Characterization of Newly Isolated Rosenblumvirus Phage Infecting Staphylococcus aureus from Different Sources

<i>Staphylococcus aureus</i> is a globally significant pathogen associated with severe infections, foodborne illnesses, and animal diseases. Its control has become increasingly challenging due to the spread of antibiotic-resistant strains, highlighting the urgent need for effective alternatives. In this context, bacteriophages have emerged as promising biocontrol agents. This study aimed to characterize the newly isolated <i>Staphylococcus</i> phage CapO46 and evaluate its efficacy in reducing <i>S. aureus</i> in milk. Identified as a new species within the <i>Rosenblumvirus</i> genus, CapO46 exhibited a podovirus-like structure and a small linear dsDNA genome (17,107 bp), with no lysogeny-related, antimicrobial resistance, or virulence genes. Host range assays demonstrated its ability to infect all 31 <i>S. aureus</i> isolates from two different countries and in diverse environmental contexts, achieving high efficiency of plating (EOP > 0.5) in 64.5% of cases. Kinetic analyses revealed rapid adsorption and a short latent period, with a burst size of approximately 30 PFU/cell. In UHT whole-fat milk, CapO46 achieved a maximum reduction of 7.2 log10 CFU/mL in bacterial load after 12 h, maintaining significant suppression (1.6 log10 CFU/mL) after 48 h. Due to its genetic safety, high infectivity across multiple isolates, and antimicrobial activity in milk, CapO46 can be considered a promising candidate for <i>S. aureus</i> biocontrol applications.