豨莶草内生抗感染细菌的分离鉴定及生长特性
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摘要
豨莶草有多种药用价值,旨在从豨莶草果实中分离和筛选出高拮抗活性的内生细菌。采用了形态学观察、生理生化试验、16S r RNA序列比对、系统发育树分析以及OD600值、药敏试验和超滤等方法对该抗感染细菌C-5-1进行了相关生物学特性评价。结果显示,C-5-1为侧孢短芽孢杆菌(Brevibacillus laterosporus),对多种病原细菌均敏感,尤其是对铜绿假单胞菌;低p H和高Na Cl盐度环境中耐受性有限,在p H≤5.0或盐度≥15%时其生长受到了完全的抑制甚至是死亡,但在p H范围7.0-11.0和盐度范围0-5%下均能生长良好,说明该菌株为耐碱,但不耐酸和盐;筛选出了对菌株C-5-1高度敏感而对5种指示病原菌耐药的抗生素,即氨苄西林;C-5-1发酵上清液中存在着两种抗菌物质,且它们的相对分子质量均小于2 000。因此豨莶草有着优良微生物资源,可以成为开发抗感染细菌药物的重要资源。
Herba siegesbeckiae has many healing values,and our aim is to isolate and screen highly active antagonistic endophytesfrom its fruits. The biological characteristics of anti-infective bacterium C-5-1 were determined by antagonistic experiment,morphologicalobservation,physiological and biochemical tests,sequence alignment of 16 S r RNA gene,analysis of phylogenetic tree,measuring OD600,drug sensitive tests,and ultra-filtration centrifugation. The results showed that the C-5-1 was identified as Brevibacillus laterosporus and wassensitive to various pathogenic bacteria,especially Pseudomonas aeruginosa. The strain C-5-1 had limited tolerance to low p H and highlysaline environments,i.e.,inhibited completely or even died in p H≤5 or more than 15% Na Cl,but it grew well in p H≥7 and 0-5% Na Cl,suggesting that C-5-1 had good alkali resistance,but not resistant to acid and salt. Screening experiment revealed that the antibiotic ampicillinwas highly sensitive to C-5-1,but resistant to 5 pathogenic bacteria. Fermentation supernatant of C-5-1 contained two antibacterial substancesand relative molecular mass of both was less than 2 000. Conclusively,H. siegesbeckiae having excellent microbial resources will be a keyresource for the development of anti-bacteria drugs.
Herba siegesbeckiae has many healing values,and our aim is to isolate and screen highly active antagonistic endophytesfrom its fruits. The biological characteristics of anti-infective bacterium C-5-1 were determined by antagonistic experiment,morphologicalobservation,physiological and biochemical tests,sequence alignment of 16 S r RNA gene,analysis of phylogenetic tree,measuring OD600,drug sensitive tests,and ultra-filtration centrifugation. The results showed that the C-5-1 was identified as Brevibacillus laterosporus and wassensitive to various pathogenic bacteria,especially Pseudomonas aeruginosa. The strain C-5-1 had limited tolerance to low p H and highlysaline environments,i.e.,inhibited completely or even died in p H≤5 or more than 15% Na Cl,but it grew well in p H≥7 and 0-5% Na Cl,suggesting that C-5-1 had good alkali resistance,but not resistant to acid and salt. Screening experiment revealed that the antibiotic ampicillinwas highly sensitive to C-5-1,but resistant to 5 pathogenic bacteria. Fermentation supernatant of C-5-1 contained two antibacterial substancesand relative molecular mass of both was less than 2 000. Conclusively,H. siegesbeckiae having excellent microbial resources will be a keyresource for the development of anti-bacteria drugs.
引文
[1]国家药典委员会.中华人民共和国药典[M].北京:化学工业出版社,2015:348.
[2]Lu Y,Qian RQ,Xiao J,et al.Kirenol attenuates experimentalautoimmune encephalomyelitis by inhibiting differentiation of Th1and Th17 cells and inducing apoptosis of effect or T cells[J].Scientific Reports,2015,5:9022.
[3]Lu Y,Qian R,Xiao J,et al.Kirenol,a compound from Herba Siegesbeckiae,induces apoptosis in human chronic myeloid leukemiaK562 cells[J].Pharmazie,2014,69(2):148-153.
[4]Hwang JH,Kim JD.Inhibitory effects of Siegesbeckiae herbaextract on angiogenesis and adipogenesis[J].Biotechnology andBioprocess Engineering,2011,1(16):144-152.
[5]柴明艳.车前草内生真菌的分离及抑菌活性测定[J].江苏农业科学,2016,44(1):353-356.
[6]周邦靖.常用中药的抗菌作用及其测定方法[M].重庆:科学技术文献出版社重庆分社,1987:100-299.
[7]Marchesi JR,Sato T,Weightman AJ,et al.Design and evaluation ofuseful bacterium-specific PCR primers that amplify genes coding forbacterial 16S r RNA[J].Applied and Environmental Microbiology,1998,64(2):795-799.
[8]史应武,娄恺,李春.植物内生菌在生物防治中的应用[J].微生物学杂志,2009,11(6):61-64.
[9]黄午阳.植物内生真菌的抗菌活性研究[J].南京中医药大学学报,2005,21(1):24-26.
[10]Laubach CA.Studies on aerobic spore-bearing non-pathogenicbacteria partⅡ.Spore-bearing organisms in water[J].Journalof Bacterial,1916,1(5):506-512.
[11]Shida O,Takagi H,Kadowaki K,et al.Proposal for two new genera,Brevibacillus gen.nov.and Aneurinibacillus gen.Nov[J].International Journal of Systematic and Evolutionary Microbiology,1996,46(4):939-946.
[12]陈潺,陈升富,王建宇,等.侧孢短芽孢杆菌的应用研究进展[J].山东农业科学,2015,47(2):149-156.
[13]Djukic M,Poehlein A,Thürmer A,et al.Genome sequence of Brevibacillus laterosporus LMG 15441,a pathogen of invertebrates[J].Journal of Bacterial,2011,193(19):5535-5536.
[14]Sharma V,Singh PK,Midha S,et al.Genome sequence ofBrevibacillus laterosporus strain GI-9[J].Journal of Bacterial,2012,194(5):1279.
[15]Hong HA,Duc LH,Cutting SM.The use of bacterial spore formersas probiotics[J].FEMS Microbiology Rev,2005,29(4):813-835.
[16]Barsby T,Kelly MT,Andersen RJ.Tupuseleiamides andbasiliskamides,new acyldipeptides and antifungal polyketidesproduced in culture by a Bacillus laterosporus isolate obtained froma tropical marine habitat[J].Journal of Natural Products,2002,65(10):1447-1451.
[17]Desjardine K,Pereira A,Wright H,et al.Tauramamide,alipopeptide antibiotic produced in culture by Brevibacillus laterosporus isolated from a marine habitat:Structure elucidationand synthesis[J].Journal of Natural Products,2007,70(12):1850-1853.
[18]Yang X,Huang E,Yuan CH.Isolation and structural elucidationof Brevibacillin,an antimicrobial lipopeptide from Brevibacillus laterosporus that combats drug-resistant Gram-positivebacteria[J].Applied and Environmental Microbiology,2016,82(9):2763-2772.
[2]Lu Y,Qian RQ,Xiao J,et al.Kirenol attenuates experimentalautoimmune encephalomyelitis by inhibiting differentiation of Th1and Th17 cells and inducing apoptosis of effect or T cells[J].Scientific Reports,2015,5:9022.
[3]Lu Y,Qian R,Xiao J,et al.Kirenol,a compound from Herba Siegesbeckiae,induces apoptosis in human chronic myeloid leukemiaK562 cells[J].Pharmazie,2014,69(2):148-153.
[4]Hwang JH,Kim JD.Inhibitory effects of Siegesbeckiae herbaextract on angiogenesis and adipogenesis[J].Biotechnology andBioprocess Engineering,2011,1(16):144-152.
[5]柴明艳.车前草内生真菌的分离及抑菌活性测定[J].江苏农业科学,2016,44(1):353-356.
[6]周邦靖.常用中药的抗菌作用及其测定方法[M].重庆:科学技术文献出版社重庆分社,1987:100-299.
[7]Marchesi JR,Sato T,Weightman AJ,et al.Design and evaluation ofuseful bacterium-specific PCR primers that amplify genes coding forbacterial 16S r RNA[J].Applied and Environmental Microbiology,1998,64(2):795-799.
[8]史应武,娄恺,李春.植物内生菌在生物防治中的应用[J].微生物学杂志,2009,11(6):61-64.
[9]黄午阳.植物内生真菌的抗菌活性研究[J].南京中医药大学学报,2005,21(1):24-26.
[10]Laubach CA.Studies on aerobic spore-bearing non-pathogenicbacteria partⅡ.Spore-bearing organisms in water[J].Journalof Bacterial,1916,1(5):506-512.
[11]Shida O,Takagi H,Kadowaki K,et al.Proposal for two new genera,Brevibacillus gen.nov.and Aneurinibacillus gen.Nov[J].International Journal of Systematic and Evolutionary Microbiology,1996,46(4):939-946.
[12]陈潺,陈升富,王建宇,等.侧孢短芽孢杆菌的应用研究进展[J].山东农业科学,2015,47(2):149-156.
[13]Djukic M,Poehlein A,Thürmer A,et al.Genome sequence of Brevibacillus laterosporus LMG 15441,a pathogen of invertebrates[J].Journal of Bacterial,2011,193(19):5535-5536.
[14]Sharma V,Singh PK,Midha S,et al.Genome sequence ofBrevibacillus laterosporus strain GI-9[J].Journal of Bacterial,2012,194(5):1279.
[15]Hong HA,Duc LH,Cutting SM.The use of bacterial spore formersas probiotics[J].FEMS Microbiology Rev,2005,29(4):813-835.
[16]Barsby T,Kelly MT,Andersen RJ.Tupuseleiamides andbasiliskamides,new acyldipeptides and antifungal polyketidesproduced in culture by a Bacillus laterosporus isolate obtained froma tropical marine habitat[J].Journal of Natural Products,2002,65(10):1447-1451.
[17]Desjardine K,Pereira A,Wright H,et al.Tauramamide,alipopeptide antibiotic produced in culture by Brevibacillus laterosporus isolated from a marine habitat:Structure elucidationand synthesis[J].Journal of Natural Products,2007,70(12):1850-1853.
[18]Yang X,Huang E,Yuan CH.Isolation and structural elucidationof Brevibacillin,an antimicrobial lipopeptide from Brevibacillus laterosporus that combats drug-resistant Gram-positivebacteria[J].Applied and Environmental Microbiology,2016,82(9):2763-2772.