title: Chimeric phage lysins act synergistically with lysostaphin to kill mastitis-causing Staphylococcus aureus in murine mammary glands.
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, 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 λSA2 endolysin endopeptidase domain fused to staphylococcal cell wall binding domains from either lysostaphin (λSA2-E-Lyso-SH3b) or the staphylococcal phage K endolysin, LysK (λSA2-E-LysK-SH3b). We demonstrate killing of 16 different S. aureus mastitis isolates, including penicillin-resistant strains, by both constructs. At 100 μg/ml in processed cow milk, λSA2-E-Lyso-SH3b and λSA2-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 λSA2-E-Lyso-SH3b, however, λSA2-E-LysK-SH3b permitted regrowth of the pathogen after 1 h. In a mouse model of mastitis, infusion of 25 μg of λSA2-E-Lyso-SH3b or λSA2-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, λSA2-E-LysK-SH3b and lysostaphin (12.5 μg 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-α) 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.
authors.affiliation: Animal Biosciences and Biotechnology Laboratory, Animal & Natural Resources Institute, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland, USA.
authors.forename: Mathias
authors.initials: M
authors.lastname: Schmelcher
authors.forename: Anne M
authors.initials: AM
authors.lastname: Powell
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Becker
authors.forename: Mary J
authors.initials: MJ
authors.lastname: Camp
authors.forename: David M
authors.initials: DM
authors.lastname: Donovan
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology / Q000627:therapeutic use; D002417:Cattle; D002418:Cattle Diseases / Q000188:drug therapy / Q000382:microbiology; D004357:Drug Synergism; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology* / Q000627:therapeutic use; D005260:Female; D008248:Lysostaphin / Q000378:metabolism / Q000494:pharmacology* / Q000627:therapeutic use; D008321:Mammary Glands, Animal / Q000382:microbiology*; D008414:Mastitis, Bovine / Q000188:drug therapy* / Q000382:microbiology; D051379:Mice; D008810:Mice, Inbred C57BL; D008826:Microbial Sensitivity Tests; D023421:Models, Animal; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D013204:Staphylococcus Phages / Q000378:metabolism; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000302:isolation & purification; D016896:Treatment Outcome
chemical_list: D000900:Anti-Bacterial Agents; D011993:Recombinant Fusion Proteins; D010450:Endopeptidases; D008248:Lysostaphin; C001143:endolysin