Project Abstract Treatment of biofilm-associated infections using antibiotics can be limited by pathogen antibiotic resistance, as well as antibiotic tolerance displayed by infections. This application seeks to develop new strategies to eliminate persistent wound infections caused by multidrug resistant pathogens. Bacteriophages are natural, abundant, and diverse with minimal toxicity, particularly when used topically, and assumed to have negligible impact on the microbiome. Due to limited cross-resistance, bacteria displaying antibiotic resistance tend to remain phage susceptible and phage cocktails (polyphages) can be developed to minimize resistance to multiple phages, thereby better assuring continued susceptibility of targeted bacteria. Thus, an aggressive mixture of bacteria- and biofilm-disrupting agents – phages and antibiotics – may be employed towards reducing antibiotic-resisting and otherwise challenging-to-treat experimental wound infections. Here we propose to develop bactericidal bacterial viruses (bacteriophages or phages) as adjuvants to antibiotic application, with an aim towards future clinical testing while retaining current standards of care. Emphasis is placed on the treatment of MDR Pseudomonas aeruginosa-infected and mixed- infected wounds. The five key phage characteristics that we developed for therapy are robust antibacterial activity, ability to function against biofilms, host range breadth including multidrug resistant and colony variant P. aeruginosa, limited cytotoxicity against host cells, and phage stability. Aim 1 will evaluate the phage cocktail therapy in mono- and poly-microbial model systems. Aim 2 utilizes eco-systems biology approaches for in vivo testing of cocktails in a complex polymicrobial infection and will investigate how phage therapy-based targeting of a pathogen may cause changes to the wound microbiota. Overall, such therapy will be used to reduce the burden of devastating infections caused by multidrug resistant P. aeruginosa. This study will also lay the foundation for a program targeting each of the major ESKAPE pathogens.