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

VT-FACS 噬菌体分离和表征的优化方法

• 批准号: 10364653
• 负责人: Natasha Jayna Sharp
• 金额: $ 24.67万
• 依托单位: GUILD ASSOCIATES, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-04 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10364653
• 关键词: Anti-Bacterial Agents; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Antimicrobial Resistance; Archives; Bacteria; Bacterial Infections; Bacteriophages; Biological Sciences; Cells; Code; Collection; Computer software; Consumption; Cost Savings; Coupled; Custom; Data; Development; Engineering; Ensure; Environment; Formulation; Future; Goals; Individual; Infection; Life; Metagenomics; Methodology; Methods; Microbial Biofilms; Multi-Drug Resistance; Mutate; Outcomes Research; Population; Preparation; Process; Production; Pseudomonas aeruginosa; Research Support; Residual state; Resistance; Sampling; Sewage; Source; Stains; System; Techniques; Technology; Testing; Therapeutic; Time; United States; Viral; Work; antibiotic resistant infections; antimicrobial; antimicrobial drug; antimicrobial resistant pathogen; bacterial resistance; base; blind; clinical application; combat; cost; cost effective; drug resistant pathogen; effective therapy; emerging pathogen; experience; global health; improved; innovation; interest; lytic replication; multi-drug resistant pathogen; pathogen; pathogenic bacteria; personalized medicine; screening; success; virtual

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.

抽象的 抗生素耐药（AMR）病原体正在以惊人的速度出现，当前的治疗选择是 变得越来越有限，昂贵，在某些情况下不存在。 200万抗生素耐药性 每年在美国发生感染 反对这些新兴的多药病原体，自2009年以来，仅批准了2种新的抗生素。 因此，替代抗菌剂的发展至关重要。一种有希望的抗生素替代品是 噬菌体（噬菌体）具有自然能力，可以选择性感染和杀死靶细菌。噬菌体正在接受 新的兴趣是一种安全有效的疗法，但是用于隔离，表征，生产的技术 并测试噬菌体以识别“合适的”噬菌体鸡尾酒候选者很耗时，昂贵且低 吞吐量。在这里，我们建议基于经过验证的VT-FACS平台的创新高通量方法 减轻或规避这些限制，从而提供时间和成本节省的开发和生产 热噬菌体鸡尾酒。 该项目的目的是进一步开发VT-FACS，这是一种高通量噬菌体隔离管道， 推进，表征和支持噬菌体的使用和快速选择作为经典的替代品 抗生素。 AIM 1将定义和验证用于筛查和预测损害病原体的可能性的方法 准备好的裂解物中的特定噬菌体用于按需选择。在AIM 2中，我们将使用VT- FACS和适应当前的工作流程，以识别最佳细菌宿主，以生产最包容和最具包容性的细菌 最高滴度噬菌体可最大程度地减少最终鸡尾酒的复杂性和生产成本。最后，AIM 3将使用VT-FACS 开发和验证高通量方法将噬菌体与抗生膜活性分离。通过这3 独立目标我们将开发基本方法，每种方法都加速了开发和生产 噬菌体作为一种抗菌疗法。对于未来工作环境中的最高吞吐量和效率， 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。 这项工作的成功完成将为选择和生产提供加快且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要活动。 该研究的最终结果支持改进和个性化的噬菌体疗法，以对抗当今的 紧急抗生素抗性威胁。