Monteferrante Carmine G, Sultan Sadaf, Ten Kate Marian T, Dekker Lennard J M, Sparbier Katrin, Peer Markus, Kostzrewa Markus, Luider Theo M, Goessens Wil H F, Burgers Peter C
Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center, Rotterdam, The Netherlands.
Department of Neurology, Erasmus University Medical Center, Rotterdam, The Netherlands.
J Antimicrob Chemother. 2016 Oct;71(10):2856-67. doi: 10.1093/jac/dkw208. Epub 2016 Jun 10.
Carbapenemase-resistant bacteria are increasingly spreading worldwide causing public concern due to their ability to elude antimicrobial treatment. Early identification of these bacteria is therefore of high importance. Here, we describe the development of a simple and robust protocol for the detection of carbapenemase activity in clinical isolates of Enterobacteriaceae, suitable for routine and clinical applications.
The final protocol involves cellular lysis and enzyme extraction from a defined amount of bacterial cells followed by the addition of a benchmark drug (e.g. the carbapenem antibiotic imipenem or ertapenem). Carbapenem inactivation is mediated by enzymatic hydrolysis (cleavage) of the β-lactam common structural motif, which can be detected using MALDI-TOF MS.
A total of 260 strains were studied (208 carbapenemase producers and 52 non-carbapenemase producers) resulting in 100% sensitivity and 100% specificity for the KPC, NDM and OXA-48-like PCR-confirmed positive isolates using imipenem as benchmark. Differences between the benchmark (indicator) antibiotics imipenem and ertapenem, buffer constituents and sample preparation methods have been investigated. Carbapenemase activity was further characterized by performing specific inhibitor experiments. Intraday and interday reproducibility (coefficient of variation) of the observed hydrolysis results were 15% and 30%, respectively. A comparative study of our extraction method and a recently published method using whole bacterial cells is presented and differences are discussed.
Using this method, an existing carbapenemase activity can be directly read from the mass spectrum as a ratio of hydrolysed product and substrate, setting an important step towards routine application in clinical laboratories.
耐碳青霉烯酶细菌在全球范围内日益扩散，因其能够逃避抗菌治疗而引起公众关注。因此，尽早识别这些细菌至关重要。在此，我们描述了一种简单且可靠的方案，用于检测肠杆菌科临床分离株中的碳青霉烯酶活性，适用于常规和临床应用。
最终方案包括从一定量的细菌细胞中进行细胞裂解和酶提取，随后添加一种基准药物（如碳青霉烯抗生素亚胺培南或厄他培南）。碳青霉烯的失活是由β-内酰胺共同结构基序的酶促水解（裂解）介导的，这可以使用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）进行检测。
共研究了260株菌株（208株碳青霉烯酶产生菌和52株非碳青霉烯酶产生菌），以亚胺培南作为基准时，对KPC、NDM和OXA-48样PCR确认的阳性分离株的灵敏度和特异性均为100%。研究了基准（指示）抗生素亚胺培南和厄他培南、缓冲液成分和样品制备方法之间的差异。通过进行特异性抑制剂实验进一步表征了碳青霉烯酶活性。观察到的水解结果的日内和日间重现性（变异系数）分别为15%和30%。本文还展示了我们的提取方法与最近发表的使用全细菌细胞的方法的比较研究，并讨论了差异。
使用该方法，可以直接从质谱图中读取现有的碳青霉烯酶活性，作为水解产物与底物的比率，这为临床实验室的常规应用迈出了重要一步。
