Engineered Superinfective Pf Phage Prevents Dissemination of Pseudomonas aeruginosa in a Mouse Burn Model

Pf is a filamentous bacteriophage integrated in the chromosome of most clinical isolates of Pseudomonas aeruginosa. Under stress conditions, mutations occurring in the Pf genome result in the emergence of superinfective variants of Pf (SI-Pf) that are capable of circumventing phage immunity; therefore, SI-Pf can even infect Pf-lysogenized P. aeruginosa. Here, we identified specific mutations located between the repressor and the excisionase genes of Pf4 phage in the P. aeruginosa PAO1 strain that resulted in the emergence of SI-Pf. Based on these findings, we genetically engineered an SI-Pf (eSI-Pf) and tested it as a phage therapy tool for the treatment of life-threatening burn wound infections caused by PAO1. In validation experiments, eSI-Pf was able to infect PAO1 grown in a lawn as well as biofilms formed in vitro on polystyrene. eSI-Pf also infected PAO1 present in burned skin wounds on mice but was not capable of maintaining a sustained reduction in bacterial burden beyond 24 h. Despite not lowering bacterial burden in burned skin tissue, eSI-Pf treatment completely abolished the capability of P. aeruginosa to disseminate from the burn site to internal organs. Over the course of 10 days, this resulted in bacterial clearance and survival of all treated mice. We subsequently determined that eSI-Pf induced a small-colony variant of P. aeruginosa that was unable to disseminate systemically. This attenuated phenotype was due to profound changes in virulence determinant production and altered physiology. Our results suggest that eSI-Pf has potential as a phage therapy against highly recalcitrant antimicrobial-resistant P. aeruginosa infections of burn wounds.

Automated summary

Pf is a filamentous bacteriophage integrated into the chromosome of Pseudomonas aeruginosa clinical isolates. Mutations occurring in the Pf genome result in the emergence of superinfective variants (SI-Pf) that can infect Pf-lysogenized P. aeruginosa. In this study, specific mutations were identified in the Pf4 phage that led to the emergence of SI-Pf, and an engineered SI-Pf (eSI-Pf) was tested as a phage therapy tool for burn wound infections caused by P. aeruginosa. The treatment with eSI-Pf resulted in bacterial clearance and survival of all treated mice, suggesting its potential as a phage therapy against recalcitrant P. aeruginosa infections.