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Ralsto nia pseudosolanacearum is an increasingly prominent multidrug-resistant phytopathogen. Phages are an effective alternative for treating R. pseudosolanacearum infections. In this study, the phage vB_RsoP_BMB116, which is specific to R . pseudosolanacearum , was isolated from Fujian, China. Electron microscopy revealed that vB_RsoP_BMB116 exhibited a Podoviridae morphotype. The double-stranded DNA genome of vB_RsoP_BMB116 spans 82,991 bp (base pairs) and encodes 119 predicted unidirectionally oriented genes, of which 40 have putative functions assigned, while the remainder encode hypothetical proteins. Genome analysis indicated that vB_RsoP_BMB116 is a new genus. Furthermore, tomato plant experiments conducted in this study demonstrated that vB_RsoP_BMB116 significantly reduces the disease incidence of tomato bacterial wilt induced by R. pseudosolanacearum , thus providing effective experimental support for its potential as a biological control agent against tomato bacterial wilt in agricultural settings. In summary, these results not only expand the genetic repertoire of phages targeting R. pseudosolanacearum but also establish a theoretical and experimental basis for developing phage-mediated biocontrol approaches to manage bacterial wilt in tomato cultivation. Key points •  The new R. pseudosolanacearum phage vB_RsoP_BMB116 (novel genus) is a key resource for microbiology and biotechnology, aiding new phage therapies and biocontrol agents •  Observed via transmission electron microscopy (TEM), the novel phage vB_RsoP_BMB116 belongs to the Podoviridae family •  In vitro assays demonstrated that it exhibits significant application potential for the prevention and control of plant bacterial diseases

Multidrug-resistant (MDR) Proteus mirabilis is a significant opportunistic pathogen, largely due to its ability to form robust biofilms that are recalcitrant to conventional antibiotic therapy. Bacteriophages represent a promising alternative, yet their therapeutic potential is contingent on a thorough characterization of their biological functions and genomic architecture. This study details the isolation and comprehensive analysis of a novel bacteriophage targeting MDR P. mirabilis . A novel lytic bacteriophage, designated Proteus phage ram_arti_1324, was isolated from sewage. Transmission electron microscopy classified it as a member of the family Podoviridae . Functional assays demonstrated its potent efficacy against established P. mirabilis biofilms, achieving a 55% reduction in total biomass in vitro conditions. Whole-genome sequencing revealed a linear, double-stranded DNA genome of 91,735 bp with a GC content of 34.7%. Genomic annotation identified 146 putative coding sequences (CDS), including key lytic genes encoding two distinct endolysins, and four tRNA genes. Crucially, the genome lacks any identifiable virulence factors, antibiotic resistance genes, or markers indicative of a lysogenic life cycle. Comparative genomic analysis against 51 known Proteus phages confirmed its novelty. Proteus phage ram_arti_1324 shares less than 95% average nucleotide identity with its closest relatives, suggesting it represents a novel viral species. This uniqueness was further supported by whole-genome alignment, which revealed a distinct arrangement of locally co-linear blocks not observed in other related phages. The genomic and functional data establish that Proteus phage ram_arti_1324 is a novel, strictly lytic bacteriophage with significant therapeutic potential against both planktonic and biofilm-associated MDR P. mirabilis . Its potent anti-biofilm activity, combined with a genome free of deleterious genes, makes it a strong candidate for further development in phage-based clinical applications. The complete genome sequence provides a valuable resource for future research into phage-host dynamics and the bioengineering of next-generation antimicrobial agents. Key Points •  Novel lytic bacteriophage Proteus phage ram_arti_1324 was isolated with genome size 91735 bp. •  Proteus phage ram_arti_1324 displayed absence of virulence factors, antibiotic resistance genes (ARGs) and deleterious genes. •  Proteus phage ram_arti_1324 displayed antibiofilm activity against MDR P. mirabilis.

Vibrio parahaemolyticus causes aquatic vibriosis. Its biofilm protects it from antibiotics; therefore, a new different method is needed to control V. parahaemolyticus for food safety. Phage therapy represents an alternative strategy to control biofilms. In this study, the lytic Vibrio phage vB_VpaP_FE11 (FE11) was isolated from the sewers of Guangzhou Huangsha Aquatic Market. Electron microscopy analysis revealed that FE11 has a typical podovirus morphology. Its optimal stability temperature and pH range were found to be 20–50 °C and 5–10 °C, respectively. It was completely inactivated following ultraviolet irradiation for 20 min. Its latent period is 10 min and burst size is 37 plaque forming units/cell. Its double-stranded DNA genome is 43,397 bp long, with a G + C content of 49.24% and 50 predicted protein-coding genes. As a lytic phage, FE11 not only prevented the formation of biofilms but also could destroy the formed biofilms effectively. Overall, phage vB_VpaP_FE11 is a potential biological control agent against V. parahaemolyticus and the biofilm it produces.

Staphylococcus aureus is a notorious foodborne pathogen since it has ability to produce variety of toxins including heat-stable enterotoxin, form biofilm, and acquire resistance to antibiotics. Biocontrol of foodborne pathogens by lytic bacteriophages garners increasing interest from both researchers and food industry. In the present study, 29 phages against S. aureus were successfully isolated from chicken, pork, and fish. Characterization of the isolates revealed that phage SA46-CTH2 belonging to Podoviridae family had a number of features suitable for food industry applications such as wide host range, short latent period, large burst size, high stress tolerance, and a genome free of virulence genes. Furthermore, phage SA46-CTH2 alone or in combination with nisin exhibited great efficacy in reducing planktonic and biofilm cells of S. aureus at various conditions tested. The combination of phage SA46-CTH2 and nisin was also found to be able to inhibit the regrowth of S. aureus at both 37 and 24 °C.Key points• A total of 29 S. aureus phages were successfully isolated from fish, pork, and chicken products.• Phage SA46-CTH2 was characterized by host range, morphology, and genome sequencing.• SA46-CTH2 significantly reduced both planktonic and biofilm cells of S. aureus.• Combination of SA46-CTH2 and nisin inhibited the regrowth of S. aureus.

Phage therapy is the use of lytic bacteriophages to cure infections caused by bacteria. The aim of this study is to isolate and to characterize the bacteriophages against Escherichia coli isolated from clinical samples. For isolation of bacteriophages, water samples were collected from the Ganges River, and phage enrichment method was followed for phage isolation. Microbiological, genomic and lyophilization experiments were carried out to characterize the bacteriophage. Galleria mellonella was used to study the potential of phages against E. coli infection. Escherichia phage myPSH1131 belonging to Podoviridae family and found to have broad host range infectivity (n = 31) to infect Enterohemorrhagic E. coli (n = 9), Enteropathogenic E. coli (n = 6), Enterotoxigenic E. coli (n = 3), Enteroaggregative E. coli (n = 3), Uropathogenic E. coli (n = 9) and one unknown E. coli. The genome size is 76,163 base pairs (97 coding regions) and their genes show high similarity to SU10 phage. Lyophilization studies showed that the use of 1M sucrose, 2% gelatin and the combination of both 0.5M sucrose plus 1% gelatin could restore phage viability up to 20 months at 4°C. For in vivo studies, it was observed that a single phage dose can reduce the E. coli infection but to achieve 100% survival rate the infected larvae should be treated with three phage doses (20 μL, 10(3) PFU/mL) at 6 hours interval. The characterized Escherichia phage myPSH1131 was found to have broad host range activity against E. coli pathogens and in vivo studies showed that multiple doses are required for effective treatment.

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

Background Acinetobacter baumannii is a significant opportunistic pathogen causing severe nosocomial infections that poses increasing clinical challenges due to escalating multidrug resistance. Phage therapy has emerged as a promising alternative strategy to combat multidrug-resistant (MDR) infections. Results In this study, a lytic phage targeting ST208/KL2-type A. baumannii strain Ac28 was isolated and purified from hospital sewage. Whole-genome sequencing and a bioinformatics analysis successfully identified a novel podovirus, designated “vB_AbaA_SWMUZ8”. A host-range analysis demonstrated a lysis rate of 58.33% against 84 clinically derived A. baumannii isolates of diverse multilocus sequence types, with particularly high efficacy against 88% of ST208 strains. Notably, vB_AbaA_SWMUZ8 lysed 100% of geographically co-localized isolates. It inhibited the growth of MDR A. baumannii strain Ac28 in vitro and improve survival rates in a Galleria mellonella infection model. Conclusions In this work, we identified a therapeutically promising lytic phage and provided evidence for the phage-based control of MDR A. baumannii infections.

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.

Background Pseudomonas aeruginosa ( P. aeruginosa ) is a leading cause of healthcare-associated infection and is prone to antibiotic resistance. Treating infections caused by antibiotic-resistant P. aeruginosa remains clinically challenging. The increasing prevalence of multidrug-resistant (MDR) bacteria has prompted the development of alternatives to antibiotics. Bacteriophage (phage) therapy has been regarded as a promising antibacterial alternative in many cases of MDR bacterial infections. This study aimed to isolate and characterize phages with therapeutic potential against MDR P. aeruginosa . Methods Phage PUTH1 was isolated from filtered sewage water collected at Peking University Third Hospital using the double-layer agar spot method. The phage morphology was characterized using transmission electron microscopy (TEM). Subsequently, key biological properties were evaluated, including temperature and pH stability, one-step growth curve, adsorption rate, and optimal multiplicity of infection (MOI). Additionally, the anti-biofilm activity was assessed using crystal violet staining assays. Finally, whole genome sequencing was conducted, followed by comprehensive bioinformatics analysis. Results Morphological and genomic characterization confirmed that the phage PUTH1 belongs to the Podoviridae family. The phage exhibits remarkable thermal stability (active up to 60 °C) and across a broad pH range (pH 4.0–9.0). Furthermore, the phage is characterized by a short latent period (approximately 30 min), a large burst size (54.5 PFU/cell), and rapid adsorption efficiency, achieving approximately 80% adsorption after 6 min. PUTH1 exhibits significant anti-biofilm activity. The genome of phage was 45,483 bp with 52.25% GC content and contained 70 putative genes; functions were predicted for 32 of them. Phylogenetic analysis revealed that PUTH1 is distinct from currently known phages and belongs to a novel species. The genome sequencing analysis suggested that it does not exist any known virulence and antibiotic resistant genes. Conclusion Therefore, our findings demonstrate that this novel phage represents a promising therapeutic candidate for treating MDR P. aeruginosa infections.

Background Phage therapy is the therapeutic use of bacteriophages to treat highly drug resistant bacterial infections. The current surge in bacteriophage therapy is motivated mainly because of the emergence of antibiotic-resistant bacteria in clinics. This study evaluated the therapeutic potential of three bacteriophages isolated against Escherichia coli ec311, Klebsiella pneumoniae kp235 and Enterobacter cloacae el140 strains using Galleria mellonella . The in vitro activity of three different phages belonging to Podoviridae and Myoviridae families was studied by the double agar overlay method against multi-drug resistant strains. Larval survivability studies were performed to evaluate the potential of phages against infection using G. mellonella . Results All the three phages were found to have potential to infect the host bacterial strains. For in vivo studies it was observed that E. coli and E. cloacae infected larvae, should be treated with three phage doses (20 μL, 10 4 PFU/mL) at 6 h interval to achieve 100% survival rate. But in the case of K. pneumoniae , a single phage dose treatment showed promising outcome. When mixed bacterial infections (all three bacterial cultures at 10 8  CFU/mL) were tested, minimum of four doses of phage cocktail (three phages) at 6 h interval was necessary to recover the larvae. All the results were confirmed by enumerating bacteria from the larvae. Conclusion Our data shows that although in vitro studies showed high infectivity of phages, for in vivo models multiple phage doses were required for effective treatment.