规诊断。
