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.

抽象的 多药耐药结核（MDR-TB）是由结核分枝杆菌（MTB）菌株引起的 在推荐的结核病中抗两种前线抗生素，异烟肼（INH）和利福平（RIF） 治疗方案。世界卫生组织（WHO）估计MDR-TB的流行 全球约有5000万人，每个人都有近500,000个新案件 年。一个更令人震惊的发展是极度药物的全球分布和全球分布 耐药性结核病（XDR-TB），定义为对INH，RIF和关键二线药物的MTB抗性。 早期识别M/XDR-TB的患者以及选择适当的抗生素的患者 分离株易感性会改善患者的预后并有助于结核病控制工作。尽管 微生物培养以确定生存能力仍然是传染病的黄金标准 诊断和表型抗生素易感性测试（AST），MTB延迟的缓慢生长AST 对患者管理或感染控制的实际实用性之外的结果。目前没有 早期，快速（<24小时）和对MTB抗生素耐药性的敏感检测的令人满意的选择。 那是迫切需要确定快速诊断的AST，以防止药物衰竭，接力， 和M/XDR-TB的死亡。 Sequella开发了快速（<1天），相对敏感（≤102个集菌的形成单元），测试 询问临床MTB分离株的代谢潜力，而无需培养 毒品。重组噬菌体设计为包含B-SMART™盒式盒子接管 MTB的代谢，并立即引导细胞合成独特核的多个副本 酸序列不存在于噬菌体或MTB中。抗生素减少B-Smarttm 信号因为它们会干扰细胞代谢（转录和翻译），因此噬菌体 无法产生信号，读数是药物的表型表征 敏感性。 B-SMART™信号序列被优化用于核酸扩增（NAA） 测试，可以通过任何NAA方法检测到。 该R21提案将通过使用剪裁 - 边缘合成基因组学方法改善其信号与噪声比并测试优化的B- MTB临床分离株中的Smart™。一旦优化了噬菌体，我们测试了 在研究实验室环境中使用各种结核病药物进行测定，我们将开发临床 在随后的应用中，实验室协议1）在患者痰中检测活MTB 样本，2）在集中实验室或护理点设置中使用，或两者兼而有之3）验证 B-SMART™AST使用FDA指南，以及4）准备商业发布的准备。