Optimizing Phagehunting Methods to Isolate and Amplify Bacteriophages
Shriya Singh, Enrique Assad-Garcia, Nacyra Assad-Garcia, Lauren Oldfield, Sanjay Vashee, and Derrick E. Fouts
J. Craig Venter Institute, Rockville, MD 20872
There are an estimated 1031 bacteriophages (phage), viruses that infect bacteria, in the biosphere, thus comprising a significant portion of the biosphere on Earth. Of these, a mere 10,733 phages have been isolated and 2,061 phages have sequenced, complete genomes, with even fewer, only 1,073, presently publicly available in the NCBI GenBank database. “Phagehunting” is the process of isolation, characterization, and genomic analyses of phages that infect bacterial hosts. There is considerable interest in using phages as diagnostics and therapeutics of multi-drug resistant (MDR) bacteria. One such MDR bacterium is Mycobacterium tuberculosis, the causative agent of TB, whose phages can be isolated on the easy-to-grow non-pathogenic Mycobacterium smegmatis. Since current phagehunting procedures of Mycobacterium phages all use identical plating conditions, we would like to explore alternative phagehunting procedures to determine if that affects the type of phages isolated. To date, no phages within the Podoviridae family have been isolated that infect Mycobacteria. With these new optimized procedures, we would like to pilot a program, contingent on the success of this study, to expose high school students to the field of molecular and synthetic genomics. We examined the precise conditions required in each step of the phagehunting process to successfully obtain phage. With the use of careful phage isolation procedures and environmental samples from various locations, such as alternative media usage and modified plaque-picking methods, we isolated potentially novel phages. One of these phages, Kalon1, has been isolated and preliminarily characterized. The success of this study may provide the foundation for an improved program for high school students to foster their interest in research and gain invaluable experiences outside of the classroom while providing bacteriophages that can serve as the base chassis of synthetically engineered phages for diagnosis and treatment of MDR pathogens. Lastly, by creating a generalizable standardized process to obtain phages, we may broaden the horizon for new exploration of novel phages and advances in phage therapy of any MDR pathogen.