Comprehensive characterization of Staphylococcus phage SAP_2 as a safe and effective biocontrol agent against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) contamination in the food chain poses a serious threat to public health, and bacteriophages have emerged as promising biocontrol agents. In this study, we isolated and characterized a broad-spectrum lytic Staphylococcus phage, SAP_2, from a livestock farm. SAP_2 showed lytic activity against 26 of 43 Staphylococcus aureus strains and 23 of 33 coagulase-negative staphylococci isolates in spot assays. It exhibited rapid adsorption, a short latent period (20 min), and a large burst size (173 PFU/cell), while remaining stable across pH 4-9, temperatures of 30-50 °C, ultraviolet irradiation, and chloroform treatment. Whole-genome analysis identified 219 open reading frames (ORFs) including eight predicted tail-associated proteins potentially involved in host recognition, with no virulence, antibiotic resistance, or lysogeny-associated genes. Phylogenetic analysis classified SAP_2 within the genus Kayvirus. In vitro, SAP_2 efficiently lysed MRSA at MOIs ≥ 0.01, compromised bacterial membrane integrity, and inhibited MRSA biofilm formation. In vivo, SAP_2 exhibited good biosafety and reduced bacterial burdens in MRSA-infected mice, with a numerically higher 7-day survival rate in the treatment group than in the model control group (66.67% vs. 33.33%). Furthermore, SAP_2 effectively reduced MRSA contamination in milk, with substantially greater antibacterial activity at 25 °C than at 4 °C. These findings support the potential of SAP_2 as a biocontrol candidate for controlling MRSA contamination in milk.