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Therapeutic Optimization of Pseudomonas aeruginosa Phages: From Isolation to Directed Evolution
Sara Bolognini1,2, Caterina Ferretti3, Claudia Campobasso1
1Department of Biology, University of Pisa, 56126 Pisa, Italy.
Abstract:
Pseudomonas aeruginosa is a major opportunistic pathogen with high levels of antibiotic resistance. Phage therapy represents a promising alternative for the treatment of difficult infections both alone and in combination with antibiotics. Here, we isolated and characterized three novel lytic myoviruses, Cisa, Nello, and Moonstruck. Genomic analysis revealed that Cisa and Nello belong to the Pbunavirus genus, while Moonstruck is a novel Pakpunavirus species. All lacked lysogeny, virulence, or resistance-associated genes, supporting their therapeutic suitability. Phage Nello and Moonstruck were active against P. aeruginosa Pa3GrPv, isolated from a patient with lung infection candidate for phage therapy. Moonstruck exhibited superior lytic activity with ciprofloxacin sub-MIC value (0.125 µg/mL), achieving bacterial suppression for 48 h. However, to improve the lytic efficacy of the phages on the clinical isolate, phage adaptation via serial passage was investigated. The killing efficacy of Nello was enhanced, whereas Moonstruck showed a less consistent improvement, suggesting phage-specific differences in evolutionary dynamics. Sequencing of the evolved phages revealed point mutations in tail-associated genes, potentially linked to a better phage-host interaction. These results support the use of phage-antibiotic combinations and directed evolution as strategies to enhance phage efficacy against drug-resistant infections. Overall, these findings support the therapeutic potential of the newly isolated phages in treating P. aeruginosa lung infections.
Insights
Three novel phages (Cisa, Nello, Moonstruck) show potential against antibiotic-resistant Pseudomonas aeruginosa lung infections. Phage adaptation enhanced efficacy, supporting phage-antibiotic combinations for treating drug-resistant bacterial infections.
Area of Science:
- Microbiology
- Bacteriology
- Virology
Background:
- Pseudomonas aeruginosa is a significant opportunistic pathogen known for high antibiotic resistance.
- Phage therapy offers a promising alternative for treating challenging infections, alone or with antibiotics.
Purpose of the Study:
- To isolate and characterize novel lytic myoviruses for potential therapeutic use against Pseudomonas aeruginosa.
- To evaluate the efficacy of isolated phages, individually and in combination with antibiotics, against a clinical isolate.
- To investigate phage adaptation strategies to enhance lytic activity against drug-resistant bacteria.
Main Methods:
- Isolation and genomic characterization of three novel lytic myoviruses: Cisa, Nello, and Moonstruck.
- Assessment of phage activity against Pseudomonas aeruginosa clinical isolate Pa3GrPv.
- Evaluation of phage-antibiotic synergy using sub-inhibitory concentrations of ciprofloxacin.
- Phage adaptation through serial passage and sequencing of evolved phages.
Main Results:
- Cisa and Nello classified under Pbunavirus, Moonstruck under Pakpunavirus; all lacked undesirable genes.
- Nello and Moonstruck demonstrated activity against the clinical isolate.
- Moonstruck showed enhanced lytic activity in combination with sub-minimum inhibitory concentration ciprofloxacin.
- Phage adaptation improved Nello's efficacy, with mutations in tail-associated genes observed in evolved phages.
Conclusions:
- Newly isolated phages Cisa, Nello, and Moonstruck possess therapeutic potential against Pseudomonas aeruginosa.
- Phage-antibiotic combinations and directed evolution are viable strategies to enhance phage efficacy.
- These findings support the development of phage therapy for drug-resistant Pseudomonas aeruginosa lung infections.
