biostudies-literatureBolognini SItalian Ministry of HealthPNRR THE e Tuscany Health Ecosystem938https://www.ebi.ac.uk/biostudies/studies/S-EPMC12300175biostudies-literatureUnknown17(7)<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 ciprofloxacVirusesTherapeutic Optimization of &lt;i&gt;Pseudomonas aeruginosa&lt;/i&gt; Phages: From Isolation to Directed Evolution.PMC12300175Project code: ECS00000017; CUP I53C22000780001Project ID: T4-AN-07 CUP: I53C22001300001Rindi LDi Luca MBolognini SMarchetti MCampobasso CFerretti CTrovato ETavanti AfalseTherapeutic Optimization of &lt;i&gt;Pseudomonas aeruginosa&lt;/i&gt; 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 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 ciprofloxac2025-01-01T00:00:00Z2025 Jun2025-08-16T03:06:45.612Z2025-08-16T03:06:45.612ZS-EPMC123001754073355610.3390/v17070938