锡林郭勒牧场人畜肠道微生物及土壤微生物多样性的比较分析
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摘要
以内蒙古锡林郭勒镶黄旗呼日敦高勒嘎查牧场内牛、山羊、绵羊、马、人的粪便和土壤为研究对象,利用Pac Bio SMRT测序平台对细菌16S rRNA基因全长进行测序,运用生物信息学分析方法对人畜肠道微生物及土壤微生物多样性进行全面评估.结果发现,人畜肠道菌群均以厚壁菌门和拟杆菌门为优势菌门,而土壤以酸杆菌门和变形菌门为优势菌门.在属水平,人畜以梭菌属、拟杆菌属为优势菌属,而土壤以Blastocatella和芽孢杆菌属为优势菌属.在种水平人以Escherichia/Shigella dysenteriae和Streptococcus salivarius为优势菌种,而4种家畜的优势菌种均为Oscillibacter valericigenes和Eubacterium coprostanoligenes.土壤以Blastocatella fastidiosa和Bacillus longiquaesitum为优势菌种.比较各样品α多样性发现,人肠道微生物的多样性显著低于其他食草动物.通过主坐标和层次聚类分析发现可将6种样品分为:组1(人)、组2(马)、组3(绵羊、牛、山羊)和组4(土壤)4组.本研究对差异OTU进行比对分析探究了造成该分组的原因.因此本研究揭示了土壤、人和家畜肠道微生物组成和结构存在显著的差异,为进一步研究不同生境中微生物多样性提供数据参考.
As an important part of ecosystem, microbes are widely distributed in various habitats. In recent years, more and more attention has been paid to the study on gut microbiota. The gut microbiota and their metabolites influence human and animal nutrition processing, metabolic balance, immune function, gastrointestinal development and other physiological activities. With the deepening of studies on human and animal gut microbiota, it has been found that some factors, such as diet, age, gender, and living environment, impacting on the composition of gut microbiota, while the differences among animal species have more significant influence on the gut microbiota composition. In relatively primitive grassland ecosystems, soil microbes interact with human and animal activities. On the one hand, microbes in the soil environment are the driving forces for the transformation and circulation of organic matter and nutrients. The improvement of soil microbial community diversity is beneficial for the soil fertility. On the other hand, human and animal activities will affect the diversity of soil microbial community. Although there are a lot of researches on soil microbiota, animal and human gut microbiota, their differences in diversity and composition within the same environment have not been studied. The advent of sequencing technology provides an effective mean for the accurate and comprehensive understanding of microbes, especially for the study of uncultivable microorganisms. The PacBio single-molecule real-time(SMRT) technology is advantageous in producing long sequence reads with high accuracy. Based on sequencing the full length 16 S rRNA genes, the microbiota composition can be identified to the species level. Therefore, it is an effective approach for studying microbial diversity. We collected 56 stool and soil samples from Xilinguole, including 6, 10, 11, 9, and 10 stool samples from human, goat, cattle, horse, and sheep, respectively, as well as 10 soil samples. Genomic DNA was extracted from the samples. After DNA extraction and quality check, the 16 S rRNA genes of all samples were amplified from the genomic DNA. The PCR products were sequenced using the PacBio RS II instrument. The QIIME software(V1.7) was used to analyze the sequencing data, and the R software(version 3.5.0) was used to further analyze and visualize the results. Firstly, it was found that the gut microbiota diversity of human was significantly lower than other samples(P<0.01). The overall composition of the human and animal gut microbiota were dominated by the Firmicutes and Bacteroidetes phyla. However, the soil microbiota was dominated by Proteobacteria and Acidobacteria. At the genus level, the sheep, goat and cattle gut microbiota were dominated by Clostridium, Bacteroides, and Oscillibacter, while the horse gut microbiota was mainly composed of Clostridium, Eubacterium, and Treponema. The soil microbiota was composed mainly of Blastocatella and Bacillus. The human gut microbiota comprised much of Veillonella, Clostridium, Escherichia/Shigella. At the species level, the human gut microbiota mainly contained Escherichia/Shigella, dysenteriae, Streptococcus salivarius. The sheep, goat, cattle, and horse gut microbiota were dominated by Oscillibacter valericigenes and Eubacterium coprostanoligenes. The major species in soil were Blastocatella fastidiosa and Bacillus longiquaesitum. Moreover, principal coordinate analysis(PCoA) and hierarchical clustering analysis showed some differences in the microbiota structure among human gut, animal gut and soil samples. The gut microbiota structure was similar among cattle, goats and sheep. They were more different from the samples collected from human, horse and soil. We classified all samples into four clusters. Cluster 1 only included human samples; cluster 2 comprised the horse samples; cluster 3 was consisted of the sheep, goat, and cattle samples; while cluster 4 contained only the soil samples. Lastly, we identified the discriminatory OTUs and assigned them taxonomically to the species level. In conclusion, there were significant differences between animal gut microbiota and soil microbiota. The soil microbiota was more complex. As an omnivore, human gut microbiota diversity was significantly lower than other herbivorous animal(namely cattle, goat, horse and sheep). Although cattle, sheep, goat and horses are all herbivorous animals, the distinct features of the digestive systems could contribute to the difference in gut microbiota composition of horse from those of sheep, goat and cattle. This study revealed the differences of gut microbiota diversity between human and other animals, as well as from the soil microbial community. This work has laid a theoretical foundation for further studies on microbial diversity in different habitats.
As an important part of ecosystem, microbes are widely distributed in various habitats. In recent years, more and more attention has been paid to the study on gut microbiota. The gut microbiota and their metabolites influence human and animal nutrition processing, metabolic balance, immune function, gastrointestinal development and other physiological activities. With the deepening of studies on human and animal gut microbiota, it has been found that some factors, such as diet, age, gender, and living environment, impacting on the composition of gut microbiota, while the differences among animal species have more significant influence on the gut microbiota composition. In relatively primitive grassland ecosystems, soil microbes interact with human and animal activities. On the one hand, microbes in the soil environment are the driving forces for the transformation and circulation of organic matter and nutrients. The improvement of soil microbial community diversity is beneficial for the soil fertility. On the other hand, human and animal activities will affect the diversity of soil microbial community. Although there are a lot of researches on soil microbiota, animal and human gut microbiota, their differences in diversity and composition within the same environment have not been studied. The advent of sequencing technology provides an effective mean for the accurate and comprehensive understanding of microbes, especially for the study of uncultivable microorganisms. The PacBio single-molecule real-time(SMRT) technology is advantageous in producing long sequence reads with high accuracy. Based on sequencing the full length 16 S rRNA genes, the microbiota composition can be identified to the species level. Therefore, it is an effective approach for studying microbial diversity. We collected 56 stool and soil samples from Xilinguole, including 6, 10, 11, 9, and 10 stool samples from human, goat, cattle, horse, and sheep, respectively, as well as 10 soil samples. Genomic DNA was extracted from the samples. After DNA extraction and quality check, the 16 S rRNA genes of all samples were amplified from the genomic DNA. The PCR products were sequenced using the PacBio RS II instrument. The QIIME software(V1.7) was used to analyze the sequencing data, and the R software(version 3.5.0) was used to further analyze and visualize the results. Firstly, it was found that the gut microbiota diversity of human was significantly lower than other samples(P<0.01). The overall composition of the human and animal gut microbiota were dominated by the Firmicutes and Bacteroidetes phyla. However, the soil microbiota was dominated by Proteobacteria and Acidobacteria. At the genus level, the sheep, goat and cattle gut microbiota were dominated by Clostridium, Bacteroides, and Oscillibacter, while the horse gut microbiota was mainly composed of Clostridium, Eubacterium, and Treponema. The soil microbiota was composed mainly of Blastocatella and Bacillus. The human gut microbiota comprised much of Veillonella, Clostridium, Escherichia/Shigella. At the species level, the human gut microbiota mainly contained Escherichia/Shigella, dysenteriae, Streptococcus salivarius. The sheep, goat, cattle, and horse gut microbiota were dominated by Oscillibacter valericigenes and Eubacterium coprostanoligenes. The major species in soil were Blastocatella fastidiosa and Bacillus longiquaesitum. Moreover, principal coordinate analysis(PCoA) and hierarchical clustering analysis showed some differences in the microbiota structure among human gut, animal gut and soil samples. The gut microbiota structure was similar among cattle, goats and sheep. They were more different from the samples collected from human, horse and soil. We classified all samples into four clusters. Cluster 1 only included human samples; cluster 2 comprised the horse samples; cluster 3 was consisted of the sheep, goat, and cattle samples; while cluster 4 contained only the soil samples. Lastly, we identified the discriminatory OTUs and assigned them taxonomically to the species level. In conclusion, there were significant differences between animal gut microbiota and soil microbiota. The soil microbiota was more complex. As an omnivore, human gut microbiota diversity was significantly lower than other herbivorous animal(namely cattle, goat, horse and sheep). Although cattle, sheep, goat and horses are all herbivorous animals, the distinct features of the digestive systems could contribute to the difference in gut microbiota composition of horse from those of sheep, goat and cattle. This study revealed the differences of gut microbiota diversity between human and other animals, as well as from the soil microbial community. This work has laid a theoretical foundation for further studies on microbial diversity in different habitats.
引文
1 Greiner T U,B?ckhed F.Microbial regulation of GLP-1 and L-cell biology.Mol Metab,2016,5:753-758
2 Rooks M G,Garrett W S.Gut microbiota,metabolites and host immunity.Nat Rev Immunol,2016,16:341
3 Liu W,Yao W,Zhu W Y.Advance on mutualism of host-microbiota in human gut(in Chinese).World Chin J Digestol,2006,14:1081-1088[刘威,姚文,朱伟云.人体与肠道微生物间的互惠共生关系.世界华人消化杂志,2006,14:1081-1088]
4 Chen W.Gut microbiota-A new concern on health regulation(in Chinese).J Food Sci Technol,2015,33:1-6[陈卫.肠道菌群:膳食与健康研究的新视角.食品科学技术学报,2015,33:1-6]
5 Xu J.Comparative study on intestinal microflora of healthy people in different ages in Sichuan Province(in Chinese).Master Dissertation.Huhhot:Inner Mongolia Agricultural University,2012[徐洁.四川地区不同年龄健康人肠道菌群的比较研究.硕士学位论文.呼和浩特:内蒙古农业大学,2012]
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7 Shin J H,Sim M,Lee J Y,et al.Lifestyle and geographic insights into the distinct gut microbiota in elderly women from two different geographic locations.J Physiol Anthropol,2016,35:31
8 Deng G H,Zha L Y,Zhang G X,et al.Comparison of human and animal fecal microbiota with Illumina sequencing of 16S rRNA tags(in Chinese).Ecol Sci,2014,33:851-857[邓冠华,查龙应,张国霞,等.高通量16S rRNA标签测序法比较人与不同动物肠道微生物组多样性.生态科学,2014,33:851-857]
9 Chen X,Zhang X J,Li Q Y,et al.Diversity of faecal bacteria in several mammals by 454 high-throughput sequencing(in Chinese).Acta Microbiol Sin,2015,55:683-690[陈秀,张晓君,李沁元,等.454焦磷酸测序分析几种哺乳动物粪便细菌多样性.微生物学报,2015,55:683-690]
10 Luo Y J,Zhong Z J,Peng G N,et al.Characterization of intestind bacterial diversity for different feeding habit animals by ERIC-PCRfingerprinting(in Chinese).Chin J Veterin Sci,2014,34:1764-1769[罗永久,钟志军,彭广能,等.不同食性动物肠道菌群的ERIC-PCR指纹图谱分析.中国兽医学报,2014,34:1764-1769]
11 Rehman A,Rausch P,Wang J,et al.Geographical patterns of the standing and active human gut microbiome in health and IBD.Gut,2015,65:238
12 Kaplan L M,Brancale J.Eat well,or get roommates who do.Cell Host Microbe,2017,21:123
13 Yang H J,Xiao Q M,Liu A Y.Soil microbial diversity and its action(in Chinese).J Nanhua Univ(Sci Technol),2005,19:21-26[杨海君,肖启明,刘安元.土壤微生物多样性及其作用研究进展.南华大学学报:自然科学版,2005,19:21-26]
14 Torsvik V,Ovreas L.Microbial diversity and function in soil:From genes to ecosystems.Curr Opin Microbiol,2002,5:240-245
15 Wang H,Cheng M,Dsouza M,et al.Soil bacterial diversity is associated with human population density in urban greenspaces.Environ Sci Technol,2018,52:5115-5124
16 Braslavsky I,Hebert B,Kartalov E,et al.Sequence information can be obtained from single DNA molecules.Proc Natl Acad Sci USA,2003,100:3960-3964
17 Tang Y,Liu X.Full-length sequencing of 16S rRNA gene and its analysis based on the SMRT sequencing technology(in Chinese).Biotechnol Bull,2017,33:34-39[唐勇,刘旭.基于SMRT测序技术的16S rRNA基因全长测序及其分析方法.生物技术通报,2017,33:34-39]
18 Ley R E,Hamady M,Lozupone C,et al.Evolution of mammals and their gut microbes.Science,2008,320:1647-1651
19 Xue Z Y.Diversity profile and phylo-functional core of the mammalian gut microbiota-with healthy human and giant panda as models(in Chinese).Doctor Dissertation.Shanghai:Shanghai Jiao Tong University,2015[薛正晟.以健康人和大熊猫为模型的哺乳动物肠道菌群多样性分布与核心功能种属的研究.博士学位论文.上海:上海交通大学,2015]
20 Petri R M,Pourazad P,Khiaosa-Ard R,et al.Temporal dynamics of in-situ fiber-adherent bacterial community under ruminal acidotic conditions determined by 16S rRNA gene profiling.PLoS One,2017,12:e0182271
21 Wang G H,Liu J J,Yu Z H,et al.Research progress of Acidobacteria ecology in soils(in Chinese).Biotechnol Bull,2016,32:14-20[王光华,刘俊杰,于镇华,等.土壤酸杆菌门细菌生态学研究进展.生物技术通报,2016,32:14-20]
22 Zeng Q C.The characteristics of soil properties and microbial under different vegetation ecozones on the Loess Plateau of China(in Chinese).Master Dissertation.Beijing:University of Chinese Academy of Sciences,2015[曾全超.黄土高原不同植被生态系统土壤微生物多样性及其影响因素研究.硕士学位论文.北京:中国科学院研究生院,2015]
23 Pryde S E,Duncan S H,Hold G L,et al.The microbiology of butyrate formation in the human colon.Fems Microbiol Lett,2002,217:133-139
24 Dumas M E,Barton R H,Toye A,et al.Metabolic profiling reveals a contribution of gut microbiota to fatty liver phenotype in insulinresistant mice.Proc Natl Acad Sci USA,2006,103:12511
25 Kniehl E,Becker A,Forster D H.Pseudo-outbreak of toxigenic Bacillus cereus isolated from stools of three patients with diarrhoea after oral administration of a probiotic medication.J Hosp Infect,2003,55:33-38
26 Wang J,Zhang H,Chen X,et al.Selection of potential probiotic lactobacilli for cholesterol-lowering properties and their effect on cholesterol metabolism in rats fed a high-lipid diet.J Dairy Sci,2012,95:1645-1654
27 Pitta D W,Indugu N,Baker L,et al.Symposium review:Understanding diet-microbe interactions to enhance productivity of dairy cows.J Dairy Sci,2018,101:1-19
28 Venkataraman A,Sieber J R,Schmidt A W,et al.Variable responses of human microbiomes to dietary supplementation with resistant starch.Microbiome,2016,4:33
29 Sakamoto M,Takagaki A,Matsumoto K,et al.Butyricimonas synergistica gen.nov.,sp.nov.and Butyricimonas virosa sp.nov.,butyric acid-producing bacteria in the family‘Porphyromonadaceae’isolated from rat faeces.Int J Syst Evol Micr,2009,59:1748
30 Zhang Y J,Li S,Gan R Y,et al.Impacts of gut bacteria on human health and diseases.Int J Mol Sci,2015,16:7493
2 Rooks M G,Garrett W S.Gut microbiota,metabolites and host immunity.Nat Rev Immunol,2016,16:341
3 Liu W,Yao W,Zhu W Y.Advance on mutualism of host-microbiota in human gut(in Chinese).World Chin J Digestol,2006,14:1081-1088[刘威,姚文,朱伟云.人体与肠道微生物间的互惠共生关系.世界华人消化杂志,2006,14:1081-1088]
4 Chen W.Gut microbiota-A new concern on health regulation(in Chinese).J Food Sci Technol,2015,33:1-6[陈卫.肠道菌群:膳食与健康研究的新视角.食品科学技术学报,2015,33:1-6]
5 Xu J.Comparative study on intestinal microflora of healthy people in different ages in Sichuan Province(in Chinese).Master Dissertation.Huhhot:Inner Mongolia Agricultural University,2012[徐洁.四川地区不同年龄健康人肠道菌群的比较研究.硕士学位论文.呼和浩特:内蒙古农业大学,2012]
6 Suzuki Y,Ikeda K,Sakuma K,et al.Association between yogurt consumption and intestinal microbiota in healthy young adults differs by host gender.Front Microbiol,2017,8:847
7 Shin J H,Sim M,Lee J Y,et al.Lifestyle and geographic insights into the distinct gut microbiota in elderly women from two different geographic locations.J Physiol Anthropol,2016,35:31
8 Deng G H,Zha L Y,Zhang G X,et al.Comparison of human and animal fecal microbiota with Illumina sequencing of 16S rRNA tags(in Chinese).Ecol Sci,2014,33:851-857[邓冠华,查龙应,张国霞,等.高通量16S rRNA标签测序法比较人与不同动物肠道微生物组多样性.生态科学,2014,33:851-857]
9 Chen X,Zhang X J,Li Q Y,et al.Diversity of faecal bacteria in several mammals by 454 high-throughput sequencing(in Chinese).Acta Microbiol Sin,2015,55:683-690[陈秀,张晓君,李沁元,等.454焦磷酸测序分析几种哺乳动物粪便细菌多样性.微生物学报,2015,55:683-690]
10 Luo Y J,Zhong Z J,Peng G N,et al.Characterization of intestind bacterial diversity for different feeding habit animals by ERIC-PCRfingerprinting(in Chinese).Chin J Veterin Sci,2014,34:1764-1769[罗永久,钟志军,彭广能,等.不同食性动物肠道菌群的ERIC-PCR指纹图谱分析.中国兽医学报,2014,34:1764-1769]
11 Rehman A,Rausch P,Wang J,et al.Geographical patterns of the standing and active human gut microbiome in health and IBD.Gut,2015,65:238
12 Kaplan L M,Brancale J.Eat well,or get roommates who do.Cell Host Microbe,2017,21:123
13 Yang H J,Xiao Q M,Liu A Y.Soil microbial diversity and its action(in Chinese).J Nanhua Univ(Sci Technol),2005,19:21-26[杨海君,肖启明,刘安元.土壤微生物多样性及其作用研究进展.南华大学学报:自然科学版,2005,19:21-26]
14 Torsvik V,Ovreas L.Microbial diversity and function in soil:From genes to ecosystems.Curr Opin Microbiol,2002,5:240-245
15 Wang H,Cheng M,Dsouza M,et al.Soil bacterial diversity is associated with human population density in urban greenspaces.Environ Sci Technol,2018,52:5115-5124
16 Braslavsky I,Hebert B,Kartalov E,et al.Sequence information can be obtained from single DNA molecules.Proc Natl Acad Sci USA,2003,100:3960-3964
17 Tang Y,Liu X.Full-length sequencing of 16S rRNA gene and its analysis based on the SMRT sequencing technology(in Chinese).Biotechnol Bull,2017,33:34-39[唐勇,刘旭.基于SMRT测序技术的16S rRNA基因全长测序及其分析方法.生物技术通报,2017,33:34-39]
18 Ley R E,Hamady M,Lozupone C,et al.Evolution of mammals and their gut microbes.Science,2008,320:1647-1651
19 Xue Z Y.Diversity profile and phylo-functional core of the mammalian gut microbiota-with healthy human and giant panda as models(in Chinese).Doctor Dissertation.Shanghai:Shanghai Jiao Tong University,2015[薛正晟.以健康人和大熊猫为模型的哺乳动物肠道菌群多样性分布与核心功能种属的研究.博士学位论文.上海:上海交通大学,2015]
20 Petri R M,Pourazad P,Khiaosa-Ard R,et al.Temporal dynamics of in-situ fiber-adherent bacterial community under ruminal acidotic conditions determined by 16S rRNA gene profiling.PLoS One,2017,12:e0182271
21 Wang G H,Liu J J,Yu Z H,et al.Research progress of Acidobacteria ecology in soils(in Chinese).Biotechnol Bull,2016,32:14-20[王光华,刘俊杰,于镇华,等.土壤酸杆菌门细菌生态学研究进展.生物技术通报,2016,32:14-20]
22 Zeng Q C.The characteristics of soil properties and microbial under different vegetation ecozones on the Loess Plateau of China(in Chinese).Master Dissertation.Beijing:University of Chinese Academy of Sciences,2015[曾全超.黄土高原不同植被生态系统土壤微生物多样性及其影响因素研究.硕士学位论文.北京:中国科学院研究生院,2015]
23 Pryde S E,Duncan S H,Hold G L,et al.The microbiology of butyrate formation in the human colon.Fems Microbiol Lett,2002,217:133-139
24 Dumas M E,Barton R H,Toye A,et al.Metabolic profiling reveals a contribution of gut microbiota to fatty liver phenotype in insulinresistant mice.Proc Natl Acad Sci USA,2006,103:12511
25 Kniehl E,Becker A,Forster D H.Pseudo-outbreak of toxigenic Bacillus cereus isolated from stools of three patients with diarrhoea after oral administration of a probiotic medication.J Hosp Infect,2003,55:33-38
26 Wang J,Zhang H,Chen X,et al.Selection of potential probiotic lactobacilli for cholesterol-lowering properties and their effect on cholesterol metabolism in rats fed a high-lipid diet.J Dairy Sci,2012,95:1645-1654
27 Pitta D W,Indugu N,Baker L,et al.Symposium review:Understanding diet-microbe interactions to enhance productivity of dairy cows.J Dairy Sci,2018,101:1-19
28 Venkataraman A,Sieber J R,Schmidt A W,et al.Variable responses of human microbiomes to dietary supplementation with resistant starch.Microbiome,2016,4:33
29 Sakamoto M,Takagaki A,Matsumoto K,et al.Butyricimonas synergistica gen.nov.,sp.nov.and Butyricimonas virosa sp.nov.,butyric acid-producing bacteria in the family‘Porphyromonadaceae’isolated from rat faeces.Int J Syst Evol Micr,2009,59:1748
30 Zhang Y J,Li S,Gan R Y,et al.Impacts of gut bacteria on human health and diseases.Int J Mol Sci,2015,16:7493