Construction and evaluation of next-generation reporter mycobacteriophages

下一代报告分枝杆菌噬菌体的构建和评估

• 批准号: 8078685
• 负责人: Graham F. Hatfull
• 金额: $ 5.78万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8078685
• 关键词: Affinity; Anabolism; Antibiotics; Antitubercular Agents; Argentina; Bacteria; Bacteriophages; Biological Assay; Biological Models; Cells; Cessation of life; Clinical; Collaborations; Complex; Cytolysis; DNA biosynthesis; Detection; Developing Countries; Development; Diagnosis; Diagnostic; Diagnostic Procedure; Diagnostic tests; Drug Tolerance; Drug resistance; Ensure; Evaluation; Flow Cytometry; Fluorescence; Gene Expression; Genes; Genus Mycobacterium; Goals; Grant; Growth; Hour; Human; Immune response; Immunocompetent; Incidence; Infection; Infectious Agent; Laboratory Research; Metabolic; Methods; Microbial Biofilms; Microscopy; Mus; Mycobacteriophages; Mycobacterium Infections; Mycobacterium smegmatis; Mycobacterium tuberculosis; Mycolic Acid; Organism; Pathogenesis; Patients; Pattern; Performance; Pharmaceutical Preparations; Physiological; Predisposition; Process; Protocols documentation; Recovery; Regulation; Reporter; Reporter Genes; Reporting; Research; Resistance; Role; Sampling; Sensitivity and Specificity; Signal Transduction; Solutions; Specimen; Speed; Sputum; System; Temperature; Testing; Time; Transportation; Tuberculosis; United States National Institutes of Health; Universities; Virus; base; clinical Diagnosis; cost; density; human mortality; macrophage; mouse model; mutant; mycobacterial; mycolate; next generation; novel diagnostics; novel strategies; parent grant; particle; resistant strain; response; sample fixation; tool; tuberculosis drugs

DESCRIPTION (provided by applicant): Tuberculosis (TB) is a major cause of human mortality with 9 million new cases and nearly two million deaths annually; approximately two billion people are infected with the causative agent, Mycobacterium tuberculosis. In Argentina there are about 12,000 cases and one thousand deaths per year. While M. tuberculosis infections can be effectively resolved with a standard 6-9 month course of antibiotics with at least three drugs, the emergence of drug resistant strains severely complicates treatment. There is a need for new diagnostic approaches that combine speed (time-to-detection), sensitivity, specificity, biosafety, rapid and accurate determination of resistance to the commonly used anti-tuberculosis drugs at a low cost to be applied in developing countries where the incidence of TB is high. Mycobacteriophages are excellent candidates for the development of diagnostic tools since they efficiently and specifically infect and replicate in Mycobacteria. We recently described the development of Fluoromycobacteriophages - reporter phages containing a fluorescent reporter gene - that provide a simple means of revealing the metabolic state of M. tuberculosis cells, and therefore their response to antibiotics. Fluorescence can be detected easily by fluorescent microscopy or by flow cytometry. The assay is responsive to antibiotics, and fluorescence is maintained for at least two weeks following fixation, increasing biosafety and facilitating storage or transportation of samples. Fluoromycobacteriophages have promising attributes in the research laboratory, and our goal is to develop the next generation of fluoromycobacteriophages that can be used for direct analysis of clinical samples, with a readout within one hour. We propose to modify the current phages such DNA replication contributes to signal amplification, which can be accomplished by the construction of lysis- defective phage mutants; this is a particularly desirable feature for use in developing countries, since they can be used at any infection temperature. Incorporation of optimized versions of fluorescent genes with an enhanced mycobacterial expression will also enhance the signal and shorten the time-to-detection. We also propose to develop a system for addition of affinity tags to phage particles to ensure efficient capture of mycobacterial cells, and thus optimize the sensitivity of detection. Finally, the construction of these optimized versions of Fluoromycobacteriophages will facilitate the testing of specific protocols for sputum processing to achieve efficient phage infection of mycobacterial cells directly in these samples. Together, these developments will result in a simple, rapid, and specific diagnostic test for tuberculosis. This research will be primarily done in Argentina at University of Buenos Aires in collaboration with Dr. Mariana Piuri as an extension of NIH Grant AI064494 (7/1/06 - 6/30/11). PUBLIC HEALTH RELEVANCE: Tuberculosis (TB) is a major cause of human mortality with 9 million new cases and nearly two million deaths annually; approximately two billion people are infected with the causative agent, Mycobacterium tuberculosis. Diagnosis of TB is complicated by the slow growth of the organism and the need for rapid drug susceptibility testing. The development of a simple, rapid, cheap, and accurate test for determining drug susceptibilities of clinical samples would dramatically enhance the control of tuberculosis across the world.

描述（由申请人提供）：结核病（TB）是人类死亡率的主要原因，每年有900万例新病例和近200万人死亡；大约20亿人感染了病因，结核分枝杆菌。在阿根廷，每年大约有12,000例病例和一千例死亡。虽然结核分枝杆菌感染可以通过至少三种药物的标准6-9个月的抗生素疗程有效解决，但耐药菌株的出现使治疗严重复杂。有必要采用新的诊断方法，以结合速度（检测时间），灵敏度，特异性，生物安全性，快速准确地确定对常用的抗结核药物的抵抗力，以低成本以低成本在TB的发生率的发展中国家中应用。分枝杆菌是开发诊断工具的绝佳候选者，因为它们在分枝杆菌中有效，专门感染和复制。我们最近描述了荧光病毒噬菌体的发展 - 含有荧光记者基因的记者噬菌体 - 提供了一种简单的方法来揭示结核分枝杆菌细胞的代谢状态，因此提供了对抗生素的反应。荧光可以通过荧光显微镜或流式细胞仪轻松检测。该测定法对抗生素有反应，固定后至少维持荧光至少两周，从而增加了生物安全并促进样品的储存或运输。研究实验室中的荧光菌病具有有希望的属性，我们的目标是开发下一代荧光菌病菌，可用于直接分析临床样本，并在一小时内进行读数。我们建议改变当前的噬菌体，这种DNA复制有助于信号扩增，这可以通过构建裂解缺陷的噬菌体突变体来实现。这是在发展中国家使用的特别理想功能，因为它们可以在任何感染温度下使用。将优化版本的荧光基因掺入具有增强的分枝杆菌表达的荧光基因也将增强信号并缩短检测时间。我们还建议开发一个用于添加亲和力标签以噬菌体颗粒的系统，以确保有效捕获分枝杆菌细胞，从而优化检测的敏感性。最后，这些优化版本的荧光病毒噬菌体的构建将促进痰液加工特定方案的测试，以直接在这些样品中直接实现分枝杆菌细胞的有效噬菌体感染。这些发展在一起将导致结核病的简单，快速且特定的诊断测试。这项研究将主要在布宜诺斯艾利斯大学的阿根廷与玛丽安娜·皮里（Mariana Piuri）博士合作，作为NIH Grant AI064494（7/1/06-6/6/30/11）的扩展。 公共卫生相关性：结核病（TB）是人类死亡率的主要原因，每年有900万例新病例和近200万人死亡；大约20亿人感染了病因，结核分枝杆菌。结核病的诊断因生物体的缓慢生长以及对快速药物易感性测试的需求而变得复杂。用于确定临床样本药物敏感性的简单，快速，廉价和准确的测试的开发将显着增强全球结核病的控制。