原始热带雨林鹦歌岭土壤抗MRSA放线菌的分离与筛选
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摘要
鹦歌岭是位于海南省乐东县的原始热带雨林,从鹦歌岭土壤中分离与筛选抗耐甲氧西林金黄色葡萄球菌(MRSA)的放线菌。采用平板稀释法分离,以MRSA作为受试菌株,测试抗菌活性,对具有抗菌活性的菌株进行16S rDNA序列测定。采用滤纸片法,测定菌株发酵粗提液对MRSA抑菌活性,并用高效液相色谱仪检测粗提液的活性物质。从鹦歌岭土壤中共分离到168株放线菌,其中10株具有较强的抗MRSA活性,发酵液粗提物抑菌圈直径在8 mm-25 mm之间;16S rDNA序列比对发现,10株菌均与链霉菌属(Streptomyces)的相似性达99%以上,初步确定是链霉菌属的放线菌。通过对比10株菌发酵液HPLC指纹图谱,发现菌株7、5和9-1的发酵液次级代谢产物丰富,活性测试表明,10株放线菌对MRSA的生长均有明显的抑制作用。
Yingge Ridge is a primitive tropical rainforest located in Ledong County,Hainan Province. In this study,the actinomycetes were isolated from the Yingge Ridge soil and screened for antibacterial activities against methicillin-resistant Staphylococcus aureus(MRSA).Actinomycete strains were isolated by plate dilution,and MRSA was used as the target to test antibacterial activities. Strains with antibacterial activity were subjected to 16S rDNA sequence determination. The antibacterial activity of crude extract of strain was determined by filter paper method,and the active substance of crude extract was detected by high performance liquid chromatography. A total of 168 strains of actinomycetes were obtained from the soil of the Yingge Ridge. Ten of them had strong anti-MRSA activity,and the inhibition zone of the crude extract of the fermentation broth was between 8 mm and 25 mm. The identity of 16S rDNA sequences from 10 strains of anti-MRSA Actinobacteria was over 99% in comparison with those of Streptomyces in Genbank. By HPLC fingerprints of 10 strains of fermentation broth,the secondary metabolites of strain 7,5,and 9-1 were abundant,and the activity test showed that the growth of MRSA was obviously inhibited,which provided a certain scientific basis for further research.
Yingge Ridge is a primitive tropical rainforest located in Ledong County,Hainan Province. In this study,the actinomycetes were isolated from the Yingge Ridge soil and screened for antibacterial activities against methicillin-resistant Staphylococcus aureus(MRSA).Actinomycete strains were isolated by plate dilution,and MRSA was used as the target to test antibacterial activities. Strains with antibacterial activity were subjected to 16S rDNA sequence determination. The antibacterial activity of crude extract of strain was determined by filter paper method,and the active substance of crude extract was detected by high performance liquid chromatography. A total of 168 strains of actinomycetes were obtained from the soil of the Yingge Ridge. Ten of them had strong anti-MRSA activity,and the inhibition zone of the crude extract of the fermentation broth was between 8 mm and 25 mm. The identity of 16S rDNA sequences from 10 strains of anti-MRSA Actinobacteria was over 99% in comparison with those of Streptomyces in Genbank. By HPLC fingerprints of 10 strains of fermentation broth,the secondary metabolites of strain 7,5,and 9-1 were abundant,and the activity test showed that the growth of MRSA was obviously inhibited,which provided a certain scientific basis for further research.
引文
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[14]彭云霞,姜怡一,段淑蓉,等.稀有放线菌的选择性分离方法[J].云南大学学报:自然科学版,2007,29(1):86-89.
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[16]陈电容.1株抗MRSA的土壤放线菌的分离与鉴定[J].生物技术,2008,18(2):24-26.
[17]Spatz SJ,Silva RF.Sequence determination of variable regions within the genomes of gallid herpsvirus-2 pathotypes[J].Archives of Virology,2007,152:1665-1678.
[18]杨静,易麟乙,刘美,等.1株抗耐甲氧西林金黄色葡萄球菌放线菌的鉴定及其代谢产物活性分析[J].西北农林科技大学学报:自然科学版,2014(8):8-14.
[19]Murata S,Okada T,Kano R,et al.Analysis of transcriptional activities of the Meq proteins present in highly virulent Marek’s disease virus strains,RB1B and Md5[J].Virus Genes,2011,43:66-71.
[20]韩秀芳.海南土壤放线菌分离与多相分类[C].第五届全国微生物资源学术暨国家微生物资源平台运行服务研讨会论文摘要集,2013.
[21]马伏宇,郭刚殷,晓敏,等.一株香蕉枯萎病拮抗链霉菌抑菌成分初步分析[J].分子植物育种,2016,14(11):3190-3194.
[22]袁维道.四株红树林底泥放线菌的多相分类坚定[D].海口:海南大学,2013.
[23]缪承杜,林海鹏,张志华,等.一株抗MRSA红树林真菌的分离和鉴定[J].热带作物学报,2010,31(11):2015-2019.
[24]宓伟,何深一.抗耐甲氧西林金黄色葡萄球菌的中药筛选[J].中国生化药物杂志,2011,32(1):44-46.
[25]唐依莉,谢修超,洪葵.红树植物根内生放线菌的分离鉴定及其生理活性的评价[J].热带生物学报,2012,3(01):32-37+41.
[26]高正航.霸王岭土壤抗MRSA活性放线菌的筛选及菌株Gda031、Gda0305发酵工艺的优化[D].海口:华南热带农业大学,2006.
[27]黄惠琴.海洋放线菌抗MRSA菌株的筛选及菌株AM105活性物质的研究[D].海口:华南热带农业大学,2005.
[2]Jones D,Lee L,Liu JL,et al.Marek’s disease virus encodes a basic-leucine zipper gene resembling the fos/jun oncogenes that is highly expressed in lymphoblasttoid tumor[J].Proceedings of the National Academy of Sciences USA,1992,89(9):4042-4046.
[3]Witter RL,Schat K.Marek’s disease[M]//Saif YM.In diseases of poultry.11th edition.Ames:Iowa State Press,2003:407-464.
[4]Frazee BW,Lynn J,Charlebois ED,et al.Highprevalence of methicillin-resistant Staphylococcus aureus in emergency department skin and soft tissue infections[J].Annals of Emergency Medicine,2005,45(3):311-320.
[5]Vanderhaeghen W.Hermans K,Haesebrouck F,et al.Methicillinresistant Staphylococcus aureus(MRSA)in food production animals[J].Epidemiology and Infection,2010,138(5):606-625.
[6]Saravolatz LD,Markowitz N,Arking L,et al.Methicillin-resistant Staphylococcus aureus epidemiologic observations during a community-acquired outbreak[J].Annals of Internal Medicine,1982,96(1):11-16.
[7]Fusco NM,Toussaint KA,Prescott WA.Antibiotic management of methicillin-resistant Staphylococcus aureus-associated acute pulmonary exacerbations in cystic fibrosis[J].Annals of Pharmacotherapy,2015,49(4):458-468.
[8]George S,Robert CM.Increasing antibiotic resistance among methicillin-resistant Staphylococcus aureus strains[J].Clinical Infectious Disease,2008,46(5):360-367.
[9]Newman DJ,Cragg GM.Natural products as sources of new drugs over the 30 years from 1981 to 2010[J].Journal of Natural Products,2012,75(3):311-335.
[10]Singh SB,Barrett JF.Empirical antibacterial drug discovery:foundation in natural products[J].Biochemical Pharmacology,2006,71(7):1006-1015.
[11]北京农业大学.微生物命名学[M].北京:农业出版社,1986.
[12]林家怡,吴世捷,庄雪影,等.海南鹦歌岭轮叶三棱栎(Trigonobalanus verticillata)群落特征与保护对策[J].生态学报,2007(6):2230-2238.
[13]方中达.植病研究方法[M].第3版.北京:中国农业出版社,1998:248-249.
[14]彭云霞,姜怡一,段淑蓉,等.稀有放线菌的选择性分离方法[J].云南大学学报:自然科学版,2007,29(1):86-89.
[15]陈国华,邵伟,乐超银.抗多重耐药金黄色葡萄球菌土壤放线菌筛选[J].安徽农业科学,2008,36(35):15369-15401.
[16]陈电容.1株抗MRSA的土壤放线菌的分离与鉴定[J].生物技术,2008,18(2):24-26.
[17]Spatz SJ,Silva RF.Sequence determination of variable regions within the genomes of gallid herpsvirus-2 pathotypes[J].Archives of Virology,2007,152:1665-1678.
[18]杨静,易麟乙,刘美,等.1株抗耐甲氧西林金黄色葡萄球菌放线菌的鉴定及其代谢产物活性分析[J].西北农林科技大学学报:自然科学版,2014(8):8-14.
[19]Murata S,Okada T,Kano R,et al.Analysis of transcriptional activities of the Meq proteins present in highly virulent Marek’s disease virus strains,RB1B and Md5[J].Virus Genes,2011,43:66-71.
[20]韩秀芳.海南土壤放线菌分离与多相分类[C].第五届全国微生物资源学术暨国家微生物资源平台运行服务研讨会论文摘要集,2013.
[21]马伏宇,郭刚殷,晓敏,等.一株香蕉枯萎病拮抗链霉菌抑菌成分初步分析[J].分子植物育种,2016,14(11):3190-3194.
[22]袁维道.四株红树林底泥放线菌的多相分类坚定[D].海口:海南大学,2013.
[23]缪承杜,林海鹏,张志华,等.一株抗MRSA红树林真菌的分离和鉴定[J].热带作物学报,2010,31(11):2015-2019.
[24]宓伟,何深一.抗耐甲氧西林金黄色葡萄球菌的中药筛选[J].中国生化药物杂志,2011,32(1):44-46.
[25]唐依莉,谢修超,洪葵.红树植物根内生放线菌的分离鉴定及其生理活性的评价[J].热带生物学报,2012,3(01):32-37+41.
[26]高正航.霸王岭土壤抗MRSA活性放线菌的筛选及菌株Gda031、Gda0305发酵工艺的优化[D].海口:华南热带农业大学,2006.
[27]黄惠琴.海洋放线菌抗MRSA菌株的筛选及菌株AM105活性物质的研究[D].海口:华南热带农业大学,2005.
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