Department of Clinical Laboratory, The Affiliated Hospital of Qingdao University, Qingdao, 266000, Shandong, China.
Department of Laboratory Medicine, The Fourth Affiliated Hospital Zhejiang University School of Medicine, Yiwu, 322000, Zhejiang, China.
J Nanobiotechnology. 2024 Nov 5;22(1):678. doi: 10.1186/s12951-024-02949-9.
The production of β-lactamases is the most prevalent resistance mechanism for β-lactam antibiotics in Gram-negative bacteria. Presently, over 4900 β-lactamases have been discovered, and they are categorized into hundreds of families. In each enzyme family, amino acid substitutions result in subtle changes to enzyme hydrolysis profiles; in contrast, certain conserved sequences retained by all of the family members can serve as important markers for enzyme family identification.
The SHV family was chosen as the study object. First, a unique 10-mer peptide was identified as SHV family's epitope by an approach of protein fingerprint analysis. Then, an SHV-specific magnetic epitope-imprinted gel polymer (MEI-GP) was prepared by an epitope surface imprinting technique, and its sorption behavior and recognition mechanism for template epitope and SHV were both elaborated. Finally, the MEI-GP was successfully applied to selectively extract SHV from bacteria, and the extracted SHV was submitted to MALDI-TOF MS for specific determination. By following this strategy, other β-lactamase families can also be specifically detected. According to the molecular weight displayed in mass spectra, the kind of β-lactamase and its associated hydrolysis profile on β-lactams can be easily identified. Based on this, an initial drug option scheme can be quickly formulated for antimicrobial therapy. From protein extraction to medication guidance reporting, the mean time to detection (MTTD) was less than 2 h, which is much faster than conventional phenotype-based methods (at least 16-20 h) and gene-based techniques (usually about 8 h).
This enzyme-specific detection strategy combined the specificity of epitope imprinting with the sensitivity of mass spectrometry, enabling β-lactamase to be selectively extracted from bacteria and clearly presented in mass spectra. Compared with other drug resistance detection methods, this technique has good specificity, high sensitivity (≤ 15 mg of bacteria), a short MTTD (less than 2 h), and simple operation, and therefore has a broad application prospect in clinical medicine.
β-内酰胺酶的产生是革兰氏阴性菌对β-内酰胺类抗生素产生耐药性的最主要机制。目前，已经发现了超过 4900 种β-内酰胺酶，它们被分为数百个家族。在每个酶家族中，氨基酸取代导致酶水解谱的细微变化；相比之下，所有家族成员保留的某些保守序列可以作为酶家族鉴定的重要标记。
选择 SHV 家族作为研究对象。首先，通过蛋白质指纹分析方法确定了一个独特的 10 肽作为 SHV 家族的表位。然后，通过表位表面印迹技术制备了一种 SHV 特异性磁性表位印迹凝胶聚合物（MEI-GP），并详细阐述了其对模板表位和 SHV 的吸附行为和识别机制。最后，成功地将 MEI-GP 应用于从细菌中选择性提取 SHV，并将提取的 SHV 提交 MALDI-TOF MS 进行特异性测定。通过这种策略，也可以特异性检测其他β-内酰胺酶家族。根据质谱中显示的分子量，可以轻松识别β-内酰胺酶的种类及其对β-内酰胺类药物的水解谱。在此基础上，可以快速制定抗菌治疗的初始药物选择方案。从蛋白质提取到药物指导报告，检测平均时间（MTTD）不到 2 小时，明显快于传统的表型检测方法（至少 16-20 小时）和基因检测技术（通常约 8 小时）。
这种酶特异性检测策略将表位印迹的特异性与质谱的灵敏度相结合，使β-内酰胺酶能够从细菌中选择性提取，并在质谱中清晰呈现。与其他耐药性检测方法相比，该技术具有良好的特异性、高灵敏度（≤15 毫克细菌）、短 MTTD（小于 2 小时）和简单的操作，因此在临床医学中有广阔的应用前景。
