We engineered novel T5-like bacteriophage (phage) with extended host ranges by editing the long-tail fibers (PB3 and PB4) to combat Salmonella Enteritidis. By replacing the long-tail fibers PB3 and PB4 regions of phage PH204 with those from phage SP76, we created phages RPA1 − 3 and RPB1 − 3, which exhibited expanded host ranges, lysing 54 strains compared to the original 30 strains. These phages retained the biological characteristics of PH204, including temperature, pH stability and adsorption rate. In milk, RPA1 − 3 and RPB1 − 3 inhibited Salmonella ZWSA605 growth, reducing bacterial counts to 1.51 log10 CFU/mL and 2.18 log10 CFU/mL after 8 h, respectively. Although the bacteriolytic activity of recombinant phages is lower than that of the parent phage, our findings suggest that these phages hold promise as candidates for future phage biocontrol applications in food.

Methicillin-resistant Staphylococcus aureus (MRSA) is a common antibiotic-resistant pathogen. MRSA and its biofilm pose a great threat to the food industry. In this study, we characterized the biological properties and antibacterial efficacy of phages through the double-layer plate method, transmission electron microscopy (TEM), whole-genome sequencing (WGS), bioinformatic analyses, fluorescence microscopy, and biofilm eradication assays. The results demonstrated that phage SPB is a virulent member of the genus Kayvirus (subfamily Twortvirinae), exhibited a broad host range spanning Staphylococcus species. It effectively lysed 97.3% (36/37) of clinical MRSA isolates and 100.0% (10/10) of coagulase-negative staphylococci strains tested. The optimal multiplicity of infection (MOI) was determined to be 1, with a latent period of 10 min. Environmental stability assays revealed that phage SPB maintained infectivity across temperatures ranging from 4°C to 50°C and pH values between 4 and 11. Genomic analysis showed that phage SPB possesses a 143,170 bp genome with a G+C content of 30.2%, encoding 218 putative coding sequences (CDSs), 3 tRNAs, and no virulence factors were identified through in software screening. Phage SPB exhibited potent inhibition of planktonic bacterial growth. Furthermore, at varying multiplicities of infection (MOIs), it significantly suppressed biofilm formation and eradicated pre-existing biofilms, with statistical significance (P < 0.001). These results suggest that phage SPB can be used as a potential antimicrobial agent to prevent and remove MRSA and its biofilm from food processing.

Objective: To isolate and characterize a novel Staphylococcus phage with activity against animal-derived methicillin-resistant Staphylococcus aureus (MRSA) infections and biofilms in Kashgar, Xinjiang. Methods: Phage SPD was isolated through enrichment from farm wastewater, purified using the double-layer plaque assay, and characterized by using transmission electron microscopy (TEM), spot assays, growth kinetics, stability tests, and whole-genome sequencing (Illumina HiSeq). Anti-biofilm activity was evaluated via crystal violet staining. Results: SPD is a virulent Podovirus characterized by an icosahedral head (~50 nm in diameter) and a short tail phage (~60 nm in length). It exhibits a broad host range (lyses 38 out of 50 tested strains, including 7 MRSA), a short latent period (~10 min), a high burst size (4,470 PFU/cell), and stability at temperatures ranging from 4 to 50 °C and pH values between 6 and 9. The 17,066 bp double-stranded DNA genome (35.1% GC content) encodes a lysin and a holin, with no virulence or antibiotic resistance genes detected. SPD effectively inhibits planktonic MRSA growth and reduces biofilm formation (>50% inhibition) as well as pre-formed biofilms (>60% clearance) in vitro . Conclusion: SPD represents a safe, novel Andhravirus phage with potent activity against MRSA and biofilms, offering a promising candidate for phage therapy, particularly in the Xinjiang region.