西藏沙棘根瘤及根际土壤放线菌分离及抗菌活性研究

doi:10.13560/j.cnki.biotech.bull.1985.2021-0288

摘要/Abstract

摘要：

探索西藏林芝与山南地区沙棘根瘤及其根际土壤样品中放线菌的多样性及抗菌活性,为天然产物药物研发提供新的微生物资源。采用稀释涂布法对10份样品中可培养放线菌进行分离,后经菌株16S rRNA序列测序,以邻接法（neighbor-joining method,N-J）构建系统发育树分析放线菌种属多样性;利用微孔板法对代表性菌株进行发酵培养及抗菌活性评价;进一步利用超高效液相色谱-质谱联用（UPLC-MS）考察活性菌株代谢产物情况。从10份样品中分离到112株放线菌,分属于7个目12科23个属,优势菌属为链霉菌属（Streptomyces）;抗菌活性结果表明有17株放线菌至少对一种检定菌有抗菌活性,阳性抑菌率为36.9%。有16株放线菌对临床常见的耐甲氧西林金黄色葡萄球菌（methicillin-resistant Staphylococcus aureus,MRSA）有较强抑制活性,其中13株放线菌对MRSA抑制率超过阳性对照药万古霉素;另外有7株放线菌对耐碳青霉烯鲍曼不动杆菌（carbapenem-resistant Acinetobacter baumannii,CRAB）有抑制活性,其中Streptomyces sp. XZ19-363对CRAB抑菌作用最强,抑制率为91.3%,UPLC-MS分析显示,具有广谱抗菌活性的菌株在不同培养基中产生了较为丰富的次级代谢产物。西藏林芝与山南地区沙棘根瘤及其根际土壤中可培养放线菌资源丰富多样,同时具有较好的抗菌活性且代谢产物丰富。

关键词: 沙棘根瘤, 根际土壤, 放线菌, 抗菌活性

Abstract:

In order to provide new microbial resources for the development of natural product drugs,the diversity and antibacterial activity of actinomycetes from the root nodules and rhizosphere soil samples of Hippophae rhamnoides in Nyingchi and Shannan of Tibet were explored. Dilution coating method was empployed to isolate the cultivable actinomycetes from 10 samples. After the 16S rRNA sequence alignment of the strains,the neighbor-joining method（N-J）was used to construct a phylogenetic tree to analyze the species diversity of actinomycetes. The microplate method was adapted to conduct fermentation and antibacterial activity evaluation of representative strains;and ultra-performance liquid chromatography-mass spectrometry（UPLC-MS）was to investigate the metabolites of active strains. A total of 112 strains of actinomycetes belonged to 23 genera,12 families and 7 orders were isolated from 10 samples,in which Streptomyces was the dominant genus. The results of antibacterial activity showed that 17 strains of estimated actinomycetes presented antibacterial activity against at least one of the tested bacteria,and the positive inhibition rate was 36.9%. Sixteen actinomycetes showed strong inhibitory activity against common clinical methicillin-resistant Staphylococcus aureus（MRSA）,13 of which had higher inhibitory rate than the positive control drug vancomycin. In addition,7 of evaluated actinomycetes demonstrated inhibitory activity against carbapenem-resistant Acinetobacter baumannii（CRAB）,from which Streptomyces sp. XZ19-363 had the strongest antibacterial activity against CRAB,with an inhibition rate of 91.3%. UPLC-MS analysis revealed that the strains with broad-spectrum antibacterial activities produced relatively abundant secondary metabolites in different media. The resources of cultivable actinomycetes from H. rhamnoides nodules and rhizosphere soils in Nyingchi and Shannan regions of Tibet both are rich and diverse,and possess promising antibacterial activities with rich metabolites.

Key words: Hippophae rhamnoides nodules, rhizosphere soil, actinobacteria, antibacterial activity

罗亚军, 孙红敏, 何宁, 袁丽杰, 解云英. 西藏沙棘根瘤及根际土壤放线菌分离及抗菌活性研究[J]. 生物技术通报, 2021, 37(11): 225-236.

LUO Ya-jun, SUN Hong-min, HE Ning, YUAN Li-jie, XIE Yun-ying. Isolation and Antibacterial Activity of Actinomycetes from the Nodules and Rhizosphere Soil of Hippophae rhamnoides in Tibet[J]. Biotechnology Bulletin, 2021, 37(11): 225-236.

图/表 9

参考文献 29

[1]	Frieri M, Kumar K, Boutin A. Antibiotic resistance[J]. J Infect Public Heal, 2017, 10(4): 369-378.
[2]	Xie CL, Xia JM, et al. Metabolomic investigations on Nesterenkonia flava revealed significant differences between marine and terrestrial actinomycetes[J]. Mar Drugs, 2018, 16(10): 356. doi: 10.3390/md16100356 URL
[3]	任建委, 杜宝中. 放线菌资源及主要次级代谢产物活性概述[J]. 西藏科技, 2020(4): 15-18, 28.
	Ren JW, Du BZ. Summary of actinomycetes resources and activities of main secondary metabolites[J]. Tibet Sci Technol, 2020(4): 15-18, 28.
[4]	徐志勇, 冯昭, 徐静. 红树林微生物抗菌活性成分研究进展[J]. 中国抗生素杂志, 2017, 42(4): 241-254.
	Xu ZY, Feng Z, Xu J. Research advances on antimicrobial activities of microbes derived from mangrove[J]. Chin J Antibiot, 2017, 42(4): 241-254.
[5]	Clardy J, et al. New antibiotics from bacterial natural products[J]. Nat Biotechnol, 2006, 24(12): 1541-1550. pmid: 17160060
[6]	Chen P, Zhang C, Ju X, et al. Community composition and metabolic potential of endophytic actinobacteria from coastal salt marsh plants in Jiangsu, China[J]. Front Microbiol, 2019, 10: 1063. doi: 10.3389/fmicb.2019.01063 pmid: 31139174
[7]	张万芹, 等. 贵州兴义喀斯特洞穴可培养放线菌多样性及抗菌活性初筛[J]. 微生物学报, 2020, 60(6): 1063-1073.
	Zhang WQ, Fang BZ, Han MX, et al. Diversity and antibacterial activity of culturable actinobacteria in Karst cave soil in Xingyi, Guizhou[J]. Acta Microbiol Sin, 2020, 60(6): 1063-1073.
[8]	祖健, 刘少伟, 庹利, 等. 湖北利川喀斯特洞穴放线菌多样性及抗菌活性研究[J]. 中国抗生素杂志, 2016, 41(3): 186-195.
	Zu J, Liu SW, Tuo L, et al. Studies on diversity and anti-microbial activity of actinobacteria isolated from Karst caves in Lichuan, Hubei[J]. Chin J Antibiot, 2016, 41(3): 186-195.
[9]	张爱梅, 韩雪英, 孙坤, 等. 高通量测序分析中国沙棘根瘤与根际土壤细菌多样性[J]. 草原与草坪, 2018, 38(2): 49-55.
	Zhang AM, Han XY, Sun K, et al. Root nodules endophytic and rhizosphere soil bacteria diversityof Hippophae rhamnoidoes subsp. sinensis based on high-throughput sequencing[J]. Grassland Turf, 2018, 38(2): 49-55.
[10]	黄路枝, 胡兆农, 郭正彦, 等. 土壤稀有放线菌的选择性分离及其抗菌活性研究[J]. 农药学学报, 2007, 9(1): 59-65.
	Huang LZ, Hu ZN, Guo ZY, et al. Study on selective isolation and antibiotic activity of rare actinomycetes from soil[J]. Chin J Pestic Sci, 2007, 9(1): 59-65.
[11]	彭云霞, 姜怡, 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.
	Peng YX, Jiang Y, et al. Selective isolation methods of rare actinomycetes[J]. J Yunnan Univ:Nat Sci Ed, 2007, 29(1): 86-89.
[12]	李利坤. 沙棘根瘤菌的分离鉴定及根瘤菌对植株生长发育的影响[D]. 长春:吉林农业大学, 2018.
	Li LK. Isolation and identification of Rhizobium from seabuckthorn and effects of Rhizobium on the growth and development of plants[D]. Changchun:Jilin Agricultural University, 2018.
[13]	张情. 沙棘根瘤形态结构及根瘤细菌的分离鉴定[D]. 杨凌:西北农林科技大学, 2019.
	Zhang Q. Morphology and structure of root nodules of Hippophae rhamnides and isolation and identification of bacteria in the nodules[D]. Yangling:Northwest A & F University, 2019.
[14]	Bauermeister A, Calil FA, das C L Pinto F, et al. Pradimicin-IRD from Amycolatopsis sp. IRD-009 and its antimicrobial and cytotoxic activities[J]. Nat Prod Res, 2019, 33(12): 1713-1720. doi: 10.1080/14786419.2018.1434639 pmid: 29451013
[15]	Nakashima T, Kimura T, et al. Nanaomycin H:a new nanaomycin analog[J]. J Biosci Bioeng, 2017, 123(6): 765-770. doi: S1389-1723(16)30650-8 pmid: 28202308
[16]	Hashizume H, Sawa R, Yamashita K, et al. Structure and antibacterial activities of new cyclic peptide antibiotics, pargamicins B, C and D, from Amycolatopsis sp. ML1-hF₄[J]. J Antibiot, 2017, 70(5): 699-704. doi: 10.1038/ja.2017.34 pmid: 28293037
[17]	Serrill JD, Tan M, Fotso S, et al. Apoptolidins A and C activate AMPK in metabolically sensitive cell types and are mechanistically distinct from oligomycin A[J]. Biochem Pharmacol, 2015, 93(3): 251-265. doi: 10.1016/j.bcp.2014.11.015 URL
[18]	Dasari VRRK, Muthyala MKK, Nikku MY, et al. Novel Pyridinium compound from marine actinomycete, Amycolatopsis alba var. nov. DVR D4 showing antimicrobial and cytotoxic activities in vitro[J]. Microbiol Res, 2012, 167(6): 346-351. doi: 10.1016/j.micres.2011.12.003 URL
[19]	周双清, 黄小龙, 等. Chelex-100快速提取放线菌DNA作为PCR扩增模板[J]. 生物技术通报, 2010(2): 123-125.
	Zhou SQ, Huang XL, Huang DY, et al. A rapid method for extracting DNA from actinomycetes by chelex-100[J]. Biotechnol Bull, 2010(2): 123-125.
[20]	张玉琴, 李文均, 等. PCR法快速识别actinobacteria的五种模板制备方法的比较[J]. 生物技术, 2004, 14(5): 37-39.
	Zhang YQ, Li WJ, Chen GZ, et al. Comparison of five PCR template preparation methods for fast identification of actinobacteria[J]. Biotechnology, 2004, 14(5): 37-39.
[21]	Tamura K, Peterson D, Peterson N, et al. MEGA5:molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[J]. Mol Biol Evol, 2011, 28(10): 2731-2739. doi: 10.1093/molbev/msr121 URL
[22]	Saitou N, Nei M. The neighbor-joining method:a new method for reconstructing phylogenetic trees[J]. Mol Biol Evol, 1987, 4(4): 406-425. pmid: 3447015
[23]	周子雄, 黄庆华, 朱爽, 等. 酶标仪快速测定抗菌物质抑菌活性方法的建立[J]. 微生物前沿, 2014, 3(2): 29-35.
	Zhou ZX, Huang QH, Zhu S, et al. Establishment of rapid determining method for antibacterial activity by microplate reader[J]. Advances in Microbiology, 2014, 3(2): 29-35. doi: 10.12677/AMB.2014.32004 URL
[24]	Omata K, Watanabe Y, Umegaki T, et al. Catalyst development for methanol synjournal using parallel reactors for high-throughput screening based on a 96 well microplate system[J]. J Japan Petrol Inst, 2003, 46(5): 328-334. doi: 10.1627/jpi.46.328 URL
[25]	Li YH, He N, Luo MN, et al. Application of untargeted tandem mass spectrometry with molecular networking for detection of enniatins and beauvericins from complex samples[J]. J Chromatogr A, 2020, 1634: 461626. doi: 10.1016/j.chroma.2020.461626 URL
[26]	Schulz B, Boyle C, Draeger S, et al. Endophytic fungi:a source of novel biologically active secondary metabolites[J]. Mycol Res, 2002, 106(9): 996-1004. doi: 10.1017/S0953756202006342 URL
[27]	张爱梅. 沙棘属植物根际可培养微生物的初步研究[D]. 兰州:西北师范大学, 2008.
	Zhang AM. Study on the cultivable microorganism of Hippophae rhizosphere[D]. Lanzhou:Northwest Normal University, 2008.
[28]	全国细菌耐药监测网. 全国细菌耐药监测网2014—2019年老年患者常见临床分离细菌耐药性监测报告[J]. 中国感染控制杂志, 2021, 20(2): 112-123.
	System CARS. Antimicrobial resistance of clinically isolated bacteria from elderly patients:surveillance report from China antimicrobial resistance surveillance system in 2014-2019[J]. Chin J Infect Control, 2021, 20(2): 112-123.
[29]	Lee CR, et al. Biology of Acinetobacter baumannii:pathogenesis, antibiotic resistance mechanisms, and prospective treatment options[J]. Front Cell Infect Microbiol, 2017, 7: 55.

采样地点 Sampling location	样品种类 Sample type	编号 No.	海拔 Altitude/m	采样时间 Sampling time	坐标 Coordinates
林芝南伊沟区	沙棘根瘤	P1	2 999	2019. 09. 21	29^o08’25.15” N,94^o12’58.71” E
林芝南伊沟区	根际土壤	E1	2 999	2019. 09. 21	29^o08’25.15” N,94^o12’58.71” E
山南市错那县	大沙棘根瘤	P2	4 515	2019. 09. 23	28^o18’48.24” N,91^o49’06.59” E
山南市错那县	根际土壤	E2	4 515	2019. 09. 23	28^o18’48.24” N,91^o49’06.59” E
山南市错那县	小沙棘根瘤	P3	4 515	2019. 09. 23	28^o18’48.24” N,91^o49’06.59” E
山南市错那县	根际土壤	E3	4 515	2019. 09. 23	28^o18’48.24” N,91^o49’06.59” E
山南市错那县	沙棘根瘤	P4	4 255	2019. 09. 23	28^o15’18.76” N,91^o45’47.52” E
山南市错那县	根际土壤	E4	4 255	2019. 09. 23	28^o15’18.76” N,91^o45’47.52” E
山南市隆子县	沙棘根瘤	P5	3 915	2019. 09. 23	28^o25’12.01” N,92^o23’48.99” E
山南市隆子县	根际土壤	E5	3 915	2019. 09. 23	28^o25’12.01” N,92^o23’48.99” E

采样地点 Sampling location	样品种类 Sample type	编号 No.	海拔 Altitude/m	采样时间 Sampling time	坐标 Coordinates
林芝南伊沟区	沙棘根瘤	P1	2 999	2019. 09. 21	29^o08’25.15” N,94^o12’58.71” E
林芝南伊沟区	根际土壤	E1	2 999	2019. 09. 21	29^o08’25.15” N,94^o12’58.71” E
山南市错那县	大沙棘根瘤	P2	4 515	2019. 09. 23	28^o18’48.24” N,91^o49’06.59” E
山南市错那县	根际土壤	E2	4 515	2019. 09. 23	28^o18’48.24” N,91^o49’06.59” E
山南市错那县	小沙棘根瘤	P3	4 515	2019. 09. 23	28^o18’48.24” N,91^o49’06.59” E
山南市错那县	根际土壤	E3	4 515	2019. 09. 23	28^o18’48.24” N,91^o49’06.59” E
山南市错那县	沙棘根瘤	P4	4 255	2019. 09. 23	28^o15’18.76” N,91^o45’47.52” E
山南市错那县	根际土壤	E4	4 255	2019. 09. 23	28^o15’18.76” N,91^o45’47.52” E
山南市隆子县	沙棘根瘤	P5	3 915	2019. 09. 23	28^o25’12.01” N,92^o23’48.99” E
山南市隆子县	根际土壤	E5	3 915	2019. 09. 23	28^o25’12.01” N,92^o23’48.99” E

培养基Culture medium	培养基成分Medium composition
PDB	成品,购于北京奥博星生物技术公司,pH 7.2
ISP 2	酵母膏4 g,麦芽糖10 g,葡萄糖4 g,pH 7.2
FM3	可溶性淀粉20 g,甘油5 g,脱脂小麦胚芽10 g,肉提取物3 g,干酵母3 g,CaCO₃3 g,pH 7.0
FM4	半乳糖3.3 g,水化糊精3.3 g,甘油1.7 g,豆胨1.7 g,玉米浆0.83 g,CaCO₃2 g,（NH₄）₂SO₄0.33 g,pH 7.0
FM5	葡萄糖10 g,牛肉膏10 g,蛋白胨1 g,NaCl 5 g,pH 7.0
M331	葡萄糖20 g,普通淀粉5 g,蛋白胨 10 g,（NH₄）₂SO₄7 g,CaCO₃ 2 g,pH自然
F1	普通淀粉20 g,葡萄糖20 g,蛋白胨3 g,牛肉膏3 g,微量元素1 mL,CaCO₃2 g,花生饼粉10 g,pH 7.2
M2	甘露醇40 g,麦芽浸粉40 g,酵母浸膏粉10 g,K₂HPO₄ 2 g,MgSO₄·7H₂O 0.5 g,FeSO₄·7H₂O 0.01 g,pH 7.2
Cazpeck	K₂HPO₄ 1 g,NaNO₃0.3 g,KCl 0.005 g,MgSO₄·7H₂O 0.005 g,FeSO₄ 0.001 g,蔗糖30 g,pH 7.0
FM10	甘油20 g,糖蜜10 g,酪蛋白5 g,蛋白胨1 g,CaCO₃4 g,pH 7.2
FM11	葡萄糖20 g,麦芽浸粉40 g,酵母浸粉4 g,K₂HPO₄5 g,NaCl 2.5 g,CaCO₃ 0.4 g,ZnSO₄0.04 g,pH 6.0
YMS	酵母浸粉4 g,麦芽浸粉10 g,可溶性淀粉4 g,pH 自然

培养基Culture medium	培养基成分Medium composition
PDB	成品,购于北京奥博星生物技术公司,pH 7.2
ISP 2	酵母膏4 g,麦芽糖10 g,葡萄糖4 g,pH 7.2
FM3	可溶性淀粉20 g,甘油5 g,脱脂小麦胚芽10 g,肉提取物3 g,干酵母3 g,CaCO₃3 g,pH 7.0
FM4	半乳糖3.3 g,水化糊精3.3 g,甘油1.7 g,豆胨1.7 g,玉米浆0.83 g,CaCO₃2 g,（NH₄）₂SO₄0.33 g,pH 7.0
FM5	葡萄糖10 g,牛肉膏10 g,蛋白胨1 g,NaCl 5 g,pH 7.0
M331	葡萄糖20 g,普通淀粉5 g,蛋白胨 10 g,（NH₄）₂SO₄7 g,CaCO₃ 2 g,pH自然
F1	普通淀粉20 g,葡萄糖20 g,蛋白胨3 g,牛肉膏3 g,微量元素1 mL,CaCO₃2 g,花生饼粉10 g,pH 7.2
M2	甘露醇40 g,麦芽浸粉40 g,酵母浸膏粉10 g,K₂HPO₄ 2 g,MgSO₄·7H₂O 0.5 g,FeSO₄·7H₂O 0.01 g,pH 7.2
Cazpeck	K₂HPO₄ 1 g,NaNO₃0.3 g,KCl 0.005 g,MgSO₄·7H₂O 0.005 g,FeSO₄ 0.001 g,蔗糖30 g,pH 7.0
FM10	甘油20 g,糖蜜10 g,酪蛋白5 g,蛋白胨1 g,CaCO₃4 g,pH 7.2
FM11	葡萄糖20 g,麦芽浸粉40 g,酵母浸粉4 g,K₂HPO₄5 g,NaCl 2.5 g,CaCO₃ 0.4 g,ZnSO₄0.04 g,pH 6.0
YMS	酵母浸粉4 g,麦芽浸粉10 g,可溶性淀粉4 g,pH 自然

发酵菌株 Fermentation strain	菌株发酵液对检定菌最大抑制率及发酵培养基 The maximum inhibition rate of fermentation broth to the tested bacteria and fermentation medium
发酵菌株 Fermentation strain	金黄色葡萄球菌ATCC 29213	耐甲氧西林金黄色葡萄球菌MRSA 181223	铜绿假单胞菌 ATCC 27853	大肠埃希菌ATCC 25922	肺炎克雷伯菌ATCC 700603	耐碳青霉烯鲍曼不动杆菌 CRAB 181236	白色念珠菌ATCC 10231
Streptomyces sp. XZ19-363	87.4%（FM4）	82.7%（FM4）	87.1%（FM4）	< 50%	50.8%（FM4）	91.3%（FM4）	< 50%
Streptomyces sp. XZ19-323	86.9%（FM3）	79.6%（FM3）	< 50%	< 50%	< 50%	< 50%	< 50%
Streptomyces sp. XZ19-034	84.6%（Cazpeck）	73.1%（Cazpeck）	< 50%	< 50%	< 50%	< 50%	< 50%
Streptomyces sp. XZ19-175	86.2%（FM3）	79.6%（FM3/FM4）	< 50%	< 50%	< 50%	< 50%	< 50%
Streptomyces sp. XZ19-147	86.4%（FM4）	81.0%（FM4）	< 50%	< 50%	< 50%	< 50%	< 50%
Streptomyces sp. XZ19-130	88.8% （ISP 2）	83.4%（ISP 2）	< 50%	< 50%	< 50%	< 50%	< 50%
Streptomyces sp. XZ19-336	88.1%（ISP 2）	83.1%（ISP 2）	< 50%	< 50%	< 50%	< 50%	78.9%（FM3）
Streptomyces sp. XZ19-098	88.3%（FM3）	82.7% （ISP 2/FM4）	< 50%	72.6%（YMS）	< 50%	< 50%	< 50%
Streptomyces sp. XZ19-152	84.1%（PDB）	82.7%（FM4）	< 50%	< 50%	< 50%	< 50%	< 50%
Streptomyces sp. XZ19-091	88.1%（FM3）	83.1%（FM4）	< 50%	< 50%	< 50%	< 50%	83.8%（FM3）
Streptomyces sp. XZ19-122	88.3%（FM3）	83.4%（ISP 2）	< 50%	< 50%	< 50%	53.0%（F1）	84.1%（FM3）
Streptomyces sp. XZ19-015	88.3% （ISP 2）	83.7%（ISP 2）	< 50%	< 50%	< 50%	64.4%（FM3）	83.8%（FM4）
Streptomyces sp. XZ19-030	88.3% （ISP 2）	82.0%（FM4）	< 50%	< 50%	< 50%	< 0.5%	81.0%（M2）
Streptomyces sp. XZ19-036	87.4% （ISP 2）	81.7%（ISP 2）	< 50%	< 50%	< 50%	< 0.5%	85.0% （FM11）
Streptomyces sp. XZ19-093	< 50%	< 50%	< 50%	< 50%	< 50%	55.1%（M331）	< 50%
Streptomyces sp. XZ19-021	< 50%	< 50%	< 50%	< 50%	< 50%	55.7%（F1）	< 50%
Streptomyces sp. XZ19-006	77.3%（M2）	71.3%（M2）	< 50%	< 50%	< 50%	51.2%（M331）	< 50%
Streptomyces sp. XZ19-003	82.2%（YMS）	65.5%（YMS）	< 50%	< 50%	< 50%	< 50%	< 50%
Promicromonospora sp. XZ19-011	< 50%	< 50%	< 50%	< 50%	< 50%	73.9%（M331）	< 50%
Micromonospora sp. XZ19-205	59.4%（YMS）	< 50%	< 50%	< 50%	< 50%	< 50%	< 50%
美罗培南	87.1%	9.50%	91. 5%	91.6%	93.5%	42.2%	-
万古霉素	83.3%	74.1%	-1.2%	37.8%	15.1%	40.0%	-
氟康唑	-	-	-	-	-	-	66.1%
伊曲康唑	-	-	-	-	-	-	44.5%
两性霉素B	-	-	-	-	-	-	64.6%