奶牛乳腺炎源金黄色葡萄球菌噬菌体P82的分离及特性研究

doi:10.16431/j.cnki.1671-7236.2022.06.032

摘要/Abstract

摘要： 【目的】为开发噬菌体"鸡尾酒"制剂防治奶牛乳腺炎提供生物学材料。【方法】以实验室分离的奶牛乳腺炎源金黄色葡萄球菌82为宿主菌，通过点滴法和双层平板法在奶牛场的污水、弃奶和粪便混合物中分离并纯化其噬菌体，通过噬菌斑及透射电子显微镜对该株噬菌体的形态特征进行观察。利用核酸酶处理分析该噬菌体核酸类型，并对其裂解谱、最佳感染复数、一步生长曲线、热稳定性、pH稳定性进行研究。【结果】分离筛选出金黄色葡萄球菌82的裂解性噬菌体P82，电镜下观察其头部为二十面体，大小约为120 nm×83 nm，有一条带可伸缩尾鞘的长尾，长约126 nm，属于有尾噬菌体目，肌尾噬菌体科。通过琼脂糖凝胶电泳鉴定其核酸类型为双链DNA （dsDNA）。噬菌体P82的最佳感染复数为0.001，对奶牛乳腺炎源金黄色葡萄球菌裂解率为36.51%（23/63），宿主谱较广；其生长潜伏期约为25 min，爆发期约为45 min，裂解量约为74 PFU/cell；在pH 4.0~12.0时活性稳定，在pH 2.0和13.0时则完全失去活性。噬菌体P82热稳定性较高，在70 ℃作用60 min后仍有裂解能力，在80 ℃作用40 min时则失去裂解能力。【结论】本研究分离的噬菌体P82效价较高、裂解能力强、增殖速度快、噬菌谱广，对不同温度和pH均有较好的耐受度，能作为专一性裂解奶牛乳腺炎源金黄色葡萄球菌的噬菌体候选株，可与其他噬菌体混合制成噬菌体"鸡尾酒"制剂用于防治奶牛乳腺炎，为奶牛乳腺炎的噬菌体疗法提供材料。

关键词: 金黄色葡萄球菌; 奶牛乳腺炎; 噬菌体

Abstract: 【Objective】 The study was aimed to provide biological materials for developing phage "cocktail" preparations for the prevention and treatment of dairy mastitis.【Method】 Staphylococcus aureus 82 isolated from cow mastitis was used as the host bacteria, the phages were isolated and purified from the sewage, abandoned milk and feces of dairy farms by drip method and double-layer plate method, the morphological characteristics of the phage were observed by plaque and transmission electron microscope.The nucleic acid type of the phage was analyzed by nuclease treatment.The pyrolysis spectrum, MOI, one-step growth curve, thermal stability and pH stability were studied and analyzed.【Result】 The lytic phage P82 was isolated and screened.Under electron microscope, the head of the lytic phage P82 was icosahedral with a size of about 120 nm×83 nm, and the length of the lytic phage P82 was about 126 nm, it belonged to the order of Endurophage, and the family of Myoendurophage.The nucleic acid type was identified as dsDNA by agarose gel electrophoresis.The biological characteristics of the phage were determined:The best MOI of P82 was 0.001, and the cleavage rate of Staphylococcus aureus was 36.51%(23/63).The host spectrum was wide.The incubation period of growth was about 25 min, the outbreak period was about 45 min, and the cleavage volume was about 74 PFU/cell.P82 was stable at pH 4.0-12.0, and had high thermal stability.It completely lost activity at pH 2.0 and 13.0.It still had cracking ability at 70 ℃ for 60 min, but lost cracking ability at 80 ℃ for 40 min.【Conclusion】 The phage P82 isolated in this study had high titer, strong cleavage ability, fast proliferation speed, wide phage spectrum and good tolerance to different temperatures and pH.It could be used as a phage candidate strain for specific cleavage of Staphylococcus aureus from cow mastitis.It could be mixed with other phages to make a phage "cocktail" for the prevention and treatment of cow mastitis and provide materials for phage therapy of cow mastitis.

Key words: Staphylococcus aureus; cow mastitis; phage

中图分类号:

S852.65

党瑞莹, 常军帅, 梁晏, 屈勇刚, 李彦芳, 程璐璐, 刘若彤, 杨盛源, 王紫阳, 张小玉, 赵玉, 周海琴, 谢枋得. 奶牛乳腺炎源金黄色葡萄球菌噬菌体P82的分离及特性研究[J]. 中国畜牧兽医, 2022, 49(6): 2318-2325.

DANG Ruiying, CHANG Junshuai, LIANG Yan, QU Yonggang, LI Yanfang, CHENG Lulu, LIU Ruotong, YANG Shengyuan, WANG Ziyang, ZHANG Xiaoyu, ZHAO Yu, ZHOU Haiqin, XIE Fangde. Isolation and Characterization of Staphylococcus aureus Phage P82 from Dairy Mastitis[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(6): 2318-2325.

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