Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence.
Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence.
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DOI：
10.1093/jac/dkr472
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发表时间：
2012-02
期刊：
影响因子：
--
通讯作者：
Robertson BD
中科院分区：
文献类型：
--
作者：
Andreu N;Fletcher T;Krishnan N;Wiles S;Robertson BD
结核病药物的开发受到结核分枝杆菌生长缓慢的阻碍。生物发光，由酶促反应产生的光，构成了细胞代谢功能的快速和高度灵敏的测量，可用作药物筛选测定中细胞活力的间接标志物。这项工作的目的是验证和标准化的使用发光M。结核菌株，以在96孔板中测试体外和巨噬细胞内抗菌药物的活性。我们已经使用了表达细菌lux操纵子的菌株，因此不需要外源底物来产生光，以及表达萤火虫荧光素酶的菌株，其需要荧光素底物。将结果与刃天青还原试验和cfu平板法所得结果进行比较。使用生物发光，我们能够将测量抗菌剂的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天内检测到对细胞内分枝杆菌的抗菌活性。结果与常规方法所得结果相当。我们已经开发了一种简单而快速的方法来筛选培养和巨噬细胞中的抗分枝杆菌药物。自发光细菌的使用也有利于生长和抑制动力学的测定。该方法具有成本效益，可以很容易地适用于更大的规模，并适合于自动化。目前的努力是针对应用该技术在体内药物筛选。
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.
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