Innovative Approaches in the Discovery of Aquatic Mycobacteriophages

Bacteriophages (phages), viruses that infect bacteria, have many applications in medicine, agriculture, molecular biology, and other fields. As antibiotic resistance becomes an increasing problem, interest in phages has grown. The traditional techniques of phage discovery are successful for some phages, but others require modified procedures to achieve detectable host infection. Mycobacterium is a diverse bacterial genus characterized by a unique cell wall containing mycolic acids, which aids in survival and pathogenesis. The aims of the present research were to isolate mycobacteriophages, use bioinformatics techniques to analyze mycobacterial prophages, and combine genetic analysis with multi-well plate host range studies to identify phages that may infect M. marinum, M. fortuitum, or M. chelonae, aquatic pathogens with importance to aquaculture, zoonotic infections, and more. Firstly, three phage discovery procedures were adapted to a multi-well plate format using the host M. smegmatis mc2155 as a model organism; the 96-well plate detection assay was at least as sensitive as the traditional plaque assay and a technique was developed for the purification of single phage types from mixed cultures in liquid medium. Secondly, phage enrichment from various environmental sources and prophage induction using a variety of techniques did not result in confirmed phage isolation for several tested strains of M. marinum, M. fortuitum, and M. chelonae, demonstrating the difficulty of phage isolation for these species. Thirdly, the emergence and evolution of prophages was investigated in 49 sequenced genomes of the M. ulcerans-M. marinum complex (MuMC), resulting in 134 identified prophages in nine genomic insertion sites. Lastly, phages from a pre-existing phage collection were used in host range studies against 10 strains of mycobacteria, using both plaque assays and multi-well plate turbidity assays (MuMC, M. fortuitum, and M. chelonae). As phages become more extensively used in medicine and other fields, it will become increasingly important to efficiently isolate phages that can infect the bacterium of interest and that have the appropriate qualities for each specific application. Phages are already known to be diverse in their morphology, physiology, and ecological roles, and it is now becoming clearer that the techniques to manipulate them must also be diverse