Isolation, genomic characterization, and biocontrol potential of a bacteriophage cocktail against Staphylococcus aureus in milk.

The objectives of this study were to isolate, characterize, and evaluate the biocontrol potential of Staphylococcus aureus-targeting bacteriophages in milk. From wastewater and raw milk samples, a total of 19 lytic phages were isolated. Among them SAKAY1298 and SAKAY1306 phages were selected for detailed phenotypic and genomic analysis based on their lytic profiles, host range and growth kinetics. Both phages exhibited icosahedral capsids with short, non-contractile tails under transmission electron microscopy. The phages exhibited moderate acid tolerance by remaining stable at pH 4 and maintained thermal stability at temperatures up to 50 °C. They also maintained high titers over one year at + 4 °C and -80 °C in tryptic soy broth and Tris-buffered saline (1 ×). Genome analyses revealed that both phages possess complete genomes (~ 17.5 kb), encoding 18 phage-related genes and lacking virulence and resistance determinants. Comparative and phylogenetic analyses confirmed assignment to the genus Rosenblumvirus, with 99% query coverage and ~ 92% whole-genome nucleotide identity to Staphylococcus phage vB_SauR_SW21 (GenBank accession OR683639.1). Significant reductions in S. aureus counts were achieved following phage cocktail treatment, reaching up to 4.31 log CFU/mL in vitro at 37 °C and 1.44 log CFU/mL in UHT milk at 4 °C (p < 0.001). Additionally, the phage cocktail fully inhibited the production of staphylococcal enterotoxin A in the in vitro assay. Altogether, the data supports the application of SA1298KAY and SA1306KAY phages have strong potential as effective biocontrol agents for reducing S. aureus in dairy applications.