Nanoluciferase reporter phage for rapid phenotypic characterization of resistance to next-generation antimycobacterial agents

纳米荧光素酶报告噬菌体用于快速表征下一代抗分枝杆菌药物的耐药性

• 批准号: 10593796
• 负责人: WILLIAM Robert JACOBS
• 金额: $ 24.17万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10593796
• 关键词: Antibiotic susceptibility; Antimycobacterial Agents; BCL2/Adenovirus E1B 19kd Interacting Protein 3-Like; Bacteriophages; Biological; Biological Assay; Bioluminescence; Caring; Clinical; Cohort Studies; Collaborations; Colony-Forming Units Assay; Complement; Complex; DNA cassette; Data; Detection; Development; Diagnosis; Diagnostic; Disease; Drug resistance; Drug resistance in tuberculosis; Drug resistant Mycobacteria Tuberculosis; Drug usage; Early identification; Energy Transfer; Ensure; Enzymes; Fluorescence; Generations; Genetic; Genotype; Genus Mycobacterium; Growth; Income; Infection; Intermediate resistance; Intervention; Laboratories; Linezolid; Measures; Mediating; Methods; Minimum Inhibitory Concentration measurement; Monitor; Morbidity - disease rate; Multi-Drug Resistance; Multidrug-Resistant Tuberculosis; Mycobacteriophages; Mycobacterium tuberculosis; Oral; Outcome; Patients; Performance; Persons; Pharmaceutical Preparations; Phenotype; Predisposition; Process; Prospective, cohort study; Public Health; Publishing; Reader; Regimen; Reporter; Research; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Rifampicin resistance; Rifampin; Sampling; Scientist; Signal Transduction; South African; Sputum; System; Techniques; Testing; Therapeutic; Time; Treatment Protocols; Treatment outcome; Tuberculosis; Tuberculosis diagnosis; Validation; Virulent; Virus; Work; World Health Organization; bactericide; clinical phenotype; clinically relevant; community transmission; cost; detection limit; detection platform; diagnostic assay; drug repurposing; drug testing; drug-sensitive; experience; experimental study; fluorophore; genome sequencing; global health; implementation science; improved; instrumentation; luminescence; mortality; nanoluciferase; next generation; novel; novel therapeutics; phage vector; phenotypic data; preservation; prevent; programs; resistant strain; side effect; success; treatment duration; tuberculosis diagnostics; tuberculosis drugs; tuberculosis treatment; whole genome

Project Summary: Mycobacterium tuberculosis (M.tuberculosis) is the causative agent of tuberculosis (TB); although a treatable disease TB remains a leading cause of morbidity and mortality globally. This in large is driven by the unparalleled increase in anti-mycobacterial drug resistance. Approximately 10 million people fell ill with TB in 2019, where 500,000 were new rifampicin resistant cases and 78% of rifampicin cases were multi-drug resistant; compromising the most effective first-line drugs. In line with recent WHO guidance, the majority of these patients are treated with all-oral short-course treatment incorporating bedaquiline and other novel agents. Emerging drug resistance to these novel drugs is a critical threat to expansion of shortened, all-oral, DR-TB treatment regimens. Current diagnostic assays do not incorporate resistance detection to these new drugs, largely as a result of our incomplete understanding of the underlying genetic mechanisms mediating resistance. As a result, a combination of genotypic and phenotypic techniques is required to monitor resistance to these new drugs. Reporter mycobacteriophage assays represent a promising approach for deriving phenotypic data in a replication-independent system, maintaining high sensitivity while reducing the time to result attendant with growth-based methods. We created a TM4 phage vector that delivers a gene cassette of the Nluc BRET-GeNL nanoluciferase reporter enzyme. The BRET-GeNL nanoluciferase phage was tested on a range of auxotrophic and virulent clinical M. tuberculosis strains, assessing cellular limit of detection and compatibility with drug susceptibility testing on a wide range of antimycobacterial drugs. We found that following a preculture period, we could identify drug susceptibility consistent with WHO-endorsed treatment concentrations to a suite of first-line and second-line drugs as well as novel and repurposed drugs (bedaquiline, pretomanid, and linezolid). The overall objective of this research is to validate the nanoluciferase phage for detection of resistance to novel anti-mycobacterial agents, will be accomplished in two Aims. Aim 1 will establish the utility of the assay in paucibacillary conditions and mixed infection. Aim2 will derive correlations between established minimum inhibitory values around critical concentrations and nanoluciferase fluorescence intensity measures, and validate these cut points in a established prospective cohort study of MDR-TB treatment. An exploratory sub Aim will trial minimally processed sputum rather than MTB culture for MTB detection and drug susceptibility testing. The proposed work will provide additional validation on the BRET-GeNL nanoluciferase phage system and offer a quantitative phenotypic reference method and complement to genotypic methods for diagnosis and antibiotic susceptibility testing.

项目摘要： 结核分枝杆菌（大麻结核）是结核病的病因（TB）；虽然可以治疗 疾病结核病仍然是全球发病率和死亡率的主要原因。这总的来说是由 抗细菌耐药性无与伦比的增加。大约1000万人因结核病生病 2019年，有500,000例新的利福平耐药病例和78％的利福平病例是多药 抵抗的;损害最有效的一线药物。与最近的WHO指导一致 这些患者接受了全口腔短道，并结合了Bedaquiline和其他新颖的治疗 代理商。对这些新型药物的新兴耐药性是对缩短，全口的扩展的关键威胁 DR-TB治疗方案。当前的诊断测定不包含对这些新的抗性检测 药物主要是因为我们对介导的基本遗传机制的不完全理解 反抗。结果，需要基因型和表型技术的组合来监测 对这些新药的抵抗力。记者分枝杆菌分析是一种有希望的方法 在独立于复制的系统中得出表型数据，保持高灵敏度，同时降低 是时候通过基于增长的方法来导致服务员了。我们创建了一个TM4噬菌体向量，该向量提供基因 NLUC BRET-GENL纳米酸酯酶报告酶的盒子。 BRET-GENL纳米酸酯酶噬菌体 在一系列可营养和有毒的临床分枝杆菌菌株上进行了测试，评估了细胞极限 在广泛的抗菌药物上进行药物敏感性测试的检测和兼容性。我们 发现在先进的时期之后，我们可以识别出与谁认可的药物敏感性 一组一线和二线药物以及新型药物的治疗浓度 （Bedaquiline，Pitermanid和lineZolid）。这项研究的总体目的是验证 纳米酸酯噬菌酶噬菌酶用于检测对新型抗细菌剂的抗性，将在 两个目标。 AIM 1将在paucibacillary条件和混合感染中确定测定的效用。 AIM2 将在临界浓度周围建立的最小抑制值和 纳米酸酯酶荧光强度度量，并在已建立的前瞻性中验证这些切点 MDR-TB治疗的队列研究。探索性子目标将试用最少处理的痰液，而不是 MTB培养物用于MTB检测和药物敏感性测试。拟议的工作将提供其他 对Bret-Genl纳米酸酯酶噬菌酶噬菌酶的验证并提供定量表型参考 方法和对诊断和抗生素易感性测试的基因型方法的补充。