基于受激拉曼散射显微技术的抗生素对细菌代谢的抑制机理研究

Rapid identification of the susceptibility of pathogenic bacteria to antibiotics to ensure the use of effective antibiotics is a key problem in clinical practice. Currently the conventional method to obtain bacterial susceptibility to antibiotics in clinical is the culture-based antimicrobial susceptibility testing (AST), which is reliable but takes long time, therefore fails to provide guidance for the early treatment of severe bacterial infections. In this project we propose a rapid AST method based on metabolic activity measurement in bacteria. We will first use stimulated Raman scattering (SRS) microscopy and deuterium labeling technology to detect and analyse water metabolism in single bacteria quantatively by measuring the carbon-deuterium (C-D) chemical bond formed from heavy water (D2O) metabolism in bacteria. With this technique, we will then study the inhibition of water metabolism by antibiotics and the mechanism of inhibiton through inhibiting lipids, proteins or other metabolites. Finally, we will develop a rapid AST based on SRS microscopy and water metabolism, and verify the feasibility of this method in clinical application. The implementation of this project will provide new technical means and theoretical basis for understanding the interaction between antibiotics and bacterial metabolism and developing new and rapid AST methods.

快速识别致病菌对抗生素药物的敏感性，以确保使用有效的抗生素，是临床上急需解决的关键问题。目前临床上获得细菌的药物敏感性的常规方法是基于体外培养的抗菌药敏试验，其优点是可靠性高，缺点是耗时长，难以对临床上重症细菌感染的早期治疗提供用药指导。本项目将利用细菌快速代谢的特点来研究通过检测细菌的水代谢来实现快速的药敏试验。拟首先利用受激拉曼散射（SRS）显微技术与氘代标记技术的结合，通过探测重水（D2O）参与细菌代谢后形成的碳-氘（C-D）化学键，实现对单细菌水代谢的高速实时检测和定量分析；然后基于该技术研究抗生素对细菌水代谢的抑制作用，揭示其是通过抑制脂质、蛋白质还是其它代谢物的合成来抑制水代谢的作用机理；最后提出基于SRS成像和水代谢的快速药敏试验方法，并验证该方法在临床上应用的可行性。本项目的实施将为理解抗生素与细菌代谢的相互作用及机理，发展快速药敏试验提供新的技术手段和理论依据。

快速识别致病菌对抗生素药物的敏感性，以确保使用有效的抗生素，是临床上急需解决的关键问题。目前临床上获得细菌药物敏感性的常规方法是基于体外培养的抗菌药敏试验，其优点是可靠性高，缺点是耗时长，难以对临床上重症细菌感染的早期治疗提供用药指导。本项目利用细菌快速代谢的特点研究了通过检测细菌的重水（D2O）代谢来实现快速药敏试验的可行性。首先利用受激拉曼散射（SRS）显微技术与氘代标记技术的结合，通过检测重水参与细菌代谢后形成的碳-氘（C-D）化学键，建立了基于SRS显微技术和重水标记的细菌水代谢定量分析方法。利用该方法研究了抗生素对细菌水代谢的抑制作用，建立了基于SRS显微技术和细菌水代谢的快速药敏试验方法。最后将所开发的快速药敏方法应用到了尿路感染和菌血症临床样品，实现了临床样本的直接药敏检测。研究结果显示：SRS与重水标记的结合可以快速检测并量化细菌的代谢活性；基于SRS代谢成像技术可以在2小时内快速测定抗生素对临床常见细菌的药物敏感性，准确度超过90%。本项目的研究为尿路感染和菌血症临床样本的快速诊断提供了新的技术手段和理论依据。