Computational front end to diverse therapeutic phage cocktails

New therapies are necessary to combat bacterial pathogens as they become increasingly resistant to antibiotics. We have developed a technology platform comprised of computational, molecular biology, and microbiology tools that together enable on-demand production of phage therapy against virtually any given bacterial isolate. Two complementary computational tools support identification and precise mapping of prophages and other integrative genetic elements (IGEs) in bacterial genomes, are used to identify prophage-laden bacteria that are close relatives of the target strain. Prophage genomes are synthesized, removing the integrase gene, through use of long amplicon PCR and Gibson assembly. Finally, the engineered prophage genomes are introduced into host bacteria for production of lytic phage. As an initial demonstration, we used this approach to produce novel engineered phage with lytic activity against the PAO1 strain of the opportunistic pathogen Pseudomonas aeruginosa. Prophages were computationally predicted in two closely-related prophage-laden P. aeruginosa strains, ATCC 39324 and ATCC 27853. Mitomycin C treatment of these strains, followed by deep sequencing, revealed that seven prophage genomes could be induced to excise from the bacterial genome, circularize, and generate phage particles that grow on P. aeruginosa PAO1. Through this method we isolated two wild-type temperate phages, and produced five mutant phages engineered for lytic activity. The ∆int phages, individually and in cocktails, showed killing of P. aeruginosa PAO1 in vitro as well as in a waxworm (Galleria mellonella) model of infection.