Microbiology, Department of Medicine, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK.
J Antimicrob Chemother. 2012 Feb;67(2):404-14. doi: 10.1093/jac/dkr472. Epub 2011 Nov 17.
Tuberculosis drug development is hampered by the slow growth of Mycobacterium tuberculosis. Bioluminescence, light produced by an enzymatic reaction, constitutes a rapid and highly sensitive measurement of cell metabolic function that can be used as an indirect marker of cell viability in drug screening assays. The aim of this work was to validate and standardize the use of luminescent M. tuberculosis strains to test the activity of antibacterial drugs in vitro and inside macrophages in a 96-well format.
We have used strains that express the bacterial lux operon and therefore do not require exogenous substrate to produce light, as well as strains expressing the firefly luciferase that need luciferin substrate. Results were compared with those obtained using the resazurin reduction assay and cfu plating.
Using bioluminescence we were able to reduce the time required to measure the MIC and bactericidal concentrations of antimicrobials to just 3 and 6 days, respectively. Furthermore, antibacterial activity against intracellular mycobacteria was detected within 2 days post-infection. Results were comparable to those obtained by conventional methods.
We have developed a simple and rapid method for screening antimycobacterial drugs in culture and in macrophages. The use of autoluminescent bacteria also facilitates the determination of growth and inhibition kinetics. The method is cost-effective, can easily be adapted to a larger scale and is amenable to automation. Current efforts are directed towards applying this technology to drug screening in vivo.
分枝杆菌缓慢的生长速度阻碍了抗结核药物的研发。生物发光是一种酶促反应产生的光，可作为细胞代谢功能的快速、高灵敏度测量指标，可作为药物筛选测定中细胞活力的间接标志物。本研究旨在验证和标准化发光分枝杆菌菌株在 96 孔格式中体外和巨噬细胞内检测抗菌药物活性的用途。
我们使用了表达细菌 lux 操纵子的菌株，因此不需要外源性底物来产生光，以及表达荧光素酶的菌株，需要 luciferin 底物。结果与使用 Resazurin 还原测定法和 CFU 平板获得的结果进行了比较。
使用生物发光，我们能够将测量 MIC 和杀菌浓度所需的时间分别缩短至 3 天和 6 天。此外，在感染后 2 天内即可检测到针对细胞内分枝杆菌的抗菌活性。结果与传统方法相当。
我们开发了一种简单快速的方法，用于在培养物和巨噬细胞中筛选抗分枝杆菌药物。自发光细菌的使用也便于确定生长和抑制动力学。该方法具有成本效益，易于扩展，并且适用于自动化。目前的努力方向是将该技术应用于体内药物筛选。
