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

项目摘要-Felix Biotech正在开发一种微流控平台，用于快速、高通量 针对致病细菌的治疗性噬菌体的筛选。联邦机构，多个 公司和美国各地主要学术医疗中心的传染病专家正在进步 噬菌体用于广泛的应用，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，但狭隘的东道主 大多数噬菌体的范围限制了作为通用工具的噬菌体的商业和临床潜力。工程学 扩大宿主范围的噬菌体可能会提供一种可能的解决方案，但研究人员缺乏必要的 了解决定寄主范围的遗传因素。收集寄主范围内遗传变异的数据 耗时、昂贵且吞吐量低。在初步研究中，费利克斯证明了1) 使用顺流聚焦装置在单个液滴中以可重复的比例可靠地结合细菌和噬菌体，2) 将细菌和噬菌体在液滴中共培养并观察噬菌体对目标细菌的特异性杀灭的能力， 优化菌噬菌体比例，对敏感菌株的≥杀灭率可达99.9%。在……里面 在概念验证阶段I SBIR中，Felix建议用独特的基于寡核苷酸的标记来标记噬菌体和细菌 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类，统一 通过将噬菌体杀死细菌的液滴与聚合酶链式反应试剂合并而获得各自的条形码(“epicPCR”)，然后 融合识别特定噬菌体和涉及的特定细菌的条形码。这些水滴会 然后裂解并对混合条形码池进行排序，为我们提供关于以下各项的信息和排序- 用于检测裂解配对的统一扩增器。然后，Felix将演示区分正确配对的能力 噬菌体/细菌在由不同噬菌体和细菌组成的10x10矩阵中。目的1.验证寡核苷酸的使用 用于识别液滴中的噬菌体/细菌配对的条形码。里程碑/成功指标：验证20个唯一 基于寡核苷酸的条形码(10个噬菌体，10个细菌)。目标2.演示条形码正确识别的能力 从10个不同的噬菌体和10个不同的细菌组成的矩阵开始识别噬菌体/细菌对。 里程碑/成功指标：≥80%符合传统斑块试验和微流控试验。 进入第二阶段的Go/No-Go标准：斑块和微流体至少80%的符合率 鉴定噬菌体/宿主对的检测足以保证进一步的优化。影响-成功证明- 概念将支持微流体设备的进一步开发，其目标产品配置文件能够 用≥95%与传统斑块分析法筛选1,000×1,000的基质。这将提供 比当前方法多几个数量级的数据，提供了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噬菌体。这些进展可能 加速使用噬菌体作为治疗细菌性疾病的可持续一线治疗方法。

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.