Microfluidics Platform for Rapid, High-throughput Screening of Therapeutic Bacteriophages Based on Patient Bacterial Isolates

项目概要-Felix Biotechnology正在开发一种微流体平台，用于快速，高通量 筛选针对致病细菌的治疗性噬菌体。多个联邦机构 公司和美国主要学术医疗中心的传染病专家正在推进 将抗生素用于广泛的应用，包括治疗多重耐药细菌， 感染和预防食源性疾病。虽然这些努力显示出巨大的希望， 大多数药物的范围限制了药物作为通用工具的商业和临床潜力。工程 具有扩大的宿主范围的噬菌体可能提供一种可能的解决方案，但研究人员缺乏必要的 了解决定宿主范围的遗传因素。收集宿主范围内遗传变异的数据 是耗时、昂贵和低吞吐量的。在初步研究中，费利克斯证明了1） 使用共流聚焦装置，在单个液滴中以可再现的比例可靠地将联合收割机细菌和噬菌体结合，2） 在液滴中共培养细菌和噬菌体并观察噬菌体特异性杀死靶细菌的能力， 和3）优化细菌与噬菌体的比率以在敏感菌株中实现≥ 99.9%杀灭的能力。在 在这一概念验证的第一阶段SBIR中，Felix提出用独特的基于谷胱甘肽的 在将它们组合在液滴中之前，对噬菌体成功杀死细菌的液滴进行分选， 通过合并液滴，其中噬菌体用PCR试剂杀死细菌，然后 融合识别所涉及的特定噬菌体和特定细菌的条形码。水滴会 然后被裂解，混合条形码库将被测序，给我们信息和序列， 用于检测裂解配对的统一扩增子。菲利克斯将展示区分正确配对的能力， 噬菌体/细菌在10 × 10的不同噬菌体和细菌的基质中。目标1。使用寡核苷酸 用于鉴定液滴中的噬菌体/细菌配对的条形码。里程碑/成功指标：确认20个独特的 基于多核苷酸的条形码（10个噬菌体，10个细菌）。目标2.展示条形码正确识别 当以10种不同的噬菌体和10种不同的细菌的基质开始时，鉴定噬菌体/细菌对。 里程碑/成功指标：传统噬斑试验和微流体试验之间的一致性≥ 80%。 进入II期的通过/不通过标准：蚀斑和微流体之间至少80%的一致性 用于鉴定噬菌体/宿主对的试验足以保证进一步的优化。影响-成功证明- 这一概念将支持进一步开发具有目标产物分布的微流体装置， 筛选1，000 x 1，000的基质，与传统噬斑试验的一致性≥ 95%。这将提供 比现有方法多几个数量级的数据，提供准确识别所需的数据量 寄主范围的遗传基础和扩大寄主范围的工程菌。这些进步可以 加速使用抗生素作为细菌性疾病的可持续一线治疗。

PROJECT SUMMARY—Felix Biotechnology is developing a microfluidics platform for rapid, high-throughput screening of therapeutic bacteriophages that target disease-causing bacteria. Federal agencies, multiple companies, and infectious disease specialists in major academic medical centers across the US are advancing the use of phages for a broad range of applications including the treatment of multi-drug resistant bacterial infections and the prevention of food-borne illnesses. While these efforts show great promise, the narrow host range of most phages limits the commercial and clinical potential of phages as a generalized tool. Engineering phage with expanded host ranges may provide a possible solution, but researchers lack the necessary understanding of the genetic factors that determine host range. Collecting data on genetic variation in host range is time consuming, expensive, and low throughput. In preliminary studies, Felix demonstrated 1) the ability to reliably combine bacteria and phage in reproducible ratios in single droplets using a co-flow focusing device, 2) the ability to co-culture bacteria and phage in the droplets and observe phage-specific killing of target bacteria, and 3) the ability to optimize the ratio of bacteria to phage to achieve ≥ 99.9% killing in susceptible strains. In this proof-of-concept Phase I SBIR, Felix proposes to tag phages and bacteria with unique oligonucleotide-based barcodes prior to combining them in droplets, sort droplets where phage successfully kills the bacteria, unify the respective barcodes (“epicPCR”) by merging droplets where phage kill bacteria with PCR reagents and then fusing the barcodes identifying the specific phage and specific bacteria that were involved. The droplets would then be lysed and the pool of hybrid barcodes would be sequenced, giving us information on and sequence- unified amplicons for detecting a lytic pairing. Felix will then demonstrate the ability to distinguish correctly paired phage/bacteria in a 10 x 10 matrix of different phages and bacteria. Aim 1. Validate the use of oligonucleotide barcodes for identifying phage/bacteria pairing in droplets. Milestone / Success Metric: Validation of 20 unique oligonucleotide-based barcodes (10 phage, 10 bacteria). Aim 2. Demonstrate the ability of barcodes to correctly identify phage/bacteria pairs when starting with a matrix of 10 different phages and 10 different bacteria. Milestone / Success Metric: ≥ 80% agreement between traditional plaquing assay and the microfluidics assay. Go/No-Go Criterion for Advancing to Phase II: At least 80% agreement between plaquing and microfluidics assays for identifying phage/host pairs is sufficient to warrant further optimization. Impact—Successful proof-of- concept would support further development of a microfluidics device with a target product profile capable of screening a matrix of 1,000 x 1,000 with ≥ 95% agreement with traditional plaquing assays. This would provide orders of magnitude more data than current methods, providing the volume of data needed to accurately identify the genetic basis of host range and engineer phages with expanded host range. These advances could accelerate the use of phages as a sustainable first-line treatment for bacterial disease.