Animal Biosciences and Biotechnology Laboratory, Animal & Natural Resources Institute, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland, USA.
Appl Environ Microbiol. 2012 Apr;78(7):2297-305. doi: 10.1128/AEM.07050-11. Epub 2012 Jan 27.
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.
葡萄球菌引起牛乳腺炎，其中金黄色葡萄球菌是导致乳业乳腺炎相关损失的主要原因（高达每年 20 亿美元）。治疗主要采用抗生素，但抗生素往往效果不佳，并且可能导致耐药性的产生。噬菌体溶菌酶（肽聚糖水解酶）是一种很有前途的替代抗菌药物来源。在这里，我们评估了两种融合蛋白，它们由链球菌 λSA2 内溶素内切酶结构域与来自溶葡萄球菌素（λSA2-E-Lyso-SH3b）或葡萄球菌噬菌体 K 内溶素 LysK 的葡萄球菌细胞壁结合结构域融合而成（λSA2-E-LysK-SH3b）。我们证明了这两种构建体可以杀死 16 种不同的金黄色葡萄球菌乳腺炎分离株，包括耐青霉素菌株。在经过处理的牛奶中，浓度为 100μg/ml 时，与缓冲液对照组相比，λSA2-E-Lyso-SH3b 和 λSA2-E-LysK-SH3b 在 3 小时内分别使金黄色葡萄球菌的细菌负荷减少了 3 和 1 个对数单位。然而，与 λSA2-E-Lyso-SH3b 不同的是，λSA2-E-LysK-SH3b 在 1 小时后允许病原体重新生长。在乳腺炎的小鼠模型中，将 25μg 的 λSA2-E-Lyso-SH3b 或 λSA2-E-LysK-SH3b 注入乳腺中，与溶葡萄球菌素相比，金黄色葡萄球菌 CFU 减少了 0.63 或 0.81 个对数单位。在平板溶菌棋盘测定中，两种嵌合体与溶葡萄球菌素对金黄色葡萄球菌均具有协同作用。在小鼠中联合测试时，λSA2-E-LysK-SH3b 和溶葡萄球菌素（每个腺体 12.5μg）导致 CFU 减少了 3.36 个对数单位。此外，大多数蛋白质处理都降低了乳腺湿重和肿瘤坏死因子-α（TNF-α）浓度，这是炎症的指标。总的来说，我们的动物模型结果表明，融合肽聚糖水解酶作为治疗金黄色葡萄球菌引起的乳腺炎的抗菌药物具有潜力。
