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The list of standard abbreviations for JDS is available at adsa.org/jds-abbreviations-24. Nonstandard abbreviations are available in the Notes. Bovine mastitis is a prevalent infectious disease in dairy herds worldwide, resulting in substantial economic losses. Staphylococcus aureus is a major cause of mastitis in animals, and its antibiotic resistance poses challenges for treatment. Recently, renewed interest has focused on the development of alternative methods to antibiotic therapy, including bacteriophages (phages), for controlling bacterial infections. In this study, 2 lytic phages, vB_SauM_JDYN (JDYN) and vB_SauM_JDF86 (JDF86), were isolated from the cattle sewage effluent samples collected from dairy farms in Shanghai. The 2 phages have a broad bactericidal spectrum against Staphylococcus of various origins. Genomic and morphological analyses revealed that the 2 phages belonged to the Myoviridae family. Moreover, JDYN and JDF86 remained stable under a wide temperature and pH range and were almost unaffected in chloroform. In this study, we prepared a phage cocktail (PHC-1) which consisted of a 1:1:1 ratio of JDYN, JDF86, and SLPW (a previously characterized phage). We found that PHC-1 showed the strongest bacteriolytic effect and the lowest frequency of emergence of bacteriophage insensitive mutants compared with monophages. Bovine mammary epithelial cells and lactating mice mastitis models were used to evaluate the effectiveness of PHC-1 in vitro and in vivo, respectively. The results demonstrated that PHC-1 treatment significantly reduced bacterial load, alleviated inflammatory response, and improved mastitis pathology. Altogether, these results suggest that PHC-1 has the potential to treat S. aureus -induced bovine mastitis and that phage cocktails can combat antibiotic-resistant S. aureus infections. [ABSTRACT FROM AUTHOR]