抗菌肽NZ2114对奶牛乳房炎源金黄色葡萄球菌细胞膜、基因组和耐药性的影响

doi:10.16431/j.cnki.1671-7236.2019.04.027

《中国畜牧兽医》 ›› 2019, Vol. 46 ›› Issue (4): 1181-1190.doi: 10.16431/j.cnki.1671-7236.2019.04.027

抗菌肽NZ2114对奶牛乳房炎源金黄色葡萄球菌细胞膜、基因组和耐药性的影响

郭英^1,2,3,4, 滕达^3,4, 王秀敏^3,4, 毛若雨^3,4, 郝娅^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. 农业农村部饲料生物技术重点实验室, 北京 100081;
4. 中国农业科学院饲料研究所, 基因工程研究室, 北京 100081

修回日期:2019-01-31 出版日期:2019-04-20 发布日期:2019-04-22
通讯作者: 王建华, 范寰 E-mail:wangjianhua@caas.cn;fanhuan13212107231@126.com
作者简介:郭英(1993-),女,山西大同人,硕士生,研究方向:抗菌肽及抗生素替代品,E-mail:2607965895@qq.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化（CAAS-ZDXT2018008）；国家农业科技创新工程——抗菌肽及抗生素替代品方向（CAAS-ASTIP-2013-FRI-02）；天津市科技计划——应用复合抗菌肽提高奶牛乳房炎治疗效果的研究（18YFZCNC01130）

Effect of Antimicrobial Peptide NZ2114 on Cell Membrane,Genome and Antimicrobial Resistance of Staphylococcus aureus Isolated from Bovine Mastitis Milk

GUO Ying^1,2,3,4, TENG Da^3,4, WANG Xiumin^3,4, MAO Ruoyu^3,4, HAO Ya^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. Key Laboratory of Feed Biotechnology, Ministry of Agriculture and Rural Affairs, Beijing 100081, China;
4. Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China

Revised:2019-01-31 Online:2019-04-20 Published:2019-04-22

摘要/Abstract

摘要：

本试验以真菌防御素Plectasin衍生肽NZ2114和奶牛乳房炎源金黄色葡萄球菌为研究对象，旨在阐明抗菌肽NZ2114对金黄色葡萄球菌的体外抗菌机制及其阻遏金黄色葡萄球菌耐药性的效果。通过16S rRNA基因鉴定对55份乳房炎奶样中的病原菌进行分离鉴定，采用纸片扩散法检测其耐药性，通过微量稀释法测定抗菌肽NZ2114对金黄色葡萄球菌的抑菌活性，借助流式细胞术和扫描电镜观察抗菌肽NZ2114对金黄色葡萄球菌细胞膜完整性及细胞形态的影响，通过凝胶阻滞和圆二色谱分析抗菌肽NZ2114对金黄色葡萄球菌基因组DNA的影响，将抗菌肽NZ2114与金黄色葡萄球菌共同孵育以研究抗菌肽NZ2114对金黄色葡萄球菌耐药性的影响，通过PCR进一步分析抗菌肽NZ2114对金黄色葡萄球菌携带的耐药基因的影响。结果表明，共获得10株金黄色葡萄球菌，分离菌对青霉素等9种抗菌药具有耐药性，且50%为多重耐药。抗菌肽NZ2114对10株金黄色葡萄球菌具有强抑菌活性，最小抑菌浓度（MIC）为0.5~1.0 μg/mL。抗菌肽NZ2114可导致金黄色葡萄球菌S7细胞皱缩、细胞内容物泄漏，甚至细胞裂解，碘化丙啶（PI）细胞膜穿透率达5%，且抗菌肽NZ2114可与金黄色葡萄球菌S7基因组DNA结合并改变其DNA结构。金黄色葡萄球菌与1/4×MIC抗菌肽NZ2114孵育12 h后发现，除对阿莫西林和磺胺异噁唑的耐药率没有降低外，对其他抗菌药的耐药率均有不同程度的降低（10%~40%），且抗菌肽NZ2114对β-内酰胺类耐药基因（blaZ）及杆菌肽类耐药基因（braRS）的消除率分别达28.57%（2/7）和22.22%（2/9）。上述结果证明，抗菌肽NZ2114对耐药金黄色葡萄球菌具有体外强抑菌活性，其干扰耐药金黄色葡萄球菌细胞膜并结合胞内DNA的作用机制为其低耐药杀菌机制奠定了细胞学基础。同时抗菌肽NZ2114对耐药菌株的耐药性及相关耐药基因均有一定消除作用。由此可见，抗菌肽NZ2114是一种极具前景的治疗金黄色葡萄球菌引起的奶牛乳房炎的抗生素替代品。

关键词: 奶牛乳房炎; 金黄色葡萄球菌; 抗菌肽NZ2114; 抗菌机制; 耐药性消除

Abstract:

The aim of this study was to elucidate antibacterial mechanism of the fungal defense Plectasin derived peptide NZ2114 against Staphylococcus aureus (S.aureus) that caused cow mastitis and its effect on inhibiting drug resistance of S.aureus in vitro.16S rRNA gene identification and disc diffusion method were used to identify the pathogenic bacteria of 55 mastitis milk samples and test their drug resistance,microdilution method was used for determining the antimicrobial activity of NZ2114 to S.aureus.The effect of NZ2114 on S.aureus cell membrane integrity and cell morphology were observed by flow cytometry and scanning electron microscope.The methods of gel retardation and circular dichroism spectrum were used to analyze the effect of NZ2114 on S.aureus genome DNA.The effect of NZ2114 on the drug resistance S.aureus was studied by incubating NZ2114 with S.aureus.The effect of NZ2114 on the drug resistance genes of S.aureus was further identified by PCR.The results showed that 10 strains of S.aureus were obtained,which were resistant to 9 antibiotics such as penicillin,and 50% of them were multidrug resistant.NZ2114 showed strong inhibitory activity against 10 strains of S.aureus,and the minimum inhibitory concentrations (MIC) were 0.5 to 1.0 μg/mL.NZ2114 could cause micromorphological change of S.aureus S7,including cell surface shrinkage,cell contents leakage,and cell lysis and propidine iodide (PI) cell membrane penetration rate was up to 5%.Additionally,NZ2114 could bind to S.aureus S7 genomic DNA and change its structure.S.aureus and 1/4×MIC NZ2114 were incubated together for 12 h,the result demonstrated that except amoxicillin and sulfanilamide,the resistance to other antibiotics of S.aureus had decreased to different extent (10% to 40%) and the elimination rate of NZ2114 to blaZ and braRS genes were 28.57% (2/7) and 22.22% (2/9),respectively.The above results proved that NZ2114 had strong inhibited activity against multidrug resistant S.aureus,and its mechanism of interfering with the membrane and intracellular DNA,which laid the cytological foundation for its low drug resistant bactericidal mechanism.At the same time,NZ2114 had elimination effect on the drug resistance and related drug resistant genes of S.aureus.Thus,NZ2114 was a promising antibiotic alternative for treatment of cow mastitis caused by S.aureus.

Key words: cow mastitis; Staphylococcus aureus; antimicrobial peptide NZ2114; antibacterial mechanism; elimination of drug resistance

中图分类号:

S859.79⁺7

郭英, 滕达, 王秀敏, 毛若雨, 郝娅, 范寰, 王建华. 抗菌肽NZ2114对奶牛乳房炎源金黄色葡萄球菌细胞膜、基因组和耐药性的影响[J]. 《中国畜牧兽医》, 2019, 46(4): 1181-1190.

GUO Ying, TENG Da, WANG Xiumin, MAO Ruoyu, HAO Ya, FAN Huan, WANG Jianhua. Effect of Antimicrobial Peptide NZ2114 on Cell Membrane,Genome and Antimicrobial Resistance of Staphylococcus aureus Isolated from Bovine Mastitis Milk[J]. , 2019, 46(4): 1181-1190.

参考文献

[1] 王丹,杨峰,李新圃,等.奶牛乳房炎病原菌诊断技术研究进展[J].中国畜牧兽医,2017,44(6):1811-1817. WANG D,YANG F,LI X P,et al.Research progress on diagnosis technology of dairy cow mastitis pathogens[J].China Animal Husbandry & Veterinary Medicine,2017,44(6):1811-1817.(in Chinese)
[2] KUMAR R,YADAV B R,SINGH R S.Genetic determinants of antibiotic resistance in Staphylococcus aureus isolates from milk of mastitic crossbred cattle[J].Current Microbiology,2010,60(5):379-386.
[3] GOMES F,HENRIQUES M.Control of bovine mastitis:Old and recent therapeutic approaches[J].Current Microbiology,2016,72(4):377-382.
[4] 张瑞强,史良玉,王迪,等.京津冀地区中小规模奶牛场隐性乳房炎状况调查[J].中国奶牛,2018,4:65-69. ZHANG R Q,SHI L Y,WANG D,et al.Investigation of current situation and causes of subclinical mastitis in small and medium sized dairy farms in Beijing-Tianjin-Hebei region[J].China Dairy Cattle,2018,4:65-69.(in Chinese)
[5] 芦燕,于景丽,赵吉.中国奶牛乳房炎病原菌群的地区分布特征[J].微生物前沿,2018,7(2):48-57. LU Y,YU J L,ZHAO J.Distribution characteristics of pathogenic communities associated with bovine mastitis across different regions of China[J].Advances in Microbiology,2018,7(2):48-57.(in Chinese)
[6] OLIVER S P,MURINDA S E.Antimicrobial resistance of mastitis pathogens[J].Veterinary Clinics of North America Food Animal Practice,2012,28(2):165-185.
[7] 祝宇,路璐,颜兴琼,等.云南地区牛源无乳链球菌分离鉴定及毒力基因和耐药性的检测[J].中国畜牧兽医,2018,45(11):3261-3270. ZHU Y,LU L,YAN X Q,et al.Isolation,identification and detection of virulence genes and drug resistance of Streptococcus agalactiae from cattle in Yunnan[J].China Animal Husbandry & Veterinary Medicine,2018,45(11):3261-3270.(in Chinese)
[8] MARTINI C L,LANGE C C,BRITO M A,et al.Characterisation of penicillin and tetracycline resistance in Staphylococcus aureus isolated from bovine milk samples in Minas Gerais,Brazil[J].Journal of Dairy Research,2017,84(2):202-205.
[9] ASLANTAS Ö,DEMIR C.Investigation of the antibiotic resistance and biofilm-forming ability of Staphylococcus aureus from subclinical bovine mastitis cases[J].Journal of Dairy Science,2016,99(11):8607-8613.
[10] 冯清,林伟东,张大帅,等.奶牛源MRSA菌株的分离鉴定和耐药性分析[J].中国畜牧兽医,2016,43(7):1862-1868. FENG Q,LIN W D,ZHANG D S,et al.Isolation,indentification and antibiotic resistance anaiysis of methicillin-resistant Staphylococcus aureus (MRSA) from row caw milk[J].China Animal Husbandry & Veterinary Medicine,2016,43(7):1862-1868.(in Chinese)
[11] WANG X M,TENG D,MAO R Y,et al.Combined systems approaches reveal a multistage mode of action of a marine antimicrobial peptide against pathogenic Escherichia coli and its protective effect against bacterial peritonitis and endotoxemia[J].Antimicrobial Agents and Chemotherapy,2017,61(1):e01056-16.
[12] ZHANG Y,TENG D,MAO R Y,et al.High expression of a plectasin-derived peptide NZ2114 in Pichia pastoris,and its pharmacodynamics,postantibiotic and synergy against Staphylococcus aureus[J].Applied Microbiology and Biotechnology,2014,98(2):681-694.
[13] BRINCH K S,TULKENS P M,VAN BAMBEKE F,et al.Intracellular activity of the peptide antibiotic NZ2114:Studies with Staphylococcus aureus and human THP-1 monocytes,and comparison with daptomycin and vancomycin[J].Journal of Antimicrobial Chemotherapy,2010,65(8):1720-1724.
[14] JIAO J,MAO R Y,TENG D,et al.In vitro and in vivo antibacterial effect of NZ2114 against Streptococcus suis type 2 infection in mice peritonitis models[J].Applied Microbiology and Biotechnology Express,2017,7(1):44.
[15] WANG X,WANG X M,TENG D et al.Increased intracellular activity of MP1102 and NZ2114 against Staphylococcus aureus in vitro and in vivo[J].Scientific Reports,2018,8(1):4204.
[16] 杨启文,HINDLER J A,徐英春.抗菌药物敏感性试验折点的解读与应用[J].中华检验医学杂志,2013,36(4):289-291. YANG Q W,HINDLER J A,XU Y C.Interpretation and application of antimicrobial susceptibility testing[J].Chinese Journal of Laboratory Medicine,2013,36(4):289-291.(in Chinese)
[17] JOSHI S,BISHT G S,RAWAT D S,et al.Interaction studies of novel cell selective antimicrobial peptides with model membranes and E.coli ATCC 11775[J].Biochimica et Biophysica Ata-Biomembranes,2010,1798(10):1864-1875.
[18] SPENGLER G,MICZÁK A,HAJDÜ E,et al.Enhancement of plasmid curing by 9-aminoacridine and two phenothiazines in the presence of proton pump inhibitor 1-(2-benzoxazolyl)-3,3,3-trifluoro-2-propanone[J].International Journal of Antimicrobial Agents,2003,22(3):223-227.
[19] ZHAO Q Y,YUAN F W,LIANG T,et al.Baicalin inhibits Escherichia coli isolates in bovine mastitic milk and reduces antimicrobial resistance[J].Journal of Dairy Science,2018,101(3):2415-2422.
[20] LIU H M,LI S L,MENG L,et al.Prevalence,antimicrobial susceptibility,and molecular characterization of Staphylococcus aureus isolated from dairy herds in Northern China[J].Journal of Dairy Science,2017,100(11):8796-8803.
[21] HIRON A,FALORD M,VALLE J,et al.Bacitracin and nisin resistance in Staphylococcus aureus:A novel pathway involving the BraS/BraR two-component system (SA2417/SA2418) and both the BraD/BraE and VraD/VraE ABC transporters[J].Molecular Microbiology,2011,81(3):602-622.
[22] VESTERGAARD M,NØHR-MELDGAARD K,BOJER M S,et al.Inhibition of the ATP synthase eliminates the intrinsic resistance of Staphylococcus aureus towards polymyxins[J].MBio,2017,8(5):e01114-17.
[23] WANG D,WANG Z,YAN Z,et al.Bovine mastitis Staphylococcus aureus:Antibiotic susceptibility profile,resistance genes and molecular typing of methicillin-resistant and methicillin-sensitive strains in China[J].Infection Genetics and Evolution,2015,31:9-16.
[24] FRAUNHOLZ M,SINHA B.Intracellular Staphylococcus aureus:Live in and let die[J].Frontiers in Cellular & Infection Microbiology,2012,2(4):43.
[25] PANTOSTI A,SANCHINI A,MONACO M.Mechanisms of antibiotic resistance in Staphylococcus aureus[J].Antibiotica,2007,2(3):323-334.
[26] YANG F,WANG Q,WANG X R,et al.Genetic characterization of antimicrobial resistance in Staphylococcus aureus isolated from bovine mastitis cases in Northwest China[J].Journal of Integrative Agriculture,2016,15(12):2842-2847.
[27] KUMAR R,YADAV B R,ANAND S K,et al.Molecular surveillance of putative virulence factors and antibiotic resistance in Staphylococcus aureus isolates recovered from intra-mammary infections of river buffaloes[J].Microbial Pathogenesis,2011,51(1):31-38.
[28] 王潇.NZ2114及其衍生肽对胞内金黄色葡萄球菌杀菌功能分析与机理研究[D].北京:中国农业科学院,2018. WANG X.In vitro and in vivo study on activity and mechanism of plectasin derivates against intracellular Staphylococcus aureus[D].Beijing:Chinese Academy of Agricultural Sciences,2018.(in Chinese)
[29] 刘爱平,王琦琛.细胞生物学荧光技术原理和应用[M].合肥:中国科学技术大学出版社,2007. LIU A P,WANG Q C.Principle and Application of Fluorescence Technique in Cell Biology[M].Hefei:University of Science and Technology of China Press,2007.(in Chinese)
[30] XI D,WANG X M,TENG D,et al.Mechanism of action of the tri-hybrid antimicrobial peptide LHP7 from lactoferricin,HP and plectasin on Staphylococcus aureus[J].BioMetals,2014,27(5):957-968.
[31] BELIK A S,TARASOVA N N,KHMEL' I A.Regulation of biofilm formation in Escherichia coli K12:Effect of mutations in HNS,StpA,lon,and rpoN genes[J].Molekuliarnaia Genetika Mikrobiologiia I Virusologiia,2008,23(4):159-162.

抗菌肽NZ2114对奶牛乳房炎源金黄色葡萄球菌细胞膜、基因组和耐药性的影响

Effect of Antimicrobial Peptide NZ2114 on Cell Membrane,Genome and Antimicrobial Resistance of Staphylococcus aureus Isolated from Bovine Mastitis Milk

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	樊艺萌, 魏媛媛, 王惠茹, 尕玉, 张艳楠, 赵卿宇, 郝智慧. 基于网络药理学与分子对接探究中兽药复方乳康颗粒治疗奶牛乳房炎的作用机制[J]. 中国畜牧兽医, 2023, 50(6): 2507-2517.
[2]	马晓姣, 赵艳坤, 邵伟, 武亚婷, 李明, 刘慧敏, 孟璐, 陈贺. 新疆部分地区生乳中金黄色葡萄球菌的分离鉴定及耐药性分析[J]. 中国畜牧兽医, 2023, 50(2): 789-797.
[3]	钟华晨, 王丽芳, 郭晨阳, 刘嘉琳, 宋洁. 内蒙古地区乳房炎奶样与环境中细菌的分离鉴定及耐药性分析[J]. 中国畜牧兽医, 2023, 50(2): 817-826.
[4]	蔡萌, 朱晓艳, 王梦玲, 刘子豪, 熊本海, 杨亮. 金黄色葡萄球菌处理对奶牛乳腺上皮细胞外泌体表征的影响[J]. 中国畜牧兽医, 2022, 49(8): 3091-3098.
[5]	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.
[6]	刘金环, 张珊玲, 宋玮, 罗万和, 陈伟. 槲皮素-替米考星混合物纳米粒的制备及对金黄色葡萄球菌小菌落变异株抗菌活性研究[J]. 中国畜牧兽医, 2022, 49(4): 1470-1478.
[7]	张静, 钱英红, 张凯, 吴金迪, 刘博, 毛伟, 曹金山. 金黄色葡菌球菌脂蛋白对M1型小鼠骨髓源巨噬细胞免疫作用的影响[J]. 中国畜牧兽医, 2021, 48(8): 3079-3086.
[8]	苏立燕, 王莉, 吴佳岂, 关舒函, 王大成, 王琳. 鸢尾黄素作为MgrA抑制剂对小鼠金黄色葡萄球菌肺炎的治疗作用[J]. 中国畜牧兽医, 2021, 48(7): 2617-2626.
[9]	杨效林, 韩润林, 毛伟, 包海霞, 刘昆, 吴金迪, 曹金山, 刘博. PGD₂/DP₁途径对细菌感染奶牛子宫内膜组织中HMGB-1和PAFR表达的影响[J]. 中国畜牧兽医, 2021, 48(6): 2169-2176.
[10]	张鹏举, 付丽云, 陈志强, 尚静雪, 张远达, 蔡小利, 战文. 公英翘芦散治疗亚临床型奶牛乳房炎的效果研究[J]. 中国畜牧兽医, 2021, 48(3): 1027-1033.
[11]	倪丽慧, 栾文静, 王雪飞, 李媛, 安雅男, 马方雪, 周宏, 王超, 梁俊超, 于录. 胰岛素联合利奈唑胺抑制糖尿病细菌性肺炎的机制研究[J]. 中国畜牧兽医, 2021, 48(1): 356-366.
[12]	姜晓文, 燕鸽, 李叔洪, 于文会, 魏佳宇, 艾莉雯, 白文. 板蓝根微粉水提物抗大肠杆菌活性及其机制的探究[J]. 中国畜牧兽医, 2020, 47(9): 2968-2978.
[13]	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.
[14]	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.
[15]	范丽霞, 苑学霞, 李鸳鸯, 邬元娟, 赵善仓, 张丙春, 王文博. 畜禽粪污中金黄色葡萄球菌和大肠埃希氏菌的LAMP检测及其耐药性分析[J]. 中国畜牧兽医, 2020, 47(7): 2325-2335.