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Pseudomonas syringae pv. actinidiae (Psa) is the causative agent of bacterial canker in kiwifruit ( Actinidia spp.). Psa biovar 3 is the most prevalent and virulent, causing frequent and severe outbreaks worldwide. While current treatments have low efficacy, bacteriophages emerge as possible environmentally safe alternative biocontrol agents. In this study, bacteriophage Brt_Psa3 was isolated from the soil of a kiwifruit orchard in Portugal. Morphologically, Brt_Psa3 forms clear plaques and has a Podoviral morphotype. The bacteriophage exhibited broad lytic activity against several plant-pathogenic Pseudomonas strains, including Psa isolates. The isolated bacteriophage has a latent period of 100 min, a burst size of 143 particles/cell, and demonstrates stability at different temperatures and pH values found in kiwifruit orchards. In addition, Brt_Psa3 exhibited tolerance to UVA irradiation during 120 min of incubation. Brt_Psa3 belongs to the Autographiviridae family and Ghunavirus genus, based on full-genome nucleotide alignment and supported by phylogenetic analysis of structural proteins. The phage contains 51 open reading frames with no antibiotic resistance genes identified, within a genome of 40.509 base pairs. In vitro experiments with kiwifruit leaves demonstrated significant reduction of Psa levels (40%) on leaf surfaces, highlighting the bacteriophage’s therapeutic potential in managing bacterial canker in kiwifruits. Keypoints •  Isolated bacteriophage Brt_Psa3 effectively infect Psa strains •  Brt_Psa3 maintains infectivity under conditions similar to kiwifruit orchard environment •  Leaf assay confirms its biocontrol potential

Pseudomonas aeruginosa is a gram-negative opportunistic pathogen that poses a significant threat due to its increasing multidrug resistance, particularly in clinical settings. This study aimed to isolate and characterize a novel bacteriophage, phiLCL12, from hospital wastewater and evaluate its potential in combination with antibiotics to combat P. aeruginosa infections and biofilm formation. Transmission electron microscopy revealed that phiLCL12 possesses a long contractile tail. The isolated phage exhibited a broad host range of 82.22% and could adsorb up to 98% of its target within 4 min. It was effective against multidrug-resistant strains at both high and low multiplicities of infection (MOIs) levels in lysis tests. Taxonomic classification was determined using PhaGCN2 and Whole genomic analysis, and the results identified phiLCL12 as a member of the Pbunavirus. In vitro experiments demonstrated that phiLCL12 significantly enhanced biofilm clearance and inhibited biofilm formation when combined with sub-inhibitory concentrations of imipenem. Furthermore, in vivo experiments using a zebrafish model showed that phage–antibiotic synergy (PAS) improved survival rate compared to antibiotic treatment alone. This study demonstrates that phiLCL12 is effective in both eradicating and preventing P. aeruginosa biofilm formation. The combination of phiLCL12 and imipenem provides a synergistic effect, significantly enhancing survival outcomes in a zebrafish model. These findings highlight the potential of phage–antibiotic synergy as a promising therapeutic strategy against biofilm-associated infections.

Antibiotic resistance has been attributed to both the overuse of current drugs and the lack of availability of newer drugs due to stringent regulatory requirements and reduced commercial incentives. Novel alternative therapies, such as phage treatments, have demonstrated promising outcomes in clinical trials, indicating their potential for treating recalcitrant infections in the future. The objective of this study was to isolate and characterize bacteriophages against a uropathogenic strain of Pseudomonas aeruginosa and evaluate the antimicrobial efficacy of combining sub-inhibitory concentrations of cefepime and meropenem with the isolated bacteriophages. The isolated phages were designated PAA and PAM, both exhibiting icosahedral heads and long non-contractile tails. Both phages maintained stability within Limited pH and temperature ranges. The optimal adsorption times for PAA and PAM were 10 and 20 min, respectively, with PAA demonstrating a short latent time and burst size of 47, while PAM exhibited a burst size of 83. The optimal multiplicity of infection for PAA was 1, and for PAM was 0.1. PAA demonstrated efficacy against 40% of the tested strains of P. aeruginosa , while PAM was effective against 64%. Both phages remained stable at subinhibitory concentrations of cefepime and meropenem and when employed with sub-inhibitory concentrations of cefepime and meropenem, exhibited synergistic effects against planktonic bacterial cells and demonstrated efficacy in both biofilm inhibition and eradication.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) has emerged as a significant global public health threat due to its increasing prevalence and dissemination, necessitating the development of novel antimicrobial agents. In this regard, bacteriophages, particularly lytic phages, offer a promising alternative to conventional antibiotics for the treatment of such resistant infections. In this study, we isolated and characterized a lytic Pseudomonas phage, Pa_WF01, from hospital sewage, which specifically targets clinical CRPA strains. The host range, multiplicity of infection (MOI), morphology, one-step growth curve, thermal and pH stability, chloroform sensitivity, and lytic activity of Pa_WF01 were evaluated. Our findings showed that the MOI, latent period, and burst size of Pa_WF01 were approximately 0.0001, 10 min, and 154 phages per cell, respectively. Furthermore, Pa_WF01 exhibited robust lytic activity across a broad range of pH values (4–12) and temperatures (4–50 °C), effectively inhibiting bacterial growth. Transmission electron microscopy (TEM) analysis supported that Pa_WF01 exhibits morphological characteristics similarity to the Schitoviridae family, and the result was further confirmed by phylogenetic analysis of complete genome sequences. Whole-genome sequencing revealed that Pa_WF01 has a double-stranded DNA genome of 73,369 bp and a GC content of 54.78%, containing 94 open reading frames (ORFs). Notably, no tRNA, virulence, or antibiotic resistance genes were identified within the genome. Phylogenetic tree analysis further classified Pa_WF01 as closely related to phages of the Litunavirus genus. In vivo, Pa_WF01 significantly improved the survival rate of mice infected with CRPA, reduced inflammatory responses, decreased bacterial loads in organs (lung, liver, and spleen), and alleviated organ damage. Additionally, in vitro analysis demonstrated that Pa_WF01 enhanced serum-mediated bactericidal activity. Taken together, these results highlight the potential of phage Pa_WF01 as a viable therapeutic alternative for treating CRPA infections in clinical practice.

Background Carbapenem-resistant Pseudomonas aeruginosa commonly leads to difficult-to-treat infections necessitating new therapeutics. Recently, bacteriophages have gained attention as promising alternatives. This study aimed to isolate, characterize virulent phages from various water sources against clinical carbapenem-resistant P. aeruginosa isolates to formulate a phage cocktail, and evaluate its in vivo efficacy using a mouse burn wound infection model. Results Biological and genomic characterization of isolated phages were determined by host range, temperature and pH stability, transmission electron microscopy analysis, and whole-genome sequencing. Three virulent phages without carrying antibiotic resistance, virulence or lysogeny-related gene included in the study and named as Baskent_P1_112 (Φ1), Baskent_P2_ICU (Φ2) and Baskent_P3_3B (Φ3). Φ1 exhibited podovirus-like morphology, while Φ2 and Φ3 displayed myovirus-like morphology. MOI values were determined as 100, 1, and 10, with corresponding burst sizes of 123, 288 and 115 PFU/CFU, respectively. All three phages were stable at temperatures between 4 and 50 °C; and pH 4–10, Φ1 and Φ3 were completely inactive at pH 2 and 12. Phages with diverse receptor binding site proteins exhibited complementary lytic activity profiles across different and overlapping sets of carbapenem-resistant P. aeruginosa isolates were used for formulation the phage cocktail thereby achieving a broad host range. The therapeutic efficacy of the phage cocktail was compared with antibiotic treatment in 45 Balb/c mice, divided into five groups. Blood and tissue samples were collected for CRP analysis, bacterial load, and histopathological examination. Wound surfaces were measured daily, and survival percentages were recorded. Conclusion Compared to the untreated control group, phage therapy significantly reduced CRP levels and bacterial loads, enhanced wound healing, and improved survival rates without any toxicity. These results demonstrate that the formulated phage cocktail is a promising alternative treatment with a protocol adaptable for on-demand clinical use.

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.

Background Pseudomonas aeruginosa is an opportunistic pathogen and one of the leading causes of nosocomial infections. Moreover, the species can cause severe infections in cystic fibrosis patients, in burnt victims and cause disease in domestic animals. The control of these infections is often difficult due to its vast repertoire of mechanisms for antibiotic resistance. Phage therapy investigation with P. aeruginosa bacteriophages has aimed mainly the control of human diseases. In the present work, we have isolated and characterized a new bacteriophage, named Pseudomonas phage BrSP1, and investigated its host range against 36 P. aeruginosa strains isolated from diseased animals and against P. aeruginosa ATCC strain 27853. Results We have isolated a Pseudomonas aeruginosa phage from sewage. We named this virus Pseudomonas phage BrSP1. Our electron microscopy analysis showed that phage BrSP1 had a long tail structure found in members of the order Caudovirales. “In vitro” biological assays demonstrated that phage BrSP1 was capable of maintaining the P. aeruginosa population at low levels for up to 12 h post-infection. However, bacterial growth resumed afterward and reached levels similar to non-treated samples at 24 h post-infection. Host range analysis showed that 51.4% of the bacterial strains investigated were susceptible to phage BrSP1 and efficiency of plating (EOP) investigation indicated that EOP values in the strains tested varied from 0.02 to 1.72. Analysis of the phage genome revealed that it was a double-stranded DNA virus with 66,189 bp, highly similar to the genomes of members of the genus Pbunavirus , a group of viruses also known as PB1-like viruses. Conclusion The results of our “in vitro” bioassays and of our host range analysis suggested that Pseudomonas phage BrSP1 could be included in a phage cocktail to treat veterinary infections. Our EOP investigation confirmed that EOP values differ considerably among different bacterial strains. Comparisons of complete genome sequences indicated that phage BrSP1 is a novel species of the genus Pbunavirus . The complete genome of phage BrSP1 provides additional data that may help the broader understanding of pbunaviruses genome evolution.

DEV is an obligatory lytic Pseudomonas phage of the N4-like genus, recently reclassified as Schitoviridae . The DEV genome encodes 91 ORFs, including a 3398 amino acid virion-associated RNA polymerase (vRNAP). Here, we describe the complete architecture of DEV, determined using a combination of cryo-electron microscopy localized reconstruction, biochemical methods, and genetic knockouts. We built de novo structures of all capsid factors and tail components involved in host attachment. We demonstrate that DEV long tail fibers are essential for infection of Pseudomonas aeruginosa but dispensable for infecting mutants with a truncated lipopolysaccharide devoid of the O-antigen. We determine that DEV vRNAP is part of a three-gene operon conserved in 191 Schitoviridae genomes. We propose these three proteins are ejected into the host to form a genome ejection motor spanning the cell envelope. We posit that the design principles of the DEV ejection apparatus are conserved in all Schitoviridae . Lokareddy et al. determine the complete structure of DEV, a lytic virus that infects Pseudomonas aeruginosa , using a combination of cryo-electron microscopy, biochemical methods, and genetic knockouts. They propose that the virion-associated RNA polymerase may be part of a genome ejection motor.

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.

Background Pan-drug resistance (PDR) is a ticking time bomb, as it causes high human hospitalizations and mortality rates. For instance, Pseudomonas aeruginosa is associated with significant rates of urinary tract infections (UTIs) due to several reasons including antibiotic resistance, biofilm formation and the presence of various virulence factors. Consequently, there is an urgent need for safe and effective alternative antibacterials. Phage therapy is a promising alternative that uses naturally occurring bacteriophages (phages). Therefore, our present study investigated the isolation and characterization of a novel virulent phage (vB_Ps_ZCPS13) against the PDR Pseudomonas aeruginosa strain (Ps13). Methods Phage vB_Ps_ZCPS13 was isolated from raw sewage water in Egypt during the springtime. The isolated phage was purified and amplified, followed by estimating its purity and genome size using pulsed-field gel electrophoresis (PFGE), morphology using transmission electron microscopy (TEM), antibacterial activity against other P. aeruginosa hosts, physiochemical stability studies, whole genome sequencing, antibiofilm activity on urinary catheters using scanning electron microscopy (SEM), and cytotoxicity assays against normal human skin fibroblast (HSF) cell lines. Results Based on vB_Ps_ZCPS13 morphology under TEM, the phage has been classified as a myovirus. In consistent with the PFGE results, DNA sequencing revealed a phage genome size of 92,443 bp, with lytic-associated genes and no antimicrobial resistance or virulence factors. Phage vB_Ps_ZCPS13 presented a wide host range of over 93% of tested clinical isolates having different multiple antibiotic resistance (MAR) indices. Furthermore, phage vB_Ps_ZCPS13 exhibited high efficiency in plaque formation (EOP ≥ 1) against 13% of the strains and exhibited low frequencies of bacteriophage insensitive mutants (BIM). The physical stability test against harsh environmental conditions revealed phage stability within a pH range of 3.0–11.0 and stable at temperatures below 70 °C. Phage vB_Ps_ZCPS13 also exposed a significant antibacterial activity in vitro across different MOIs, with the highest reduction in bacterial growth observed at lower MOIs. Furthermore, vB_Ps_ZCPS13 demonstrated potent biofilm inhibition and clearance capabilities, effectively eradicating P. aeruginosa from the urinary catheter surface. Moreover, the phage presented no cytotoxicity against normal human skin fibroblast (HSF) cell lines at high titer. Conclusions Our study offers an effective phage as a therapeutic candidate against PDR Gram-negative P. aeruginosa infections, including catheter-associated urinary tract infections.