Pseudomonas aeruginosa is an opportunistic pathogen that causes >50,000 infections in hospitalized immunocompromised patients and >2,500 deaths in the United States annually. With the increasing rise of antibiotic resistance acquired by this bacterium, the World Health Organization (WHO) listed P. aeruginosa as a critical Global Priority Pathogen (GPP) in need of new therapeutic methods. Hence, the main objectives of this research are to isolate and characterize P. aeruginosa lytic phages and to investigate their combined application with silver nanoparticles to combat P. aeruginosa biofilms.

Six sewage water samples were enriched for phages using nine Pseudomonas spp strains. Twenty bacteriophages were isolated based on plaque morphology and tested against 38 P. aeruginosa clinical isolates at a multiplicity of infection (MOI) of 1. Five phages were found to have a broad host range profile and inhibited the growth of multiple P. aeruginosa clinical isolates. Subsequent screening experiments were conducted to determine the phages' virulence on all 38 strains and one phage showed high virulence being able to infect 22/38 strains efficiently. P. aeruginosa strain PA14 was selected for all downstream phage characterization and host interaction experiments, such as adsorption efficiency, one-step growth curve, bacteriophage insensitive mutant (BIM) frequency, minimum inhibitory concentration (MIC) and minimum biofilm eradication concentration (MBEC) experiments. One single phage, Chandler, had a comparable biocontrol efficacy against planktonic PA14 cells versus a cocktail of three phages. Therefore, only Chandler was chosen for downstream biofilm assay experiments. During genomic analysis it was discovered that Chandler belongs to the family Myoviridae, has a genome size of approximately 66 kbp and contains no lysogenic genes. Through adsorption kinetics experiments, it was determined that Chandler has a large burst size of 144 and latent period of 30 minutes. Silver nanoparticles sensitivity experiment revealed that the chosen phage is not affected by silver nanoparticles even after being exposed to silver nanoparticles for 24 hours at 200 RPM at a concentration of 10 ng/mL and 10 7 PFU/mL. The checkerboard and MBEC assays revealed that when a combined application of phage and silver nanoparticles are used against PA14 biofilms at a concentration of 10 9 PFU/mL and 10 ng/mL respectfully, had comparable efficacy in biofilm degradation as 10 3 PFU/mL and 10 ng/mL. There was a 75% reduction in biofilm and the biofilm was only able to recover to 25% of its original biomass when treated with both phage and silver nanoparticles together. Biofilm assays are quite variable, and the results indicate that there is not a notable synergistic effect. However, the reduction in biofilm biomass and viability shows promising results for downstream applications and future experiments. Thus, these findings indicate that when using a biological entity and a chemical compound such as silver nanoparticles can present some challenges and the dynamics may be more complex than previously expected.