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 变得越来越有限，昂贵，在某些情况下，不存在。200万人对抗生素耐药 在美国每年发生感染，尽管迫切需要具有活性的试剂， 针对这些新出现的多重耐药病原体，自2009年以来仅批准了两种新的抗生素。 因此，开发替代抗菌剂至关重要。抗生素的一个有前途的替代品是 噬菌体（噬菌体），其具有选择性感染和杀死靶细菌的天然能力。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，但用于分离、表征、生产 并且测试噬菌体以鉴定“合适的”噬菌体混合物候选物是耗时的、昂贵的和低成本的。 吞吐量在这里，我们提出了创新的高通量方法的基础上，一个成熟的VT-FACS平台 其减轻或规避这些限制，从而在开发和生产中节省时间和成本 治疗性噬菌体鸡尾酒 该项目的目标是进一步开发VT-FACS，一种高通量噬菌体分离管道， 为了推进、表征和支持噬菌体的使用和快速选择， 抗生素目标1将定义和验证筛选和预测隔离病原体可能性的方法- 在制备和存档的裂解物中的特异性噬菌体，用于根据需要进行选择。在Aim 2中，我们将使用VT- 流式细胞仪和调整目前的工作流程，以确定最佳的细菌宿主生产的最具包容性和 最高滴度噬菌体，以最小化最终混合物的复杂性和生产成本。最后，Aim 3将使用VT-FACS， 开发和验证分离具有噬菌体膜活性的噬菌体的高通量方法。通过这3 独立的目标，我们将制定基本的方法，每一个加速发展和生产 噬菌体作为抗菌疗法。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，具有固有的可扩展性，并且具有自动化的潜力。 这项工作的顺利完成将提供快速和具有成本效益的手段， 病原体特异性噬菌体，具有噬菌体治疗所需的宿主范围和次级活性。的 研究的最终结果支持改进和个性化的噬菌体治疗方案，以对抗当今的 抗生素耐药性的威胁

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.