Engineered Superinfective Pf Phage Prevents Dissemination of Pseudomonas aeruginosa in a Mouse Burn Model
Federico I Prokopczuk1, Hansol Im1, Javier Campos-Gomez2
1Department of Microbiology, Heersink School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA.
Abstract:
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. IMPORTANCE Pseudomonas aeruginosa is a major cause of burn-related infections. It is also the most likely bacterial infection to advance to sepsis and result in burn-linked death. Frequently, P. aeruginosa strains isolated from burn patients display a multidrug-resistant phenotype necessitating the development of new therapeutic strategies and prophylactic treatments. In this context, phage therapy using lytic phages has demonstrated exciting potential in the control P. aeruginosa infection. However, lytic phages can present a set of drawbacks during phage therapy, including the induction of bacterial resistance and limited bacteria-phage interactions in vivo. Here, we propose an alternative approach to interfere with P. aeruginosa pathogenesis in a burn infection model, i.e., by using an engineered superinfective filamentous phage. Our study demonstrates that treatment with the engineered Pf phage can prevent sepsis and death in a burn mouse model.
Insights
Engineered superinfective phages (eSI-Pf) show promise for treating Pseudomonas aeruginosa burn wound infections. While not reducing bacterial load, eSI-Pf prevented sepsis and death in mice by attenuating bacterial virulence.
Area of Science:
- Microbiology
- Virology
- Infectious Diseases
Background:
- Pseudomonas aeruginosa is a leading cause of severe burn infections, often multidrug-resistant.
- Current phage therapy faces challenges like bacterial resistance and limited in vivo efficacy.
- Superinfective filamentous phages (SI-Pf) can overcome phage immunity in P. aeruginosa.
Purpose of the Study:
- To engineer a superinfective filamentous phage (eSI-Pf) for treating P. aeruginosa burn wound infections.
- To evaluate the efficacy of eSI-Pf in a mouse model of burn wound infection.
- To investigate the mechanism by which eSI-Pf impacts bacterial pathogenesis.
Main Methods:
- Identification of mutations conferring superinfectivity in Pf4 phage.
- Genetic engineering of an enhanced superinfective phage (eSI-Pf).
- In vitro and in vivo testing of eSI-Pf in P. aeruginosa burn wound infection models.
- Assessment of bacterial burden, dissemination, and survival in treated mice.
- Analysis of bacterial phenotypic changes induced by eSI-Pf.
Main Results:
- eSI-Pf infected P. aeruginosa in vitro and in vivo, including biofilms and burn wounds.
- eSI-Pf did not sustain bacterial load reduction beyond 24 hours but prevented systemic dissemination.
- All eSI-Pf treated mice survived and showed bacterial clearance within 10 days.
- eSI-Pf induced an attenuated small-colony variant of P. aeruginosa with altered physiology and virulence.
Conclusions:
- Engineered superinfective phages (eSI-Pf) represent a novel therapeutic strategy for multidrug-resistant P. aeruginosa burn infections.
- eSI-Pf prevents sepsis and mortality by inducing bacterial attenuation rather than direct bacterial killing.
- This approach offers a promising alternative to conventional antibiotics and standard lytic phage therapy.
