High-content assessment of Pseudomonas aeruginosa bacteriophage efficacy reveals host genetic factors involved in phage specificity

Pseudomonas aeruginosa infects immunocompromised and hospitalized individuals, resulting in over 500,000 annual deaths. With emerging multidrug resistance and stagnating antibiotic development, alternative antimicrobials are desperately needed. Bacteriophages (phages) offer a promising, effective, and safe alternative. We developed and optimized a high-content liquid assay screen and a stringent assessment of efficacy to isolate and characterize seven novel P. aeruginosa phages. Phages were screened individually and in cocktail, inhibiting the growth of over 90% (50/55) of multidrug-resistant clinical strains and [~]75% (102/137) of animal, environmental, and human isolates. When tested in a mouse bacteremia model, the phage cocktail successfully eradicated P. aeruginosa. A proteome-wide bi-directional BLAST identified eight proteins that influenced phage infection. The functional analysis of the corresponding genes reveals their putative roles involving genome modification and transcriptional regulation, metabolic processes, and structural components essential for phage docking. Collectively, we have developed a rigorous high-content approach to identify effective phages, which, coupled with functional genomics, revealed genes that affect phage-bacteria interaction. Author SummaryIn this study, we explored the potential of bacteriophages (phages) isolated from municipal and hospital wastewater sources for combating multidrug-resistant Pseudomonas aeruginosa, an opportunistic pathogen known for posing significant clinical challenges. A rigorous stepwise screen aimed at enhancing specificity against a broad set of 55 clinical P. aeruginosa strains allowed us to isolate diverse class phages that can target over 90% of the clinical isolates. Our phage efficacy assessments employed a colorimetric MTT assay to measure the metabolic activity of P. aeruginosa strains in response to phage exposure. Notably, the phages demonstrated broad coverage against the P. aeruginosa library, with individual phages showing varying degrees of efficacy and a cocktail exhibiting superior inhibitory properties. Further validation using a mouse bacteremia model confirmed the exceptional efficacy of the cocktail, supported by a complete attenuation of clinical signs of infection and a significant reduction of bacterial loads across all organs, supporting their utility as potential phage therapy. Finally, a comprehensive comparative genomic analysis of target bacteria combined with phage efficacy revealed novel genes that are potentially involved in phage infection. These findings provide a foundation for understanding phage-host interactions and pave the way for the development of targeted phage therapies against antibiotic-resistant bacterial infections.