抗菌肽NZ2114对奶牛乳房炎源停乳链球菌的杀菌作用研究

doi:10.16431/j.cnki.1671-7236.2020.08.030

中国畜牧兽医 ›› 2020, Vol. 47 ›› Issue (8): 2603-2614.doi: 10.16431/j.cnki.1671-7236.2020.08.030

抗菌肽NZ2114对奶牛乳房炎源停乳链球菌的杀菌作用研究

张清娟^1,2,3,4, 马炫炫^3,4, 滕达^3,4, 杨娜^3,4, 王秀敏^3,4, 毛若雨^3,4, 郝娅^3,4, 单玉雪^1,2,3,4, 刘鹤^3,4, 范寰², 王建华^3,4

1. 天津师范大学生命科学学院, 天津 300387;
2. 天津市畜牧兽医研究所, 天津 300381;
3. 中国农业科学院饲料研究所基因工程研究室, 北京 100081;
4. 农业农村部饲料生物技术重点实验室, 北京 100081

收稿日期:2020-02-27 出版日期:2020-08-20 发布日期:2020-08-15
通讯作者: 范寰, 王建华 E-mail:fanhuan13212107231@126.com;wangjianhua@caas.cn
作者简介:张清娟(1996-),女,甘肃兰州人,硕士生,研究方向:抗菌肽及抗生素替代品,E-mail:qingjuanzz@163.com
基金资助:
中国农业科学院重大产出科研选题"饲用抗生素替代关键产品创制与产业"（CAAS-ZDXT2018008）；中国农业科学院科技创新工程"抗菌肽及抗生素替代品方向"（CAAS-ASTIP-2013-FRI-02）；天津市科技计划"复合抗菌肽提高奶牛乳房炎治疗效果的研究"（18YFZCNC01130）

Study on Antibacterial Effect of Antimicrobial Peptide NZ2114 Against Streptococcus dysgalactiae Isolated from Bovine Mastitis

ZHANG Qingjuan^1,2,3,4, MA Xuanxuan^3,4, TENG Da^3,4, YANG Na^3,4, WANG Xiumin^3,4, MAO Ruoyu^3,4, HAO Ya^3,4, SHAN Yuxue^1,2,3,4, LIU He^3,4, FAN Huan², WANG Jianhua^3,4

1. College of Life Sciences, Tianjin Normal University, Tianjin 300387, China;
2. Tianjin Animal Science and Veterinary Research Institute, Tianjin 300381, China;
3. Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China;
4. Key Laboratory of Feed Biotechnology, Ministry of Agriculture and Rural Affairs, Beijing 100081, China

Received:2020-02-27 Online:2020-08-20 Published:2020-08-15

摘要/Abstract

摘要： 为探究真菌防御素Plectasin衍生肽NZ2114对奶牛乳房炎源停乳链球菌的体外杀菌效果及其作用机制，本试验采用微量肉汤稀释法检测停乳链球菌CVCC 3938耐药性，通过NZ2114对停乳链球菌CVCC 3938最小抑菌浓度（minimum inhibitory concentration，MIC）及在胰蛋白胨大豆肉汤（tryptic soy broth，TSB）培养基和全脂灭菌（ultra-high temperature instantaneous sterilization，UHT）牛奶中的杀菌动力学曲线的测定评价其抗菌特性；借助扫描电镜、透射电镜和流式细胞仪观察NZ2114作用下的细菌形态变化；使用凝胶阻滞、荧光光谱和圆二色谱进一步分析NZ2114对细菌基因组DNA的作用。结果显示，停乳链球菌对氧氟沙星和四环素均产生耐药性，临床分离菌株则呈现多重耐药性，NZ2114对受试停乳链球菌和金黄色葡萄球菌的MIC值为0.11~0.45 μmol/L。抗菌肽在TSB培养基和全脂灭菌牛奶中均具有抑菌活性，且在短时间内（0.5~3 h）可使停乳链球菌菌落数下降3个数量级。扫描电镜、透射电镜和流式细胞仪结果表明，抗菌肽NZ2114能够破坏停乳链球菌细胞膜，导致其内容物泄漏。凝胶阻滞、荧光光谱和圆二色谱证实抗菌肽穿过细胞膜后与细菌基因组结合，破坏了DNA，以此达到杀菌目的。上述结果表明，抗菌肽NZ2114对奶牛乳房炎源停乳链球菌杀菌活性强，其破坏菌体细胞膜后可直接作用于胞内的基因组DNA并改变其二级结构。由此可见，抗菌肽NZ2114是一种极具前景的治疗停乳链球菌引起乳腺炎的抗生素替代品。

关键词: 奶牛乳房炎; 停乳链球菌; 抗菌肽NZ2114; 杀菌活性; 作用机制

Abstract: In order to investigate the bactericidal effect and mechanism of Plectasin-derived peptide NZ2114 against Streptococcus dysgalactiae (S.dysgalactiae) isolated from bovine mastitis,the drug resistance and minimum inhibitory concentration (MIC) were tested by broth dilution method,the MIC and the bactericidal kinetics curve in tryptic soy broth (TSB) and ultra-high temperature instantaneous sterilization (UHT) environment were determined for evaluating the antibacterial properties of NZ2114,the morphological changes of S.dysgalactiae treated with NZ2114 were observed by scanning electron microscope (SEM),transmission electron microscope (TEM) and flow cytometer.The effects of NZ2114 on the genomic DNA were further analyzed by gel retardation,fluorescence spectrum and circular dichroism spectrum.The results showed that all S.dysgalactiae were resistant to ofloxacin and tetracycline,but the clinical isolates showed more antibiotic resistance,the values of MIC were only 0.11 to 0.45 μmol/L.NZ2114 had bacteriostatic activity in TSB and UHT milk medium,and reduced the number of S.dysgalactiae colonies by 3 orders of magnitude in a short time (0.5 to 3 h).The results of SEM,TEM and flow cytometer showed that NZ2114 could cause the leakage of S.dysgalactiae content and kill the bacteria.Gel retardation analysis,fluorescence and circular dichroism all showed that NZ2114 could bind to the genomic DNA and destroy DNA.The above results showed that antibacterial peptide NZ2114 had strong bactericidal activity against S.dysgalactiae isolated from bovine mastitis,it could directly affect the genomic DNA and change its secondary structure after destroying the cell membrane.Therefore,antimicrobial peptide NZ2114 was a promising alternative antibiotic for the treatment of mastitis caused by S.dysgalactiae.

Key words: bovine mastitis; Streptococcus dysgalactiae; antimicrobial peptide NZ2114; antibacterial activity; mode of action

中图分类号:

S852.61⁺1

张清娟, 马炫炫, 滕达, 杨娜, 王秀敏, 毛若雨, 郝娅, 单玉雪, 刘鹤, 范寰, 王建华. 抗菌肽NZ2114对奶牛乳房炎源停乳链球菌的杀菌作用研究[J]. 中国畜牧兽医, 2020, 47(8): 2603-2614.

ZHANG Qingjuan, MA Xuanxuan, TENG Da, YANG Na, WANG Xiumin, MAO Ruoyu, HAO Ya, SHAN Yuxue, LIU He, FAN Huan, WANG Jianhua. Study on Antibacterial Effect of Antimicrobial Peptide NZ2114 Against Streptococcus dysgalactiae Isolated from Bovine Mastitis[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(8): 2603-2614.

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抗菌肽NZ2114对奶牛乳房炎源停乳链球菌的杀菌作用研究

Study on Antibacterial Effect of Antimicrobial Peptide NZ2114 Against Streptococcus dysgalactiae Isolated from Bovine Mastitis

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