High-Throughput Bacteriophage Isolation and Characterization

The utility of phage therapy to overcome the problem of multi-drug resistant bacteria has received renewed attention. However, narrow host tropism of phages is problematic as phages must be identified for each bacterial pathogen, and a phage cocktail is required to ensure adequate species coverage. The logistical requirements needed to develop phage cocktails are immense, often requiring the isolation, characterization and host range analysis of hundreds of phages but, to date, methodologies for the identification and selection of desirable phages has changed little since the their first discovery in the 1960s. Therefore, the development of high-throughput methodologies capable of circumventing logistical constraints is highly desirable. Our Phase II goal is to demonstrate that the Viral-Tagging Fluorescence Assisted Cell Sorting methodology developed during Phase I can be refined to isolate and characterize 300 phages against 100 bacteria strains in an expedited/high-throughput manner. VT-FACS enables clonality to be achieved in a single step, and preferentially selects for broad-host range phages. Furthermore, a high-throughput luciferasebased ATP assay enables the 30.000 bacterial/phage reactions to be completed within ~50h thereby significantly reducing the time required to identify a potential phage cocktail. This innovative technology will significantly advance the identification of phages for disease treatment.