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

摘要 耐多药结核病（MDR-TB）由结核分枝杆菌（Mtb）菌株引起， 在推荐的结核病治疗方案中， 治疗方案。世界卫生组织（世卫组织）估计， 全球约有5000万人，每增加近50万例新病例， 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INH、RIF和关键二线药物耐药的结核分枝杆菌。 早期识别耐多药/广泛耐药结核病患者并选择适合他们的抗生素 分离株的易感性将改善患者的预后，并有助于结核病控制工作。而 培养微生物以确定存活力仍然是传染病的金标准 诊断和表型抗生素敏感性试验（AST），Mtb的缓慢生长延迟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），敏感检测结核分枝杆菌耐药性。 因此，迫切需要鉴定快速诊断AST以防止药物失效、复发， 和死于耐多药/广泛耐药结核病。 Sequella开发了一种快速（<1天）、相对敏感（≤102个菌落形成单位）的检测方法， 询问暴露于TB时未培养的临床Mtb分离株的代谢潜力 毒品重组噬菌体工程化以包含B-SMART™盒， Mtb的代谢，并立即指导细胞合成多个拷贝的独特的核酸 在噬菌体或Mtb中不存在的氨基酸序列。抗生素降低B-SMARTTM 信号，因为它们干扰细胞代谢（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， 易感性B-SMART™信号序列针对核酸扩增（NAA）进行优化 测试，并可以通过任何NAA方法检测。 该R21建议将通过使用切割-切割来提高B-SMART™中使用的噬菌体的灵敏度。 边缘合成基因组学方法，以提高其信噪比和测试优化的B- SMART™在结核分枝杆菌临床分离株中的应用。一旦噬菌体被优化，我们测试了 在研究实验室环境中，我们将开发一种临床 用于1）检测患者痰液中肝Mtb的后续应用中的实验室方案 样本，2）在中心实验室或护理点环境中使用，或两者，3）验证 使用FDA指南的B-SMART™ AST，以及4）准备商业发布。

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.