胰蛋白酶和糜蛋白酶与抗生素联用的体外抗菌活性研究

[目的]本研究旨在提供蛋白酶治疗奶牛乳房炎的参考数据,了解胰蛋白酶和糜蛋白酶的体外微生物敏感性。[方法]通过测定细菌的A600绘制不同细菌的生长曲线,探讨了0.5C浓度胰糜蛋白复合酶(胰、糜蛋白酶各0.08 mg？mL-1)、1C浓度胰糜蛋白复合酶(胰、糜蛋白酶各0.16 mg？mL-1)、2C浓度胰糜蛋白复合酶(胰、糜蛋白酶各0.32 mg？mL-1)及其与头孢噻呋钠、磷酸替米考星和硫氰酸红霉素联用对5种常见奶牛乳房炎致病菌(金黄色葡萄球菌、大肠杆菌、巴氏杆菌、无乳链球菌、停乳链球菌)生长的影响。同时,采用电子显微镜扫描技术,观察胰糜蛋白复合酶与头孢噻呋钠协同对大肠杆菌超微结构的影响。[结果]经胰糜蛋白复合酶处理的细菌生长缓慢。1C浓度胰糜蛋白复合酶单独使用时对5种细菌的抑菌效果最佳;2C浓度胰糜蛋白复合酶与各种抗生素配合使用的协同抑菌效果最强。扫描电镜下,胰糜蛋白复合酶对细菌细胞壁结构有明显的损伤,表现为处理组细菌的细胞壁完整程度较差。0.08 mg？mL-1胰糜蛋白复合酶与0.312 5 μg？mL-1头孢噻呋钠联用可显著破坏细菌细胞壁结构。[结论]胰糜蛋白复合酶对5种常见奶牛乳房炎致病菌有不同程度的抑菌效果,其与头孢噻呋钠联用后可通过破坏细菌表面结构而发挥协同抑菌作用。
[Objectives]With the purpose of providing protease reference data of dairy cow mastitis treatment,microbial sensitivity test in vitro is an important step. [Methods]The antimicrobial activities of different concentrations of the trypsin and chymotrypsin complex,such as 0.5C(trypsinand chymotrypsin are both 0.08 mg？mL-1),1C(trypsin and chymotrypsin are both 0.16 mg？mL-1),2C(trypsin and chymotrypsin are both 0.32 mg？mL-1)and its combination antibacterial effects with antibiotics ceftiofur sodium phosphate,tilmicosin phosphate and erythromycin thiocyanate on the growth of 5 kinds of pathogenbacterials associated with bovine mastitis,namely,Staphylococcus aureus,Escherichia coli,Pasteurella,Streptococcus agalactiae,Streptococcus dysgalactiae were studied. Meanwhile,the ultrastructure of E.coli treated with the combination of the complex of trypsin and chymotrypsin,and ceftiofur sodium was observed by using of the scanning electron microscopy. [Results]The results showed that the bacterials treated by the complex of trypsin and chymotrypsin grew much more slowly during 14 h of experiment. The concentration of the complex of trypsin and chymotrypsin at the dosage of 1C displayed the best antimicrobial effect on five kinds of the tested bacterials. The concentration of the complex of trypsin and chymotrypsin at the dosage of 2C combined with different antibiotics exhibited the best antimicrobial effect on five tested bacterials. By scanning electron microscopy,the structure of bacterial cell walls was damaged by the complex of trypsin and chymotrypsin compared with that of the control group. The combination of the complex of trypsin and chymotrypsin of 0.08 mg？mL-1 with ceftiofur sodiumat the dosage of 0.312 5 μg？mL-1,can significantly destroy the structure of bacterial cell walls. [Conclutions]The results indicate that the complex of trypsin and chymotrypsin shows obvious antimicrobial effect on the five tested pathogenic bacteria associated with bovine mastitis. The combination of the complex of trypsin and