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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.

Background Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide variety of infections, and belongs to the group of ESKAPE pathogens that are the leading cause of healthcare-associated infections and have high level of antibiotic resistance. The treatment of infections caused by antibiotic-resistant P. aeruginosa is challenging, which makes it a common target for phage therapy. The successful utilization of phage therapy requires a collection of well characterized phages. Methods Phage fMGyn-Pae01 was isolated from a commercial phage therapy cocktail. The phage morphology was studied by transmission electron microscopy and the host range was analyzed with a liquid culture method. The phage genome was sequenced and characterized, and the genome was compared to closest phage genomes. Phage resistant bacterial mutants were isolated and whole genome sequencing and motility, phage adsorption and biofilm formation assays were performed to the mutants and host bacterium. Results The genomic analysis revealed that fMGyn-Pae01 is a lytic, phiKZ-like jumbo phage with genome size of 277.8 kb. No genes associated with lysogeny, bacterial virulence, or antibiotic resistance were identified. Phage fMGyn-Pae01 did not reduce biofilm formation of P. aeruginosa , suggesting that it may not be an optimal phage to be used in monophage therapy in conditions where biofilm formation is expected. Host range screening revealed that fMGyn-Pae01 has a wide host range among P. aeruginosa strains and its infection was not dependent on O-serotype. Whole genome sequencing of the host bacterium and phage resistant mutants revealed that the mutations had inactivated either a flagellar or rpoN gene, thereby preventing the biosynthesis of a functional flagellum. The lack of functional flagella was confirmed in motility assays. Additionally, fMGyn-Pae01 failed to adsorb on non-motile mutants indicating that the bacterial flagellum is the phage-binding receptor. Conclusion fMGyn-Pae01 is a phiKZ-like jumbo phage infecting P. aeruginosa . fMGyn-Pae01 uses the flagellum as its phage-binding receptor, supporting earlier suggestions that flagellum might be utilized by phiKZ but differs from some other previous findings showing that phiKZ-like phages use the type-IV pili as the phage-binding receptor.

Pseudomonas aeruginosa ( P. aeruginosa ) is an important zoonotic pathogen. It is also the primary causative agent of systemic infections in the endangered Moschus berezovskii . The emergence of multidrug-resistant strains of P. aeruginosa has made these infections increasingly difficult to control, and bacteriophages are considered important alternatives or adjuncts to antibiotic therapy. This study isolated P. aeruginosa strains that induce suppurative infections in Moschus berezovskii from a farm in Shaanxi Province, China. The bacteriophages vB_PaeP_FMD5 (FMD5) and vB_PaeM_H24-1 (H24-1) were isolated using these bacteria as hosts. The safety and practicality of the two phages were analyzed through methods such as biological characteristic assessment, whole genome sequencing analysis, and animal experiments. FMD5 is classified within the Podoviridae family, whereas H24-1 belongs to Myxoviridae. Biological characterization revealed that both FMD5 and H24-1 exhibit tolerance to temperature, pH, chloroform, and Ultraviolet(UV) exposure. The optimal multiplicity of infection (OMOI) for FMD5 and H24-1 were 0.01 and 0.1, respectively, and the burst sizes from the one-step growth curve were 200 PFU/cell and 150 PFU/cell, respectively. In vitro inhibitory assays demonstrated that FMD5, H24-1, and their cocktail exerted a favorable inhibitory effect for up to 11 hours. Whole genome sequencing confirmed that both phages possess double-stranded DNA genomes, with FMD5 having a length of 72,254 bp and a G+C content of 55.16%, containing 91 ORFs(Open Reading Frame)​, whereas H24-1 has a genome length of 66,281 bp, a G+C content of 56.26%, and encompasses 94 ORFs. No drug-resistance genes, virulence factors, or lysogenic genes were identified in either phage. Phylogenetic analysis of conserved genes revealed that FMD5 is closely related to the previously published Pseudomonas phage LP14 (LP14), while H24-1 is closely related to the previously published Pseudomonas phage vB_PaeM_LS1(LS1), but both are newly discovered bacteriophages. In a mouse model of bacteremia treated with bacteriophages, both individual phages and the cocktail exhibited favorable therapeutic effects. The two novel bacteriophages isolated in this study exhibit efficient and stable characteristics. They demonstrate sound therapeutic effects against bacteremia in mice caused by multidrug-resistant P. aeruginosa , suggesting their great potential as alternatives or adjuncts to antibiotic therapy for treating infection.

Carbapenem-resistant Acinetobacter baumannii (CRAB) has become a critical concern that necessitates the development of novel antimicrobial approaches. One of the most promising innovative approaches for combating CRAB infections is using effective and lytic bacteriophages (phages), known as phage therapy. Therefore, we recovered and characterized a polyvalent lytic Salmonella_phage_VB_ST-SA173, producing lytic activity against 6 CRAB clinical isolates and 3 multidrug-resistant (MDR) Salmonella serovars. Throughout pH 2–10, and thermal stability at up to 60 °C, the phage maintained its stability and lytic activity against the tested isolates. The presence of a tailed phage with a characteristic prolate head and a contractile tail was detected by the transmission electron microscope (TEM). According to the Oxford nanopore sequencing data, the genome of Salmonella_phage_VB_ST-SA173 was 53,636 bp in size, contained 45.9% G + C, and had 53 opening reading frames (ORFs). According to the TEM, ORFs, and BLASTn analysis findings, it was proved that the Salmonella_phage_VB_ST-SA173 belongs to the Loughboroughvirus genus. The efficacy of the phage-formulated Carbopol 940 hydrogel in wound healing was assessed preclinically in an infected burn wound animal model with a CRABa clinical isolate. The survival rate was enhanced in the phage-treated group compared to the untreated control groups. Histopathological analysis showed improved wound healing in the form of apparently healthy skin with apparently normal epidermal and dermis layers. In conclusion, depending on its in vitro and physicochemical traits, the phage-loaded hydrogel is expected to be a promising candidate for clinical trials against human CRAB-related skin infections. Key points • A polyvalent Loughboroughvirus phage showed lytic activity against CRAB and Salmonella serovars. • The phage showed stability at a wide range of pH and temperature. • The phage hydrogel enhanced healing in the burn-wound animal model infected with CRABa.

Pseudomonas aeruginosa is an opportunistic pathogen that forms persistent biofilms in the lungs of cystic fibrosis and other chronic pulmonary disease patients, contributing to antibiotic tolerance, recurrent infection, and clinical decline. The rise of multidrug-resistant P. aeruginosa underscores the urgent need for alternative therapies. Bacteriophages (phages) offer a powerful therapeutic approach by directly lysing bacteria, diminishing biofilm structures, and overcoming mechanisms that limit antibiotic efficacy. In this study, a library of 61 distinct P. aeruginosa phages was isolated and screened against 64 clinical isolates, identifying eight with broad host range and high lytic activity. These phages, including PA-319, PA-575, and PA-711, effectively prevented P. aeruginosa colonization on A549 human lung epithelial cells, inhibited bacterial biofilm formation as well as compromised established biofilms, surpassing the effects of high-concentration antibiotics. Genomic and transmission electron microscopy analyses revealed functional heterogeneity, including nucleus-forming and non-nucleus-forming jumbo phages and depolymerase-encoding genes. Our phage library provides a valuable resource for advancing research, developing combinatorial phage therapies, and optimizing treatment strategies against chronic, drug-resistant P. aeruginosa infections.

The paper covers the history of the discovery and description of phiKZ, the first known giant bacteriophage active on Pseudomonas aeruginosa. It also describes its unique features, especially the characteristic manner of DNA packing in the head around a cylinder-shaped structure (“inner body”), which probably governs an ordered and tight packaging of the phage genome. Important properties of phiKZ-like phages include a wide range of lytic activity and the blue opalescence of their negative colonies, and provide a background for the search and discovery of new P. aeruginosa giant phages. The importance of the phiKZ species and of other giant phage species in practical phage therapy is noted given their broad use in commercial phage preparations.

Phage Lydia, a newly isolated siphovirus infecting Pseudomonas aeruginosa, was characterized with respect to its basic kinetic properties and subjected to comparative bioinformatic analysis with related phages. The phage exhibited a restricted host range, with lytic activity observed against 7 of 30 tested isolates. The genome of phage Lydia consists of a 61,986 bp dsDNA molecule and contains 89 predicted genes. Bioinformatic analysis suggests the presence of a DNA modification system, but no apparent genes associated with lysogeny or antibiotic resistance were identified. Taxonomic classification places Lydia within the Mesyanzhinovviridae family, Rabinowitzvirinae subfamily, and Yuavirus genus, with the closest relation to Pseudomonas virus M6. Comprehensive bioinformatic studies, including structural modelling and analysis of phage proteins, as well as comparative taxonomic, phylogenomic, and pangenomic analyses of the Mesyanzhinovviridae family, revealed relationships between proteins of Mesyanzhinovviridae phages, proteins from other phage groups, encapsulins, and a gene transfer agent (GTA) particle from Rhodobacter capsulatus. These analyses uncovered patterns of evolutionary history within the family, characterized by genetic exchange events alongside the maintenance of a common genomic architecture, leading to the emergence of new groups within the family.

Virulent bacteriophages (or phages) are viruses that specifically infect and lyse a bacterial host. When multiple phages co-infect a bacterial host, the extent of lysis and dynamics of bacteria–phage and phage–phage interactions are expected to vary. The objective of this study is to identify the factors influencing the interaction of two virulent phages with different Pseudomonas aeruginosa growth states (planktonic, an infected epithelial cell line, and biofilm) by measuring the bacterial time-kill and individual phage replication kinetics. A single administration of phages effectively reduced P. aeruginosa viability in planktonic conditions and infected human lung cell cultures, but phage-resistant variants subsequently emerged. In static biofilms, the phage combination displayed initial inhibition of biofilm dispersal, but sustained control was achieved only by combining phages and the meropenem antibiotic. In contrast, adherent biofilms showed tolerance to phage and/or meropenem, suggesting a spatio-temporal variation in the phage–bacterial interaction. The kinetics of adsorption of each phage to P. aeruginosa during single or co-administration were comparable. However, the phage with the shorter lysis time depleted bacterial resources early and selected a specific nucleotide polymorphism that conferred a competitive disadvantage and cross-resistance to the second phage. The extent and strength of this phage–phage competition and genetic loci conferring phage resistance are, however, P. aeruginosa genotype-dependent. Nevertheless, adding phages sequentially resulted in their unimpeded replication with no significant increase in bacterial host lysis. These results highlight the interrelatedness of phage–phage competition, phage resistance, and specific bacterial growth state (planktonic/biofilm) in shaping the interplay among P. aeruginosa and virulent phages.

It was recently shown that bacteria use, apart from CRISPR–Cas and restriction systems, a considerable diversity of phage resistance systems 1 – 4 , but it is largely unknown how phages cope with this multilayered bacterial immunity. Here we analysed groups of closely related Bacillus phages that showed differential sensitivity to bacterial defence systems, and discovered four distinct families of anti-defence proteins that inhibit the Gabija, Thoeris and Hachiman systems. We show that these proteins Gad1, Gad2, Tad2 and Had1 efficiently cancel the defensive activity when co-expressed with the respective defence system or introduced into phage genomes. Homologues of these anti-defence proteins are found in hundreds of phages that infect taxonomically diverse bacterial species. We show that the anti-Gabija protein Gad1 blocks the ability of the Gabija defence complex to cleave phage-derived DNA. Our data further reveal that the anti-Thoeris protein Tad2 is a ‘sponge’ that sequesters the immune signalling molecules produced by Thoeris TIR-domain proteins in response to phage infection. Our results demonstrate that phages encode an arsenal of anti-defence proteins that can disable a variety of bacterial defence mechanisms. A study reports the discovery and characterization of four distinct families of phage-encoded anti-defence proteins that inhibit a variety of bacterial defence systems.

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.