生物技术通报 ›› 2021, Vol. 37 ›› Issue (11): 225-236.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0288
罗亚军1(), 孙红敏2, 何宁2, 袁丽杰1(), 解云英2()
收稿日期:
2021-03-11
出版日期:
2021-11-26
发布日期:
2021-12-03
作者简介:
罗亚军,男,硕士,研究方向:微生物的分离鉴定、抗菌活性及天然产物挖掘;E-mail: 基金资助:
LUO Ya-jun1(), SUN Hong-min2, HE Ning2, YUAN Li-jie1(), XIE Yun-ying2()
Received:
2021-03-11
Published:
2021-11-26
Online:
2021-12-03
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。
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.
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 | 29o08’25.15” N,94o12’58.71” E |
根际土壤 | E1 | 2 999 | 2019. 09. 21 | 29o08’25.15” N,94o12’58.71” E | |
山南市错那县 | 大沙棘根瘤 | P2 | 4 515 | 2019. 09. 23 | 28o18’48.24” N,91o49’06.59” E |
根际土壤 | E2 | 4 515 | 2019. 09. 23 | 28o18’48.24” N,91o49’06.59” E | |
山南市错那县 | 小沙棘根瘤 | P3 | 4 515 | 2019. 09. 23 | 28o18’48.24” N,91o49’06.59” E |
根际土壤 | E3 | 4 515 | 2019. 09. 23 | 28o18’48.24” N,91o49’06.59” E | |
山南市错那县 | 沙棘根瘤 | P4 | 4 255 | 2019. 09. 23 | 28o15’18.76” N,91o45’47.52” E |
根际土壤 | E4 | 4 255 | 2019. 09. 23 | 28o15’18.76” N,91o45’47.52” E | |
山南市隆子县 | 沙棘根瘤 | P5 | 3 915 | 2019. 09. 23 | 28o25’12.01” N,92o23’48.99” E |
根际土壤 | E5 | 3 915 | 2019. 09. 23 | 28o25’12.01” N,92o23’48.99” E |
表1 样品采集信息
Table 1 Sample collection information
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 | 29o08’25.15” N,94o12’58.71” E |
根际土壤 | E1 | 2 999 | 2019. 09. 21 | 29o08’25.15” N,94o12’58.71” E | |
山南市错那县 | 大沙棘根瘤 | P2 | 4 515 | 2019. 09. 23 | 28o18’48.24” N,91o49’06.59” E |
根际土壤 | E2 | 4 515 | 2019. 09. 23 | 28o18’48.24” N,91o49’06.59” E | |
山南市错那县 | 小沙棘根瘤 | P3 | 4 515 | 2019. 09. 23 | 28o18’48.24” N,91o49’06.59” E |
根际土壤 | E3 | 4 515 | 2019. 09. 23 | 28o18’48.24” N,91o49’06.59” E | |
山南市错那县 | 沙棘根瘤 | P4 | 4 255 | 2019. 09. 23 | 28o15’18.76” N,91o45’47.52” E |
根际土壤 | E4 | 4 255 | 2019. 09. 23 | 28o15’18.76” N,91o45’47.52” E | |
山南市隆子县 | 沙棘根瘤 | P5 | 3 915 | 2019. 09. 23 | 28o25’12.01” N,92o23’48.99” E |
根际土壤 | E5 | 3 915 | 2019. 09. 23 | 28o25’12.01” N,92o23’48.99” 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,CaCO3 3 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,CaCO3 2 g,(NH4)2SO4 0.33 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)2SO4 7 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,CaCO3 2 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,NaNO3 0.3 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,CaCO3 4 g,pH 7.2 |
FM11 | 葡萄糖20 g,麦芽浸粉40 g,酵母浸粉4 g,K2HPO4 5 g,NaCl 2.5 g,CaCO3 0.4 g,ZnSO4 0.04 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 |
表2 12种发酵培养基种类
Table 2 12 kinds of fermentation medium
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,CaCO3 3 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,CaCO3 2 g,(NH4)2SO4 0.33 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)2SO4 7 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,CaCO3 2 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,NaNO3 0.3 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,CaCO3 4 g,pH 7.2 |
FM11 | 葡萄糖20 g,麦芽浸粉40 g,酵母浸粉4 g,K2HPO4 5 g,NaCl 2.5 g,CaCO3 0.4 g,ZnSO4 0.04 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 |
图1 基于16S rRNA各属代表菌株基因序列构建的菌株N-J系统进化树 分支点上的数值为1 000次自展值分析所得,标尺0.020进化距离
Fig. 1 Phylogenetic tree of strain N-J based on gene sequences of representative strains of 16S rRNA genera The value at the bifurcation point is obtained by 1 000 times of self-xpanding analysis. Scale 0.020 evolution distance
图4 46株代表性菌株发酵产物抗菌活性结果 A:金黄色葡萄球菌(ATCC 29213);B:耐甲氧西林金黄色葡萄球菌(MRSA)181223;C:铜绿假单胞菌(ATCC 27853);D:大肠埃希菌(ATCC 25922);E:肺炎克雷伯菌(ATCC 700603);F:耐碳青霉烯鲍曼不动杆菌(CRAB)181236;G:白色念珠菌(ATCC 10231)。热图(Heatmap):在7种检定菌96孔菌液板中,每孔分别加入在12种培养基发酵后的菌液,生长24 h的抑菌效果。图中绿色表示抗菌活性较好,红色表示几乎没有抗菌活性,由红色向绿色过渡为浅紫绿色,表示发酵液对检定菌活性较小或没有活性。1-12的培养基分别为PDB、ISP 2、FM3、FM4、FM5、M331、F1、M2、Cazpeck、FM10、FM11、YMS
Fig. 4 Antibacterial activities of fermentation products of 46 representative strains A:Staphylococcus aureus(ATCC 29213). B:Methicillin resistant S. aureus(MRSA)181223. C:Pseudomonas aeruginosa(ATCC 27853). D:Escherichia coli(ATCC 25922). E:Klebsiella pneumoniae(ATCC 700603). F:Carbapenem resistant Acinetobacter baumannii(crab)181236. G:Candida albicans(ATCC 10231). Heatmap:In the 96 well plates of 7 kinds of bacteria,the bacteria liquid fermented in 12 kinds of media was added to each well,and the growth time was 24 h. In the figure,green indicates good antibacterial activity,red indicates almost no antibacterial activity,and the transition from red to green to light purple green indicates that the fermentation broth has little or no antibacterial activity against the tested bacteria. The medium of 1-12 is PDB,ISP 2,FM3,FM4,FM5,M331,F1,M2,Cazpeck,FM10,FM11,and YMS
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 | ||||||
---|---|---|---|---|---|---|---|
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% |
表3 部分菌株发酵提取物对检定菌抑制率
Table 3 Inhibition rate of fermentation extracts of some strains to the tested strains
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 | ||||||
---|---|---|---|---|---|---|---|
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% |
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