High-throughput phage isolation using Viral-Tagging and Fluorescence-Activated Cell Sorting

Bacteriophages (phages), viruses which specifically infect and lyse bacteria, are attractive alternatives to antibiotic therapy as they are naturally occurring, amplify in the host, are inexpensive to produce and display narrow host ranges. This latter aspect is of particular interest to therapy as, unlike broad-spectrum antibiotics which kill the both the pathogen and endogenous bacterial flora, phages do not display off target effects. However, this selective host range typically necessitates that therapeutic phages must be developed for each bacterial pathogen, and that each preparation must contain a cocktail of phages 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. Therefore, the development of high-throughput technologies capable of mitigating these constraints is highly desirable. Our Phase I goal is to develop rapid high-throughput technologies for the development of phage cocktails. This will be accomplished by: 1) automated isolation of clonal phage by viral-tagging and fluorescence-assisted cell sorting, 2) preferential isolation of broad-host range lytic phages, and 3) high-throughput, microtiter plate based host range analyses based on luminescence.