Antibiotic-resistant Pseudomonas aeruginosa presents a critical global health challenge, particularly in hospital-acquired infections. Bacteriophages offer a promising therapeutic avenue due to their ability to target and lyse resistant strains. This study characterizes Pseudomonas phage Banzai, a newly isolated Pbunavirus (family Lindbergviridae) with lytic activity against multiple P. aeruginosa isolates, including multidrug-resistant strains. Genomic analysis revealed a 66,189 bp genome, lacking antibiotic resistance or virulence factors, and suggested a headful packaging mechanism and the presence of a bidirectional component in the replication. In vivo experiments using Galleria mellonella showed therapeutic potential, significantly improving larval survival (87% at 24 h). Host range analysis revealed activity against 13 of 30 P. aeruginosa isolates, including members of O1, O3, O5 and O6 in silico predicted serogroups. Phylogenomic analyses place phage Banzai within the genus Pbunavirus, sharing 94.8% intergenomic similarity with its closest relatives, supporting its classification as a novel species. These findings highlight phage Banzai as a potential candidate for phage therapy, demonstrating genomic stability, a strictly lytic lifestyle, and in vivo efficacy.

Bacteriophages are powerful drivers of microbial evolution and are increasingly explored as alternatives to antibiotics against multidrug-resistant pathogens such as Pseudomonas aeruginosa. Here, we describe the isolation, phenotypic characterization and genomic, structural and evolutionary analysis of Pseudomonas phage Adele, a lytic myovirus representing a novel species within the genus Pakpunavirus (family Vandenendeviridae). Phage Adele exhibits a short latent period of 20 min, a burst size of 59 ± 11 virions per infected cell and a high virulence index, efficiently lysing non-O11 Pseudomonas aeruginosa strains and reducing biofilm biomass. In vivo, Adele confers marked protection in a Galleria mellonella infection model. Phylogenetic reconstruction, synteny analysis and structural modeling demonstrate the relatedness of Vandenendeviridae to phages of the Andersonviridae and Vequintavirinae clades, pointing to a stable, ancestral virion architecture that has undergone lineage-specific elaborations, including the duplication and divergence of tail tube proteins. The tail assembly chaperone gene employs a conserved −1 programmed ribosomal frameshift. Phage Adele encodes an elaborate set of metabolic reprogramming and anti-defense systems, reflecting extensive horizontal gene transfer. The combination of a conserved structural architecture and mosaic genome establishes Adele as an exemplary system for studying modular evolution in phages, alongside its demonstrated therapeutic efficacy.

Enterococcus faecalis is an important opportunistic pathogen associated with persistent oral infections, biofilm formation, and antimicrobial tolerance, which makes phages targeting this species of both therapeutic and evolutionary interest. Here, we describe the isolation and characterization of Abitsa, a novel lytic Enterococcus phage representing a new species within the genus Efquatrovirus. Abitsa formed small plaques and displayed siphovirus-like morphology. It showed an optimal multiplicity of infection of 0.01, a short latent period of 10 min and a burst size of 28 ± 5 virions per infected cell. The phage efficiently suppressed planktonic growth of E. faecalis over a broad multiplicity of infection (MOI) range and significantly disrupted pre-formed biofilms, with the strongest effect observed at MOI 0.1, resulting in an 82.14% reduction in biofilm biomass. Abitsa remained stable at pH 4–8, at 5–50 °C for 1 h, and in up to 75% chloroform, but exhibited a narrow host range, lysing only one additional clinical E. faecalis isolate among 34 tested enterococcal isolates. Genome analysis showed that Abitsa has a 41,581-bp linear genome lacking lysogeny-associated genes and represents a novel Efquatrovirus species. Comparative structural and phylogenetic analyses additionally supported mosaic evolution and extensive domain shuffling in receptor-binding and lysis-related proteins of Efquatrovirus-like phages. Together, these results identify Abitsa as a biologically unusual and evolutionarily informative lytic phage with antibiofilm activity.