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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.

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.

Mobile genetic elements, such as phages and plasmids, are diverse and drive bacterial evolution through horizontal gene transfer. Phage-plasmids, of which many carry antibiotic resistance genes or virulence factors, are both phages and plasmids and have life cycles of temperate phages and plasmids. This makes accurate classification difficult as current computational tools typically classify them as one or the other. We addressed this problem by developing tyPPing, a new and highly precise method, to systematically identify, separate, and catalog phage-plasmids. We demonstrated that tyPPing is highly accurate and broadly compatible. It provides a reliable foundation for all future studies involving phages and plasmids, ranging from agriculture environments to pathogenic strains of clinical settings.

Validated methods for phage selection, host range expansion, and lytic activity determination are indispensable for maximizing phage therapy outcomes. In this review, we describe some relevant methods, highlighting their advantages and disadvantages, and categorize them as preliminary or confirmatory methods where appropriate. Experimental conditions, such as the composition and consistency of culture media, have an impact on bacterial growth and, consequently, phage propagation and the selection of phage-resistant mutants. The phages require different experimental conditions to be tested to fully reveal their characteristics and phage therapy potential in view of their future use in therapy. Phage lytic activity or virulence should be considered as a result of the phage, its host, and intracellular/environmental factors, including the ability of a phage to recognize receptors on the bacterial cell surface. In vitro quantitative and qualitative measurements of phage characteristics, further validated by in vivo experiments, could be incorporated into one system or mathematical model/formula, which could predict a potential successful outcome of clinical applications.

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.

ABSTRACT Antibiotic resistance is a major public health challenge worldwide, whose implications for global health might be devastating if novel antibacterial strategies are not quickly developed. As natural predators of bacteria, (bacterio)phages may play an essential role in escaping such a dreadful future. The rising problem of antibiotic resistance has revived the interest in phage therapy and important developments have been achieved over the last years. But where do we stand today and what can we expect from phage therapy in the future? This is the question we set to answer in this review. Here, we scour the outcomes of human phage therapy clinical trials and case reports, and address the major barriers that stand in the way of using phages in clinical settings. We particularly address the potential of phage resistance to hinder phage therapy and discuss future avenues to explore the full capacity of phage therapy. There is a remarkable potential of phage therapy for the control of antibiotic resistant infections within the One Health approach, thus, the challenges currently faced and the potential solutions in development must be considered.

Bacteriophages (phages) are viruses that infect bacteria. The “predator–prey” interactions are recognized as a potentially effective way to treat infections. Phages, as well as phage-derived proteins, especially enzymes, are intensively studied to become future alternative or supportive antibacterials used alone or in combination with standard antibiotic regimens treatment. There are many publications presenting phage therapy aspects, and some papers focused separately on the application of phage-derived enzymes. In this review, we discuss advantages and limitations of both agents concerning their specificity, mode of action, structural issues, resistance development, pharmacokinetics, product preparation, and interactions with the immune system. Finally, we describe the current regulations for phage-based product application.

Bacteriophages (phages), as natural antibacterial agents, are being rediscovered because of the growing threat of multi- and pan-drug-resistant bacterial pathogens globally. However, with an estimated 1031 phages on the planet, finding the right phage to recognize a specific bacterial host is like looking for a needle in a trillion haystacks. The host range of a phage is primarily determined by phage tail fibers (or spikes), which initially mediate reversible and specific recognition and adsorption by susceptible bacteria. Recent significant advances at single-molecule and atomic levels have begun to unravel the structural organization of tail fibers and underlying mechanisms of phage–host interactions. Here, we discuss the molecular mechanisms and models of the tail fibers of the well-characterized T4 phage’s interaction with host surface receptors. Structure–function knowledge of tail fibers will pave the way for reprogramming phage host range and will bring future benefits through more-effective phage therapy in medicine. Furthermore, the design strategies of tail fiber engineering are briefly summarized, including machine-learning-assisted engineering inspired by the increasingly enormous amount of phage genetic information.

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.