Therapeutic Optimization of <i>Pseudomonas aeruginosa</i> Phages: From Isolation to Directed Evolution

<i>Pseudomonas aeruginosa</i> 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...

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Main Authors: Sara Bolognini, Caterina Ferretti, Claudia Campobasso, Elisabetta Trovato, Magda Marchetti, Laura Rindi, Arianna Tavanti, Mariagrazia Di Luca
Format: Article
Language:English
Published: MDPI AG 2025-06-01
Series:Viruses
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Online Access:https://www.mdpi.com/1999-4915/17/7/938
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Summary:<i>Pseudomonas aeruginosa</i> 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 <i>Pbunavirus</i> genus, while Moonstruck is a novel <i>Pakpunavirus</i> species. All lacked lysogeny, virulence, or resistance-associated genes, supporting their therapeutic suitability. Phage Nello and Moonstruck were active against <i>P. aeruginosa</i> 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 <i>P. aeruginosa</i> lung infections.
ISSN:1999-4915