Synthetic Genomics to Improve a Phage-Based Diagnostic for Multi-Drug Resistant Bacteria

合成基因组学改进基于噬菌体的多重耐药细菌诊断

• 批准号: 9808575
• 负责人: Sanjay Vashee
• 金额: $ 24.81万
• 依托单位: J. CRAIG VENTER INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-03 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9808575
• 关键词: Antibiotic Resistance; Antibiotic Therapy; Antibiotic susceptibility; Antibiotics; Bacteria; Bacterial Infections; Bacteriophages; Base Sequence; Bioinformatics; Biological Assay; Cells; Cessation of life; Clinical; Clinical Microbiology; Clinical Trials; Collaborations; Colony-forming units; Communicable Diseases; Consult; DNA; DNA-Directed RNA Polymerase; Detection; Development; Diagnostic; Diagnostic tests; Doctor of Philosophy; Drug resistance; Drug resistance in tuberculosis; Engineering; Epidemiology; Exposure to; Extreme drug resistant tuberculosis; Failure; Frequencies; Future; Generations; Genetic; Genetic Transcription; Genome; Genome engineering; Genomic approach; Genomics; Goals; Gold; Growth; Guidelines; Herpesviridae; Infection Control; Institutes; Knowledge; Laboratories; Laboratory Research; Location; Metabolic; Metabolism; Methods; Molecular; Monitor; Multi-Drug Resistance; Multidrug-Resistant Tuberculosis; Multiple Bacterial Drug Resistance; Mycobacteriophages; Mycobacterium smegmatis; Mycobacterium tuberculosis; Mycobacterium tuberculosis H37Rv; Noise; Nucleic Acid Amplification Tests; Nucleic Acids; Outcome; Patient-Focused Outcomes; Patients; Peptide Signal Sequences; Pharmaceutical Preparations; Phenotype; Plasmids; Polymerase Gene; Predisposition; Preparation; Prevalence; Protocols documentation; Public Health; Recombinants; Relapse; Reporter; Research; Resistance; Rifampin; Sampling; Side; Signal Transduction; Speed; Sputum; Test Result; Testing; Therapeutic; Time; Translations; Treatment Protocols; Validation; Vertebral column; Work; World Health Organization; Yeasts; antimicrobial; bacterial resistance; base; compare effectiveness; design; improved; isoniazid; microorganism culture; novel; pathogenic bacteria; point of care; prevent; promoter; reconstruction; resistant strain; success; synthetic genomics; tool; tuberculosis diagnostics; tuberculosis drugs

Abstract Multi-drug resistant TB (MDR-TB) is caused by Mycobacterium tuberculosis (Mtb) strains that are resistant to two front-line antibiotics, isoniazid (INH) and rifampin (RIF), in the recommended TB treatment regimen. The World Health Organization (WHO) estimates the prevalence of MDR-TB at approximately 50 million people worldwide, expanding by nearly 500,000 new cases each year. A more alarming development is the increase in and global distribution of extremely drug- resistant TB (XDR-TB), defined as Mtb resistant to INH, RIF and key second-line drugs. Early recognition of patients with M/XDR-TB and selection of appropriate antibiotics to which their isolates are susceptible would improve patient outcomes and assist in TB control efforts. While culturing of microorganisms to determine viability remains the gold standard for infectious disease diagnostics and phenotypic antibiotic susceptibility test (AST), the slow growth of Mtb delays AST results beyond practical utility for patient management or infection control. There are presently no satisfactory options for early, rapid (< 24 hr.), and sensitive detection of Mtb antibiotic resistance. Thus, there is a desperate need to identify a rapid diagnostic AST to prevent drug failure, relapse, and death from M/XDR-TB. Sequella developed a rapid (<1 day), relatively sensitive (≤102 colony forming units), test to interrogate the metabolic potential of clinical Mtb isolates without culture when exposed to TB drugs. Recombinant phage engineered to contain the B-SMART™ cassette take over the metabolism of Mtb and immediately direct the cell to synthesize multiple copies of a unique nucleic acid sequence not otherwise present in either the phage or Mtb. Antibiotics reduce B-SMARTTM signal because they interfere with cellular metabolism (transcription and translation), thus phage are not able to produce the signal and the readout is a phenotypic characterization of drug susceptibility. B-SMART™ signal sequence is optimized for nucleic acid amplification (NAA) testing and can be detected by any NAA method. This R21 proposal will improve the sensitivity of the phage used in B-SMART™ by using a cutting- edge synthetic genomics approach to improve its signal to noise ratio and test the optimized B- SMART™ in Mtb clinical isolates. Once the phage is optimized and we test the sensitivity of the assay with the various TB drugs in a research laboratory setting, we will develop a clinical laboratory protocol in a subsequent application for 1) detection of live Mtb in patient sputum samples, 2) use in either centralized laboratories or a point-of-care setting, or both, 3) validation the B-SMART™ AST using FDA guidelines, and 4) preparation for commercial launch.

摘要 耐多药结核病(MDR-TB)是由结核分枝杆菌(Mtb)菌株引起的 对推荐结核病患者中的两种一线抗生素异烟肼(INH)和利福平(RIF)产生耐药性 治疗方案。世界卫生组织(WHO)估计耐多药结核病的流行率 全球约有5000万人，每个新增病例增加近50万 年。一个更令人震惊的事态发展是极端毒品的增加和全球分布- 耐药结核病(XDR-TB)，定义为对异烟肼、RIF和关键二线药物耐药的结核分枝杆菌。 早期识别耐药/广泛耐药结核患者并选择适当的抗生素 分离出易受感染的菌株将改善患者的预后，并有助于结核病控制工作。而当 微生物培养以确定生存能力仍然是传染病的黄金标准 诊断和表型药敏试验(AST)，结核分枝杆菌生长缓慢延迟AST 结果超出了患者管理或感染控制的实际效用。目前没有 早期、快速(24小时)和敏感地检测结核分枝杆菌抗生素耐药性的令人满意的选择。 因此，迫切需要确定一种快速诊断AST，以防止药物失效，复发， 以及死于耐多药/广泛耐药结核病。 塞奎拉开发了一种快速(&lt；1天)、相对敏感(≤102菌落形成单位)的测试 未经培养的结核分枝杆菌临床分离株暴露于结核时的代谢潜力 毒品。重组噬菌体被设计成包含B-SMART™盒接管 Mtb的代谢，并立即指示细胞合成一种独特的核的多个副本 噬菌体或结核分枝杆菌中不存在的酸序列。抗生素降低B-SMARTTM 信号，因为它们干扰细胞新陈代谢(转录和翻译)，因此噬菌体 不能产生信号，读出的是药物的表型特征 敏感度。B-SMART™信号序列优化用于核酸扩增 检测并可用任何NAA方法检测。 这一R21方案将通过使用切割-™来提高B-SMART-TMART中使用的噬菌体的敏感性- 边缘合成基因组学方法提高其信噪比并测试优化后的B-DNA序列 SMART™在结核分支杆菌临床分离株中的应用一旦噬菌体被优化，我们就测试了 在实验室里用各种结核病药物进行化验，我们将开发一种临床 用于1)检测患者痰中活结核分枝杆菌的后续应用中的实验室方案 样品，2)在集中实验室或护理点环境中使用，或两者兼而有之，3)验证 使用食品和药物管理局指南的B-SMART™AST，以及4)商业投放的准备工作。