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
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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0.5～1.0μg/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。
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.16 S 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.
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.16 S 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.
引文
[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 ?,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,HAJD,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,N?HR-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.
[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 ?,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,HAJD,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,N?HR-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.