Optimized methods for isolation and characterization of bacteriophage by VT-FACS

摘要 抗生素耐药(AMR)病原体正在以惊人的速度出现，目前的治疗方案是 变得越来越有限、昂贵，在某些情况下，甚至是不存在的。200万人对抗生素产生耐药性 感染在美国每年都会发生，尽管迫切需要有活动能力的药物 自2009年以来，针对这些新出现的多重耐药病原体，只批准了2种新的抗生素。 因此，开发替代抗菌剂是至关重要的。抗生素的一个有希望的替代品是 噬菌体：具有选择性感染和杀死目标细菌的天然能力的噬菌体。噬菌体正在接收 作为一种安全有效的治疗方法，重新引起了人们的兴趣，但分离、鉴定、生产技术 而对噬菌体进行测试以确定适合的噬菌体鸡尾酒是耗时、昂贵和低成本的 吞吐量。在这里，我们提出了基于成熟的VT-FACS平台的创新的高吞吐量方法 减轻或绕过这些限制，从而在开发和生产中节省时间和成本 治疗用的噬菌体鸡尾酒。 本项目的目标是进一步开发VT-FACS这一高通量噬菌体分离管道， 推进、鉴定和支持使用和快速选择噬菌体作为经典噬菌体的替代品 抗生素。目标1将定义和验证筛选和预测暴露病原体可能性的方法- 在准备好的和存档的裂解物中的特定噬菌体，以供按需选择。在目标2中，我们将使用VT- FACS和适应当前的工作流程，以确定生产最具包容性和 最高效价噬菌体，将最终鸡尾酒的复杂性和生产成本降至最低。最后，Aim 3将使用VT-FACS来 开发和验证高通量分离具有抗生物被膜活性的噬菌体的方法。通过这3个步骤 独立目标我们将开发必要的方法，各自加速开发和生产 噬菌体作为一种抗菌疗法。为了在未来的工作环境中实现最高的吞吐量和效率， 所提出的方法可以串联应用，本质上是可扩展的，并且具有自动化的潜力。 这项工作的成功完成将提供快速和具有成本效益的手段，以选择和生产 病原体特定的噬菌体，具有所需的宿主范围和噬菌体治疗所需的次级活性。这个 研究的最终结果支持改进和个性化的噬菌体疗法选择，以对抗当今的 迫在眉睫的抗生素耐药性威胁。

Abstract Antibiotic-resistant (AMR) pathogens are emerging at alarming rates and current treatment options are becoming increasingly limited, expensive, and in some cases, nonexistent. Two million antibiotic resistant infections occur annually in the U.S and, although there is an urgent and immediate need for agents with activity against these emerging multidrug-resistant pathogens, only 2 new antibiotics have been approved since 2009. Therefore, the development of alternative antibacterial agents is crucial. A promising alternative to antibiotics is bacteriophage (phage) which have a natural ability to selectively infect and kill target bacteria. Phage is receiving renewed interest as a safe and effective therapy, but techniques for the isolation, characterization, production and testing of phages to identify ‘suitable’ phage cocktail candidates are time consuming, expensive and low throughput. Here we propose innovative high-throughput methodologies based on a proven VT-FACS platform which mitigate or circumvent these constraints providing time- and cost-savings in development and production of therapeutic phage cocktails. The goal of this project is further development of VT-FACS, a high-throughput phage isolation pipeline, to advance, characterize and support the use and rapid selection of phage as an alternative to classical antibiotics. Aim 1 will define and validate methods for screening and predicting likelihood of insolating pathogen- specific phage in prepared and archived lysates for informed on-demand selection. In Aim 2 we will use VT- FACS and adapt current workflow to identify the optimal bacterial host for production of the most inclusive and highest titer phage to minimize final cocktail complexity and production cost. Finally, Aim 3 will use VT-FACS to develop and validate high-throughput methods that isolate phage with antibiofilm activity. Through these 3 independent aims we will develop essential methods which each accelerate development and production of bacteriophage as an antibacterial therapy. For highest throughput and efficiency in a future working environment, the proposed methods can be applied in tandem, are inherently scalable and have potential to be automated. Successful completion of this work will provide expedited and cost-effective means to select and produce pathogen specific phage with the required host-range and secondary activities necessary for phage therapy. The ultimate outcome of the research supports improved and personalized phage therapy options to combat today’s urgent antibiotic resistance threat.