Chimeric Phage Lysins Act Synergistically with Lysostaphin To Kill Mastitis-Causing Staphylococcus aureus in Murine Mammary Glands

Staphylococci cause bovine mastitis, with Staphylococcus aureus being responsible for the majority of the mastitis-based losses to the dairy industry (up to $2 billion/annum). Treatment is primarily with antibiotics, which are often ineffective and potentially contribute to resistance development. Bacteriophage endolysins (peptidoglycan hydrolases) present a promising source of alternative antimicrobials. Here we evaluated two fusion proteins consisting of the streptococcal lambda SA2 endolysin endopeptidase domain fused to staphylococcal cell wall binding domains from either lysostaphin (lambda SA2-E-Lyso-SH3b) or the staphylococcal phage K endolysin, LysK (lambda SA2-E-LysK-SH3b). We demonstrate killing of 16 different S. aureus mastitis isolates, including penicillin- resistant strains, by both constructs. At 100 mu g/ml in processed cow milk, lambda SA2-E-Lyso-SH3b and lambda SA2-E-LysK-SH3b reduced the S. aureus bacterial load by 3 and 1 log units within 3 h, respectively, compared to a buffer control. In contrast to lambda SA2-E-Lyso-SH3b, however, lambda SA2-E-LysK-SH3b permitted regrowth of the pathogen after 1 h. In a mouse model of mastitis, infusion of 25 mu g of lambda SA2-E-Lyso-SH3b or lambda SA2-E-LysK-SH3b into mammary glands reduced S. aureus CFU by 0.63 or 0.81 log units, compared to >2 log for lysostaphin. Both chimeras were synergistic with lysostaphin against S. aureus in plate lysis checkerboard assays. When tested in combination in mice, lambda SA2-E-LysK-SH3b and lysostaphin (12.5 mu g each/gland) caused a 3.36-log decrease in CFU. Furthermore, most protein treatments reduced gland wet weights and intramammary tumor necrosis factor alpha (TNF-alpha) concentrations, which serve as indicators of inflammation. Overall, our animal model results demonstrate the potential of fusion peptidoglycan hydrolases as antimicrobials for the treatment of S. aureus-induced mastitis.