Phenotypic and genomic characterization of vB_SauP-INT105, an exopolysaccharide depolymerase-encoding lytic short-tailed phage with antibiofilm activity against Staphylococcus aureus

Introduction Staphylococcus aureus is a major opportunistic pathogen associated with dairy production systems, where it contributes to intramammary infections and may enter the food chain through contaminated milk. The increasing prevalence of antimicrobial-resistant strains underscores the need for alternative biocontrol strategies. Here, we report the isolation and comprehensive characterization of a novel lytic bacteriophage, vB_SauP-INT105, obtained from wastewater in Extremadura, Spain. Methods and results Plaque morphology revealed a distinctive three-zone halo pattern suggestive of phage-encoded exopolysaccharide depolymerase activity, confirmed by bioinformatic prediction of two candidate depolymerase-encoding ORFs. Electron microscopy showed a bacteriophage with an icosahedral head and a short non-contractile tail. The phage exhibited lytic activity against 86.5% of 37 dairy-associated S. aureus isolates tested. One-step growth analysis revealed a latency period of ~40 minutes and a burst size of ~23 PFU/cell. The phage showed robust stability across a wide range of temperatures and pH values relevant to dairy-processing conditions, retaining viability over 20 months at 4 °C. Genome sequencing revealed a 17.45 kb dsDNA genome encoding 20 ORFs, with no detectable genes associated with virulence, lysogeny, or antibiotic resistance. Phylogenetic analysis placed INT105 as a novel species within the Rosenblumvirus genus. Antibiofilm assays demonstrated significant reductions in viable cell counts in two S. aureus strains with contrasting biofilm phenotypes. Conclusion These results establish INT105 as a genomically safe, environmentally robust and functionally effective phage with strong potential for biocontrol applications in dairy-production environments and broader food-safety and clinical contexts.