Chimeric phage lysins act synergistically with lysostaphin to kill mastitis causing staphylococcus aureus in murine mammary glands

Authors: Schmelcher, Mathias; Powell, Anne; Becker, Stephen; CAMP, MARY - US Department Of Agriculture (USDA); Donovan, David

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/14/2012
Publication Date: 1/27/2012
Citation: Schmelcher, M., Powell, A.M., Becker, S.C., Camp, M.J., Donovan, D.M. 2012. Chimeric phage lysins act synergistically with lysostaphin to kill mastitis causing staphylococcus aureus in murine mammary glands. Applied and Environmental Microbiology. 78(7):2297-305.

Interpretive Summary: Problem: Bovine mastitis is the most costly disease in animal agriculture, causing losses of up to $2 billion per year. Staphylococcus aureus is among the leading causes of mastitis in cattle. Traditional treatment for mastitis, most commonly antibiotics, has been shown to be only marginally effective and contribute to development of antibiotic resistance, with multi-drug resistant S. aureus strains (MRSA) being of increasing concern. Therefore, alternative antimicrobials for the treatment of bovine mastitis are urgently needed. Accomplishments: We demonstrated activity of two bacteriophage endolysin-derived chimeric fusion proteins against multiple S. aureus mastitis isolates, including antibiotic resistant strains. Both proteins killed mastitis causing staphylococci in cow milk, which appears as an important requirement for an anti-mastitis drug to be infused into a cow’s mammary glands, and reduced bacterial numbers in a mouse model of bovine mastits. Due to the demonstrated synergistic action between one of the chimeras and the staphylococcal bacteriocin Lysostaphin, a combination treatment using both agents reduced bacterial numbers in said model more efficiently than any of the lysins applied individually. Furthermore, inflammation in the glands was reduced by most protein treatments, as apparent by lower gland wet weights and intramammary TNF-a concentrations. Contribution of Accomplishment to Solving the Problem: These findings provide new evidence that bacteriophage endolysins have a high potential as alternative antimicrobials for the control of pathogenic bacteria such as S. aureus. It is widely accepted that, as opposed to antibiotics, the chance of resistance development against phage endolysins is low. Despite efforts to find them, bacterial strains resistant to phage lysins have not been reported yet. Particularly combinations of Lysostaphin and the chimeric phage lysins investigated here hold promise as anti-staphylococcal drugs for the treatment of bovine mastitis.

Technical Abstract: 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 that are often ineffective and potentially contribute to resistance development. Bacteriophage endolysins (peptidoglycan hydrolases) present a promising source of alternative antimicrobials. Here we evaluate two fusion proteins consisting of the streptococcal 'SA2 endopeptidase domain fused to staphylococcal cell wall binding domains from either lysostaphin ('SA2-E-Lyso-SH3b) or the phage K endolysin, LysK ('SA2-E-LysK-SH3b). We demonstrate killing of 16 different S. aureus mastitis isolates including penicillin-resistant strains by both enzymes. At 100 µg/ml in processed cow milk, 'SA2-E-Lyso-SH3b reduced the S. aureus bacterial load by 3 log units within 3 hours, whereas 'SA2-E-LysK-SH3b was less effective and permitted re-growth of the pathogen after 1 hour. In a mouse model of mastitis, infusion of 25 µg of 'SA2-E-Lyso-SH3b or 'SA2-E-LysK-SH3b into mammary glands reduced S. aureus CFUs 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, 'SA2-E-LysK-SH3b and lysostaphin (12.5 µg each/gland) caused a 3.36 log decrease in CFUs. Furthermore, most protein treatments reduced gland wet weights and intramammary TNF-a concentrations serving as indicators of inflammation. Overall, our animal model results demonstrate the potential of optimized peptidoglycan hydrolases as potential antimicrobials for the treatment of mastitis.