屎肠球菌对哺乳期仔猪结肠微生物群落优势门属的影响
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摘要
【目的】分析屎肠球菌(Enterococcus faecium)活菌对哺乳期仔猪结肠微生物群落优势门属的持续影响,为揭示益生菌调节仔猪肠道健康的作用机制及人为调控仔猪肠道微生物以促进其健康提供参考依据。【方法】给健康新生仔猪灌服无菌脱脂乳(对照组)或屎肠球菌制剂(益生菌组),持续灌服6 d,分别采集7和21日龄仔猪结肠内容物,提取总细菌DNA后进行PCR扩增,采用Illumina MiSeq高通量测序技术检测16S rRNA序列的V3～V4可变区,分析肠道微生物群落α多样性及其在门和属分类水平上的结构特点。【结果】给新生仔猪灌服屎肠球菌并不影响7和21日龄仔猪结肠微生物群落α多样性。与对照组相比,益生菌组7日龄仔猪结肠微生物群落优势门的相对丰度无显著变化(P>0.05,下同),21日龄仔猪拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes)的相对丰度也无显著差异,但变形杆菌门(Proteobacteria)相对丰度呈显著增加趋势(P<0.05,下同);在属水平上,益生菌组7日龄仔猪结肠微生物群落中相对丰度变化明显的优势属均隶属于拟杆菌门,其中,普雷沃氏菌属(Prevotella)相对丰度显著增加,而拟杆菌属(Bacteroides)相对丰度极显著减少(P<0.01,下同),丁酸单胞菌属(Butyricimonas)相对丰度显著减少;21日龄仔猪结肠微生物群落中66.67%优势属的相对丰度呈减少趋势,其中以厚壁菌门4个属的变化尤其明显,即巨型球菌属(Megasphaera)相对丰度极显著减少,乳杆菌属(Lactobacillus)、产琥珀酸菌属(Succiniclasticum)和粪杆菌属(Faecalibacterium)相对丰度显著减少。【结论】新生仔猪口服屎肠球菌能对其结肠微生物群落优势门或属组成产生持续影响,表现为促进有益于结肠健康菌属的生长,而抑制具有潜在致病性菌属的增殖,为今后定向研究调控仔猪肠道健康的肠道共生微生物提供明确范围。
【Objective】The purpose of the study was to analyze the continuous influence of viable Enterococcus faecium on dominant phyla and genera of colonic microbial communities of suckling piglets,in order to provide scientific reference for fully understanding the mechanism of probiotics regulating intestinal health of piglets,and also regulating intestinal microorganism of piglets artificially to promote their health.【Method】Healthy newborn piglets were fed with sterile skim milk(as the control group),or E. faecium preparation(as the probiotic group)for continuously 6 d. Colonic contents of piglets aged 7 and 21 days were collected. Total DNA extraction and PCR amplification were performed successively. Illumina MiSeq high-throughput sequencing technology was utilized to detect the V3-V4 variable regions of 16 S rRNA sequence. Finally,alpha diversity of intestinal microbial community and structural characteristics at phylum and genus level were analyzed.【Result】The results showed that no effect of E. faecium on alpha diversity of colonic microbial communities in 7-and 21-day-old piglets was observed. Compared with the control group,there was no significant change in relative abundance of the dominant phyla in colonic microbial communities of 7-day-old piglets in the probiotics group(P>0.05,the same below). The relative abundance of Bacteroidetes and Firmicutes in 21-day-old piglets showed no significantly difference either,but the relative abundance of Proteobacteria was significantly increased(P<0.05,the same below). At the level of genus,the dominant genera whose relative abundance changed obviously in the colonic microbial communities in probiotics group of 7-day-old piglets were all belonging to phylum Bacteroidetes. Of which,the relative abundance of genus Prevotella increased remarkably. In contrast,the relative abundant of Bacteroides decreased extremely(P<0.01,the same below),and Butyricimonas reduced significantly. The relative abundance of 66.67% dominant genera in the colonic microbial communities of 21-day-old piglets showed a decreasing trend,especially in the four genera of phylum Firmicutes. The abundance of genus Megasphaera downregulated extremely,and Lactobacillus,Succiniclasticum and Faecalibacterium declined significantly.【Conclusion】Newborn piglets oral ingestion of viable probiotic E. faecium has a sustained impact on the composition at the genus or phylum level of colonic microbial community,which is demonstrated by promoting the growth of bacteria beneficial to colon health and simultaneously inhibiting the proliferation of potentially pathogenic bacteria. These findings provide a clear target for future studies on intestinal symbiotic microorganisms modulating intestinal health of piglets.
【Objective】The purpose of the study was to analyze the continuous influence of viable Enterococcus faecium on dominant phyla and genera of colonic microbial communities of suckling piglets,in order to provide scientific reference for fully understanding the mechanism of probiotics regulating intestinal health of piglets,and also regulating intestinal microorganism of piglets artificially to promote their health.【Method】Healthy newborn piglets were fed with sterile skim milk(as the control group),or E. faecium preparation(as the probiotic group)for continuously 6 d. Colonic contents of piglets aged 7 and 21 days were collected. Total DNA extraction and PCR amplification were performed successively. Illumina MiSeq high-throughput sequencing technology was utilized to detect the V3-V4 variable regions of 16 S rRNA sequence. Finally,alpha diversity of intestinal microbial community and structural characteristics at phylum and genus level were analyzed.【Result】The results showed that no effect of E. faecium on alpha diversity of colonic microbial communities in 7-and 21-day-old piglets was observed. Compared with the control group,there was no significant change in relative abundance of the dominant phyla in colonic microbial communities of 7-day-old piglets in the probiotics group(P>0.05,the same below). The relative abundance of Bacteroidetes and Firmicutes in 21-day-old piglets showed no significantly difference either,but the relative abundance of Proteobacteria was significantly increased(P<0.05,the same below). At the level of genus,the dominant genera whose relative abundance changed obviously in the colonic microbial communities in probiotics group of 7-day-old piglets were all belonging to phylum Bacteroidetes. Of which,the relative abundance of genus Prevotella increased remarkably. In contrast,the relative abundant of Bacteroides decreased extremely(P<0.01,the same below),and Butyricimonas reduced significantly. The relative abundance of 66.67% dominant genera in the colonic microbial communities of 21-day-old piglets showed a decreasing trend,especially in the four genera of phylum Firmicutes. The abundance of genus Megasphaera downregulated extremely,and Lactobacillus,Succiniclasticum and Faecalibacterium declined significantly.【Conclusion】Newborn piglets oral ingestion of viable probiotic E. faecium has a sustained impact on the composition at the genus or phylum level of colonic microbial community,which is demonstrated by promoting the growth of bacteria beneficial to colon health and simultaneously inhibiting the proliferation of potentially pathogenic bacteria. These findings provide a clear target for future studies on intestinal symbiotic microorganisms modulating intestinal health of piglets.
引文
陈曦,李英英,宋铁英.2016.单一益生菌(屎肠球菌)介导的断奶仔猪肠道菌群变化规律[J].福建农业学报,31(10):1091-1097.[Chen X,Li Y Y,Song T Y.2016.Changes induced by probiotic,Endterococcus faecium,in gut microbiota of post-weaning piglet[J].Fujian Journal of A-gricultural Sciences,31(10):1091-1097.]
方磊涵,毕玉霞,王振,贾悟松.2017.绞股蓝多糖对断奶仔猪肠道微生物区系的影响[J].河南农业科学,46(12):122-125.[Fang L H,Bi Y X,Wang Z,Jia W S.2017.Effects of gynostemma pentaphyllum polysaccharides on intestinal microflora in weaned piglet[J].Journal of Henan A-gricultural Sciences,46(12):122-125.]
黄怡,郭乾鹏,梁世忠,秦津,李卫芬.2016.屎肠球菌对仔猪肠道健康的影响[J].动物营养学报,28(4):968-973.[Huang Y,Guo Q P,Liang S Z,Qin J,Li W F.2016.Influence of Enterococcus faecium on gut health of piglets[J].Chinese Journal of Animal Nutrition,28(4):968-973.]
黄怡,黄琴,崔志文,李卫芬,余东游.2012.新生仔猪口服屎肠球菌对其肠道菌群组成及化学屏障的影响[J].中国兽医学报,32(7):1007-1010.[Huang Y,Huang Q,Cui ZW,Li W F,Yu D Y.2012.Roles of Enterococcus faecium in composition of the intestinal microflora and chemistry barrier in newborn piglets by oral administration[J].Chinese Journal of Veterinary Science,32(7):1007-1010.]
李媛媛,郭乾鹏,梁世忠,向明,黄怡.2018.热灭活屎肠球菌及其活菌对仔猪腹泻率、消化酶活性和脏器指数的影响[J].黑龙江畜牧兽医,(15):1-5.[Li Y Y,Guo Q P,Liang S Z,Xiang M,Huang Y.2018.Effect of heatkilled Enterococcus faecium and its viable bacteria on diarrhea rate,digestive enzyme activity and visceral index in piglets[J].Heilongjiang Animal Science and Veterinary Medicine,(15):1-5.]
茹文红,段亚飞,郑晓婷,董宏标,刘青松,张家松.2018.室内培育不同产地卤虫的成虫肠道菌群结构分析[J].南方农业学报,49(9):1841-1848.[Ru W H,Duan Y F,Zheng X T,Dong H B,Liu Q S,Zhang J S.2018.Structure of intestinal tract bacterial communities of adult Artemia parthenogenetica from different producing areas under indoor culture[J].Journal of Southern Agriculture,49(9):1841-1848.]
佟延南,李芳远,李忠琴,赵光军,李高俊,王德强.2018.不同养殖阶段罗非鱼肠道微生物多样性的动态分析[J].南方农业学报,49(7):1415-1422.[Tong Y N,Li F Y,Li ZQ,Zhao G J,Li G J,Wang D Q.2018.Dynamic analysis of intestinal microbial diversity in tilapia at different culture stages[J].Journal of Southern Agriculture,49(7):1415-1422.]
夏围围,贾仲君.2014.高通量测序和DGGE分析土壤微生物群落的技术评价[J].微生物学报,54(12):1489-1499.[Xia W W,Jia Z J.2014.Comparative analysis of soil microbial communities by pyrosequencing and DGGE[J].Acta Microbiologica Sinica,54(12):1489-1499.]
曾燕,邓家波,牛李丽,蒲阳,李杨,勾堃,曾东,倪学勤.2018.采用实时荧光定量PCR法分析小熊猫胃肠道菌群[J].湖南农业大学学报(自然科学版),44(6):638-644.[Zeng Y,Deng J B,Niu L L,Pu Y,Li Y,Gou K,Zeng D,Ni XQ.2018.Analysis of the bacteria communities along the gastrointestinal tract of a red panda using real-time fluorescence quantitative PCR[J].Journal of Hunan Agricultural University(Natural Sciences),44(6):638-644.]
Aktas B,De Wolfe T J,Safdar N,Darien B J,Steele J L.2016.The impact of Lactobacillus casei on the composition of the cecal microbiota and innate immune system is strain specific[J].PLoS One,11(5):e0156374.
Alain B P E,Chae J P,Balolong M P,Bum K H,Kang D K.2014.Assessment of fecal bacterial diversity among healthy piglets during the weaning transition[J].Journal of General and Applied Microbiology,60(4):140-146.
Boquet P,Munro P,Fiorentini C,Just I.1998.Toxins from anaerobic bacteria:Specificity and molecular mechanisms of action[J].Current Opinion in Microbiology,1(1):66-74.
Chiang M L,Chen H C,Chen K N,Lin Y C,Lin Y T,Chen M J.2015.Optimizing production of two potential probiotic Lactobacilli strains isolated from piglet feces as feed additives for weaned piglets[J].Asian-Australasian Journal of Animal Science,28(8):1163-1170.
De Filippo C,Cavalieri D,Di Paola M,Ramazzotti M,Poullet J B,Massart S,Collini S,Pieraccini G,Lionetti P.2010.Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa[J].Proceedings of the National Academy of Sciences of the United States of America,107(33):14691-14696.
Estrada A,Drew M D,van Kessel A G.2001.Effect of the dietary supplementation of fructo-oligosaccharides and Bifidobacteria longum to early weaned pigs on performance and fecal bacterial populations[J].The Canadian Veterinary Journal,81(1):141-148.
Eyeraert N,van Cruchten S,Westr?m B,Bailey M,van Ginneken C,Thymann T,Pieper R.2017.A review on early gut maturation and colonization in pigs,including biological and dietary factors affecting gut homeostasis[J].Animal Feed Science and Technology,233:89-103.
Gill S R,Pop M,DeBoy R T,Eckburg P B,Turnbaugh P J,Samuel B S,Gordon J I,Relman D A,Fraser-Liggett CM,Nelson K E.2006.Metagenomic analysis of the human distal gut microbiome[J].Science,312(5778):1355-1359.
Gorvitovskaia A,Holmes S P,Huse S M.2016.Interpreting prevotella and Bacteroides as biomarkers of diet and lifestyle[J].Microbiome,4:15.doi:10.1186/s40168-016-0160-7.
Herlemann D P R,Labrenz M,Jürgens K,Bertilsson S,Waniek J J,Andersson A F.2011.Transitions in bacterial communities along the 2000 km salinity gradient of the Baltic Sea[J].The ISME Journal,5(10):1571-1579.
Hooper L V,Gordon J I.2001.Commensal host-bacterial relationship[J].Science,292(5519):1115-1118.
Huang C H,Qiao S Y,Li D F,Piao X S,Ren J P.2004.Effects of Lactobacilli on the performance,diarrhea incidence,VFA concentration and gastrointestinal microbial flora of weaning pigs[J].Asian-Australasian Journal of Animal Science,17(3):401-409.
Kim H B,Borewicz K,White B A,Singer R S,Sreevatsan S,Tu Z J,Isaacson R E.2011.Longitudinal investigation of the age-related bacterial diversity in the feces of commercial pigs[J].Veterinary Microbiology,153(1-2):124-133.
Liu H,Zhang J,Zhang S H,Yang F J,Thacker P A,Zhang GL,Qiao S Y,Ma X.2014.Oral administration of Lactobacillus fermentum I5007 favors intestinal development and alters the intestinal microbiota in formula-fed piglets[J].Journal of Agricultural and Food Chemistry,62(4):860-866.
Mach N,Berri M,EstelléJ,Levenez F,Lemonnier G,Denis C,Leplat J,Chevaleyre C,Billon Y,DoréJ,Rogel-Gaillard C,Lepage P.2015.Early-life establishment of the swine gut microbiome and impact on host phenotypes[J].Environmental Microbiology Reports,7(3):554-569.
Pieper R,Janczyk P,Urubschurov V,Korn U,Pieper B,Souffrant W B.2009.Effect of a single oral administration of Lactobacillus plantarum DSMZ 8862/8866 before and at the time point of weaning on intestinal microbial communities in piglets[J].International Journal of Food Microbiology,130(3):227-232.
Reid G.2004.When microbe meets human[J].Clinical Infectious Diseases,39(6):827-830.
Ulger Toprak N,Bozan T,Birkan Y,Isbir S,Soyletir G.2015.Butyricimonas virosa:The first clinical case of bacteraemia[J].New Microbes and New Infections,4:7-8.
方磊涵,毕玉霞,王振,贾悟松.2017.绞股蓝多糖对断奶仔猪肠道微生物区系的影响[J].河南农业科学,46(12):122-125.[Fang L H,Bi Y X,Wang Z,Jia W S.2017.Effects of gynostemma pentaphyllum polysaccharides on intestinal microflora in weaned piglet[J].Journal of Henan A-gricultural Sciences,46(12):122-125.]
黄怡,郭乾鹏,梁世忠,秦津,李卫芬.2016.屎肠球菌对仔猪肠道健康的影响[J].动物营养学报,28(4):968-973.[Huang Y,Guo Q P,Liang S Z,Qin J,Li W F.2016.Influence of Enterococcus faecium on gut health of piglets[J].Chinese Journal of Animal Nutrition,28(4):968-973.]
黄怡,黄琴,崔志文,李卫芬,余东游.2012.新生仔猪口服屎肠球菌对其肠道菌群组成及化学屏障的影响[J].中国兽医学报,32(7):1007-1010.[Huang Y,Huang Q,Cui ZW,Li W F,Yu D Y.2012.Roles of Enterococcus faecium in composition of the intestinal microflora and chemistry barrier in newborn piglets by oral administration[J].Chinese Journal of Veterinary Science,32(7):1007-1010.]
李媛媛,郭乾鹏,梁世忠,向明,黄怡.2018.热灭活屎肠球菌及其活菌对仔猪腹泻率、消化酶活性和脏器指数的影响[J].黑龙江畜牧兽医,(15):1-5.[Li Y Y,Guo Q P,Liang S Z,Xiang M,Huang Y.2018.Effect of heatkilled Enterococcus faecium and its viable bacteria on diarrhea rate,digestive enzyme activity and visceral index in piglets[J].Heilongjiang Animal Science and Veterinary Medicine,(15):1-5.]
茹文红,段亚飞,郑晓婷,董宏标,刘青松,张家松.2018.室内培育不同产地卤虫的成虫肠道菌群结构分析[J].南方农业学报,49(9):1841-1848.[Ru W H,Duan Y F,Zheng X T,Dong H B,Liu Q S,Zhang J S.2018.Structure of intestinal tract bacterial communities of adult Artemia parthenogenetica from different producing areas under indoor culture[J].Journal of Southern Agriculture,49(9):1841-1848.]
佟延南,李芳远,李忠琴,赵光军,李高俊,王德强.2018.不同养殖阶段罗非鱼肠道微生物多样性的动态分析[J].南方农业学报,49(7):1415-1422.[Tong Y N,Li F Y,Li ZQ,Zhao G J,Li G J,Wang D Q.2018.Dynamic analysis of intestinal microbial diversity in tilapia at different culture stages[J].Journal of Southern Agriculture,49(7):1415-1422.]
夏围围,贾仲君.2014.高通量测序和DGGE分析土壤微生物群落的技术评价[J].微生物学报,54(12):1489-1499.[Xia W W,Jia Z J.2014.Comparative analysis of soil microbial communities by pyrosequencing and DGGE[J].Acta Microbiologica Sinica,54(12):1489-1499.]
曾燕,邓家波,牛李丽,蒲阳,李杨,勾堃,曾东,倪学勤.2018.采用实时荧光定量PCR法分析小熊猫胃肠道菌群[J].湖南农业大学学报(自然科学版),44(6):638-644.[Zeng Y,Deng J B,Niu L L,Pu Y,Li Y,Gou K,Zeng D,Ni XQ.2018.Analysis of the bacteria communities along the gastrointestinal tract of a red panda using real-time fluorescence quantitative PCR[J].Journal of Hunan Agricultural University(Natural Sciences),44(6):638-644.]
Aktas B,De Wolfe T J,Safdar N,Darien B J,Steele J L.2016.The impact of Lactobacillus casei on the composition of the cecal microbiota and innate immune system is strain specific[J].PLoS One,11(5):e0156374.
Alain B P E,Chae J P,Balolong M P,Bum K H,Kang D K.2014.Assessment of fecal bacterial diversity among healthy piglets during the weaning transition[J].Journal of General and Applied Microbiology,60(4):140-146.
Boquet P,Munro P,Fiorentini C,Just I.1998.Toxins from anaerobic bacteria:Specificity and molecular mechanisms of action[J].Current Opinion in Microbiology,1(1):66-74.
Chiang M L,Chen H C,Chen K N,Lin Y C,Lin Y T,Chen M J.2015.Optimizing production of two potential probiotic Lactobacilli strains isolated from piglet feces as feed additives for weaned piglets[J].Asian-Australasian Journal of Animal Science,28(8):1163-1170.
De Filippo C,Cavalieri D,Di Paola M,Ramazzotti M,Poullet J B,Massart S,Collini S,Pieraccini G,Lionetti P.2010.Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa[J].Proceedings of the National Academy of Sciences of the United States of America,107(33):14691-14696.
Estrada A,Drew M D,van Kessel A G.2001.Effect of the dietary supplementation of fructo-oligosaccharides and Bifidobacteria longum to early weaned pigs on performance and fecal bacterial populations[J].The Canadian Veterinary Journal,81(1):141-148.
Eyeraert N,van Cruchten S,Westr?m B,Bailey M,van Ginneken C,Thymann T,Pieper R.2017.A review on early gut maturation and colonization in pigs,including biological and dietary factors affecting gut homeostasis[J].Animal Feed Science and Technology,233:89-103.
Gill S R,Pop M,DeBoy R T,Eckburg P B,Turnbaugh P J,Samuel B S,Gordon J I,Relman D A,Fraser-Liggett CM,Nelson K E.2006.Metagenomic analysis of the human distal gut microbiome[J].Science,312(5778):1355-1359.
Gorvitovskaia A,Holmes S P,Huse S M.2016.Interpreting prevotella and Bacteroides as biomarkers of diet and lifestyle[J].Microbiome,4:15.doi:10.1186/s40168-016-0160-7.
Herlemann D P R,Labrenz M,Jürgens K,Bertilsson S,Waniek J J,Andersson A F.2011.Transitions in bacterial communities along the 2000 km salinity gradient of the Baltic Sea[J].The ISME Journal,5(10):1571-1579.
Hooper L V,Gordon J I.2001.Commensal host-bacterial relationship[J].Science,292(5519):1115-1118.
Huang C H,Qiao S Y,Li D F,Piao X S,Ren J P.2004.Effects of Lactobacilli on the performance,diarrhea incidence,VFA concentration and gastrointestinal microbial flora of weaning pigs[J].Asian-Australasian Journal of Animal Science,17(3):401-409.
Kim H B,Borewicz K,White B A,Singer R S,Sreevatsan S,Tu Z J,Isaacson R E.2011.Longitudinal investigation of the age-related bacterial diversity in the feces of commercial pigs[J].Veterinary Microbiology,153(1-2):124-133.
Liu H,Zhang J,Zhang S H,Yang F J,Thacker P A,Zhang GL,Qiao S Y,Ma X.2014.Oral administration of Lactobacillus fermentum I5007 favors intestinal development and alters the intestinal microbiota in formula-fed piglets[J].Journal of Agricultural and Food Chemistry,62(4):860-866.
Mach N,Berri M,EstelléJ,Levenez F,Lemonnier G,Denis C,Leplat J,Chevaleyre C,Billon Y,DoréJ,Rogel-Gaillard C,Lepage P.2015.Early-life establishment of the swine gut microbiome and impact on host phenotypes[J].Environmental Microbiology Reports,7(3):554-569.
Pieper R,Janczyk P,Urubschurov V,Korn U,Pieper B,Souffrant W B.2009.Effect of a single oral administration of Lactobacillus plantarum DSMZ 8862/8866 before and at the time point of weaning on intestinal microbial communities in piglets[J].International Journal of Food Microbiology,130(3):227-232.
Reid G.2004.When microbe meets human[J].Clinical Infectious Diseases,39(6):827-830.
Ulger Toprak N,Bozan T,Birkan Y,Isbir S,Soyletir G.2015.Butyricimonas virosa:The first clinical case of bacteraemia[J].New Microbes and New Infections,4:7-8.