抗菌肽NZ2114对来源于奶牛乳房炎的无乳链球菌及其生物膜的消杀作用

doi:10.16431/j.cnki.1671-7236.2020.07.035

中国畜牧兽医 ›› 2020, Vol. 47 ›› Issue (7): 2284-2294.doi: 10.16431/j.cnki.1671-7236.2020.07.035

抗菌肽NZ2114对来源于奶牛乳房炎的无乳链球菌及其生物膜的消杀作用

单玉雪^1,2,3,4, 杨娜^3,4, 滕达^3,4, 王秀敏^3,4, 毛若雨^3,4, 郝娅^3,4, 马炫炫^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:2020-01-07 出版日期:2020-07-20 发布日期:2020-07-18
通讯作者: 范寰(1971-),女,天津人,研究员,研究方向:饲料微生物,E-mail:fanhuan13212107231@126.com;王建华(1961-),男,湖北武汉人,研究员,研究方向:抗菌肽及抗生素替代品,E-mail:wangjianhua@caas.cn E-mail:fanhuan13212107231@126.com;wangjianhua@caas.cn
作者简介:单玉雪(1993-),女,河北承德人,硕士生,研究方向:抗菌肽及抗生素替代品,E-mail:syx1098840130@126.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）

The Disinfection Effect of Antimicrobial Peptide NZ2114 on Streptococcus agalactiae Isolated from Bovine Mastitis and Its Biofilm

SHAN Yuxue^1,2,3,4, YANG Na^3,4, TENG Da^3,4, WANG Xiumin^3,4, MAO Ruoyu^3,4, HAO Ya^3,4, MA Xuanxuan^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:2020-01-07 Online:2020-07-20 Published:2020-07-18

摘要/Abstract

摘要： 为探究抗菌肽NZ2114对无乳链球菌的体外抑制效果和相关机制，本试验以无乳链球菌ATCC 13813为研究对象，通过最小抑菌浓度（minimum inhibitory concentration，MIC）、杀菌动力学、抗生素后效应和电镜学试验对抗菌肽NZ2114杀菌特性和杀菌机制进行评价，并进一步分析其对无乳链球菌生物膜和持留菌的抑制和消除作用。结果显示，NZ2114对无乳链球菌ATCC 13813的MIC为0.23 μmol/L，对3株奶牛乳腺炎临床分离菌株CAU-FRI 1、2和3的MIC均为0.11 μmol/L，万古霉素对以上4株受试菌株的MIC值均为0.67 μmol/L，因此NZ2114的抗菌活性是万古霉素的2.91和6.09倍。同时，2×MIC、4×MIC浓度的NZ2114可在0.5 h内杀灭99.9%细菌，且持续药效作用可达270 min。扫描电镜结果显示，NZ2114处理后，细菌表面出现皱缩甚至破裂，细菌产生的生物膜消除。NZ2114在2×MIC下，24 h对早期生物膜抑制率为99.9%；在32×MIC下，24 h对成熟生物膜消除率达99.9%。此外，NZ2114在16×MIC时，对生物膜内的菌体具有99.9%以上的杀灭效果；万古霉素无法清除的持留菌经0.5×MIC的NZ2114处理后，也可以达到99%以上的消除率。激光共聚焦结果显示，NZ2114处理后的生物膜厚度从28.48 μm减少到10.32 μm，证明了其强效杀菌和抑制生物膜的作用。上述结果表明，NZ2114对无乳链球菌ATCC 13813杀菌活性高、速度快、抑制/去除生物膜能力强，并对膜内菌及持留菌具有高效杀菌作用，且作用显著高于万古霉素。因此，NZ2114具有开发成为治疗由无乳链球菌引起的奶牛乳房炎新型制剂的潜力。

关键词: 抗菌肽NZ2114; 奶牛乳房炎; 无乳链球菌; 杀菌活性; 生物膜

Abstract: In order to explore the antimicrobial activity and antimicrobial mechanism of antimicrobial peptide NZ2114 against Streptococcus agalactiae (S.agalactiae),S.agalactiae ATCC 13813 was used.The antimicrobial properties and mechanism of antimicrobial peptide NZ2114 were evaluated by means of minimum inhibitory concentration (MIC),time-killing curves,post antibiotic effect (PAE) and electron microscopy.The inhibition and elimination effects of S.agalactiae on biofilms and persister bacteria were further analyzed.The results showed that the MIC value of NZ2114 against S.agalactiae ATCC 13813 was 0.23 μmol/L,while the values of NZ2114 against CAU-FRI 1,2 and 3 strains,which were clinical isolated from mastitis cow,were all 0.11 μmol/L.The MIC values of vancomycin against the above-mentioned four strains were all 0.67 μmol/L,so the antimicrobial activity of NZ2114 were 2.91 and 6.09 times than that of vancomycin,respectively.Meanwhile,2×MIC and 4×MIC of NZ2114 could kill 99.9% S.agalactiae in 0.5 h and the pharmacodynamic time was up to 270 min.The result of scanning electron microscope (SEM) showed that the surface of the bacteria appeared to shrink or even break after the treatment of NZ2114,and the biofilms were eliminated.NZ2114 effectively inhibited early biofilms by 99.9% in 2×MIC,and eradicated mature biofilms by 99.9% in 32×MIC.At the same time,the bacteria (approximately 99.9%) in biofilm were efficiently killed by NZ2114 in 16×MIC,and that of the persisters which were resistant to vancomycin were eliminated by 99% in 0.5×MIC of NZ2114.Confocal laser scanning microscopy (CLSM) also demonstrated the potent antimicrobial and anti-biofilm activity of NZ2114 (biofilm from 28.48 μm reduced to 10.32 μm).The above results showed that NZ2114 had high bactericidal activity against S.agalactiae ATCC 13813 with fast speed,strong ability to inhibit/eradicate the biofilm,and high bactericidal activity against persisters in early and mature biofilms,and its effects were all significantly higher than those of vancomycin.NZ2114 had the potential to develop as a novel antibacterial agent for mastitis treatment caused by S.agalactiae.

Key words: antimicrobial peptide NZ2114; cow mastitis; Streptococcus agalactiae; antimicrobial activity; biofilm

中图分类号:

S859.79⁺7

单玉雪, 杨娜, 滕达, 王秀敏, 毛若雨, 郝娅, 马炫炫, 范寰, 王建华. 抗菌肽NZ2114对来源于奶牛乳房炎的无乳链球菌及其生物膜的消杀作用[J]. 中国畜牧兽医, 2020, 47(7): 2284-2294.

SHAN Yuxue, YANG Na, TENG Da, WANG Xiumin, MAO Ruoyu, HAO Ya, MA Xuanxuan, FAN Huan, WANG Jianhua. The Disinfection Effect of Antimicrobial Peptide NZ2114 on Streptococcus agalactiae Isolated from Bovine Mastitis and Its Biofilm[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(7): 2284-2294.

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抗菌肽NZ2114对来源于奶牛乳房炎的无乳链球菌及其生物膜的消杀作用

The Disinfection Effect of Antimicrobial Peptide NZ2114 on Streptococcus agalactiae Isolated from Bovine Mastitis and Its Biofilm

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