Generation of a novel nucleic acid-based reporter system to detect phenotypic susceptibility to antibiotics in Mycobacterium tuberculosis.
Generation of a novel nucleic acid-based reporter system to detect phenotypic susceptibility to antibiotics in Mycobacterium tuberculosis.
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作者：
Mulvey MC;Sacksteder KA;Einck L;Nacy CA
我们设计、构建并评价了一个原型新型报告系统，该系统由两个功能盒组成：（i）在分枝杆菌中活性启动子的转录控制下的SP 6 RNA聚合酶基因和（ii）指导表达其他未表达序列的共有SP 6聚合酶启动子。我们将报告系统纳入到一个分枝杆菌噬菌体交付到活结核分枝杆菌，并引入导致合成的SP 6聚合酶依赖的替代标记RNA，我们检测到的逆转录酶PCR（RT-PCR）。报告者证实了药物敏感和耐药M的敏感性特征。结核菌株暴露于一线抗结核药物，并且需要暴露于靶向细菌代谢过程的抗菌剂短至16小时以准确读取反应。报告系统将细菌表型翻译成可通过快速且灵敏的核酸检测解释的语言。作为一种仅对活菌M起作用的表型测定。在结核病方面，它可用于快速评估对任何药物的耐药性，包括耐药机制未知或由许多潜在的已知（和未知）遗传改变引起的药物。检测缓慢生长细菌的抗生素耐药性的能力（即，结核分枝杆菌（Mycobacterium tuberculosis）受到两个因素的阻碍，即检测时间（数周至数月）和耐药机制（许多药物未知），从而延迟了耐药或耐多药结核病（TB）患者的适当治疗。本文中描述的新技术使用了由感染M.结核病在不到一天的时间内记录表型抗生素敏感性。
We designed, constructed, and evaluated a prototype novel reporter system comprised of two functional cassettes: (i) the SP6 RNA polymerase gene under transcriptional control of a promoter active in mycobacteria and (ii) the consensus SP6 polymerase promoter that directs expression of an otherwise unexpressed sequence. We incorporated the reporter system into a mycobacteriophage for delivery into viable Mycobacterium tuberculosis, and introduction led to synthesis of an SP6 polymerase-dependent surrogate marker RNA that we detected by reverse transcriptase PCR (RT-PCR). The reporter confirmed the susceptibility profile of both drug-susceptible and drug-resistant M. tuberculosis strains exposed to first-line antitubercular drugs and required as little as 16 h of exposure to antibacterial agents targeting bacterial metabolic processes to accurately read the reaction. The reporter system translated the bacterial phenotype into a language interpretable by rapid and sensitive nucleic acid detection. As a phenotypic assay that works only on viable M. tuberculosis, it could be used to rapidly assess resistance to any drug, including drugs for which the mechanism of resistance is unknown or which result from many potential known (and unknown) genetic alterations. The ability to detect antibiotic resistance of slow-growing bacteria (i.e., Mycobacterium tuberculosis) is hampered by two factors, the time to detection (weeks to months) and the resistance mechanism (unknown for many drugs), delaying the appropriate treatment of patients with drug-resistant or multidrug-resistant tuberculosis (TB). The novel technique described in this article uses a unique surrogate nucleic acid marker produced by phage that infects M. tuberculosis to record phenotypic antibiotic susceptibility in less than a day.