Supplementary material.docx

Bovine mastitis (<b>BM</b>) is a prevalent infectious disease in dairy herds worldwide, resulting in substantial economic losses.<i> Staphylococcus aureus</i> is a major cause of mastitis in animals, and its antibiotic resistance poses challenges for treatment. Recently, there has been a renewed interest in the development of alternative methods to antibiotic therapy, including bacteriophages (phages), for controlling bacterial infections. In this study, two lytic phages (designated as<b> JDYN </b>for vB_SauM_JDYN and <b>JDF86</b> for vB_SauM_JDF86) were isolated from the cattle sewage effluent samples collected from dairy farms in Shanghai. The two phages have a broad bactericidal spectrum against <i>Staphylococcus</i> of various origins. Genomic and morphological analyses revealed that the two phages belonged to the <i>Myoviridae</i> family. Moreover, JDYN and JDF86 remained stable under a wide range of temperatures or pH and were almost unaffected in chloroform. In this study, we prepared a phage cocktail designated “PHC-1” which consisted of a 1:1:1 ratio of JDYN, JDF86 and SLPW (a previously characterized phage). PHC-1 showed the strongest bacteriolytic effect and the lowest frequency of emergence of bacteriophage insensitive mutants compared to monophages. The bovine mammary epithelial cells (<b>MAC-T</b> cells) and lactating mice mastitis model were used to evaluate the effectiveness of PHC-1 <i>in vitro</i> and <i>in vivo</i>, 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 <i>S. aureus</i>-induced bovine mastitis and that phage cocktails can combat antibiotic-resistant <i>S. aureus</i> infections.