宏基因组学揭示二氟沙星对牦牛瘤胃微生态及CAZy谱影响
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摘要
反刍动物瘤胃微生物结构和功能关系到生态系统正常的物质循环、能量流动。为了探讨抗生素对瘤胃微生态的影响,揭示抗生素对反刍动物瘤胃微生物影响的规律,以牦牛为研究对象,进行二氟沙星(DIF)处理,采集瘤胃液,提取DNA,采用宏基因组学方法分析DIF对瘤胃微生物组成及碳水化合物酶类(CAZy)的影响。结果显示,拟杆菌门(Bacteroidetes)、变形菌门(Proteobacteria)等相对丰度下降,而厚壁菌门(Firmicutes)、广古菌门(Euryarchaeota)等增加。普雷沃菌属(Prevotella)、拟杆菌属(Bacteroides)等相对丰度下降,而金黄杆菌属(Chryseobacterium)、链球菌属(Streptococcus)等增加;CAZy酶类中,CBM17、CBM25等丰度上调(P<0.01或P<0.05);GH53、CE8、CE13、CBM21、CBM26及对接蛋白(dockerin)等下调(P<0.01或P<0.05)。结论:DIF影响瘤胃微生物结构,并不同程度改变各优势门、属的相对丰度,抑制部分有益菌的作用;改变CAZy酶中部分家族基因丰度,影响瘤胃微生物对淀粉及果胶的降解作用,降低瘤胃微生物降解植物纤维素的效率。
The structure and function of rumen microbes in ruminants are important for the normal material cycle and energy flow in the ecosystems.To explore the effects of antibiotics on rumen micro-ecology,yaks were used in the present research,and were randomly divided into the treatment group and the control group.The treatment group was given difloxacin(DIF).The rumen fluid samples of the treated yaks were collected and DNA was extracted and sequenced.The sequencing data was analyzed by the metagenomic method to identify the effects of antibiotics on rumen microbial composition and carbohydrate enzymes(CAZy).The results showed that the relative abundance of Bacteroidetes and Proteobacteria decreased,while Firmicutes and Euryarchaeota increased.The relative abundance of Prevotella and Bacteroides decreased,while Chryseobacterium and Streptococcus increased.Among the CAZys,the abundance of CBM17 and CBM25 was up-regulated(P<0.01 and P<0.05 respectively).The abundance of GH14,GH53,CE8,CE13,CBM21,CBM26 and dockerin were all down-regulated(P<0.01 or P<0.05).In conclusion:DIF affected the rumen microbial structure,changed the relative abundance of the dominant phylum and genus,and inhibited the function of some beneficial bacteria.It also changed the abundance of some gene families in CAZy,which hindered the degradation of starch and pectin by rumen microbes and reduced the efficiency of microbial degradation of plant cellulose.
The structure and function of rumen microbes in ruminants are important for the normal material cycle and energy flow in the ecosystems.To explore the effects of antibiotics on rumen micro-ecology,yaks were used in the present research,and were randomly divided into the treatment group and the control group.The treatment group was given difloxacin(DIF).The rumen fluid samples of the treated yaks were collected and DNA was extracted and sequenced.The sequencing data was analyzed by the metagenomic method to identify the effects of antibiotics on rumen microbial composition and carbohydrate enzymes(CAZy).The results showed that the relative abundance of Bacteroidetes and Proteobacteria decreased,while Firmicutes and Euryarchaeota increased.The relative abundance of Prevotella and Bacteroides decreased,while Chryseobacterium and Streptococcus increased.Among the CAZys,the abundance of CBM17 and CBM25 was up-regulated(P<0.01 and P<0.05 respectively).The abundance of GH14,GH53,CE8,CE13,CBM21,CBM26 and dockerin were all down-regulated(P<0.01 or P<0.05).In conclusion:DIF affected the rumen microbial structure,changed the relative abundance of the dominant phylum and genus,and inhibited the function of some beneficial bacteria.It also changed the abundance of some gene families in CAZy,which hindered the degradation of starch and pectin by rumen microbes and reduced the efficiency of microbial degradation of plant cellulose.
引文
[1]胡燕,白继庚,胡先明,等.我国抗生素滥用现状、原因及对策探讨[J].中国社会医学杂志,2013,30(2):128-130.
[2]王云鹏,马越.养殖业抗生素的使用及其潜在危害[J].中国抗生素杂志,2008,33(9):519-523.
[3]Vaccaro E,Giorgi M,Longo V,et al.Inhibition of cytochrome p450enzymes byenrofloxacin in the sea bass(Dicentrarchus labrax)[J].Aquat Toxicol,2003,62(1):27-33.
[4]Jianying Hu,Wanfeng Wang,Zhu Z,et al.Quantitative Structureactivity Relationship Model for Prediction of Genotoxic Potential for Quinolone Antibacterials[J].Environ Sci Technol,2007,41(13):4806-4812.
[5]Kemper N.Veterinary antibiotics in the aquatic and terrestrial environment[J].Ecol Indic,2008,8(1):1-13.
[6]Burgess C.Livestock Drugs Infiltrate Dust:Another Hazard for Farmers[J].ENVIRON HEALTH PERSP,2003,111(13):a717-a717.
[7]Moreno-Bondi M C,Marazuela M D,Herranz S,et al.An overview of sample preparation procedures for LC-MS multiclass antibiotic determination in environmental and food samples[J].Anal Bioanal Chem,2009,395(4):921-46.
[8]Sarmah A K,Meyer M T,Boxall A B.A global perspective on the use,sales,exposure pathways,occurrence,fate and effects of veterinary antibiotics(VAs)in the environment[J].Chemosphere,2006,65(5):725-759.
[9]邓玉英,陈桂先,曾年英.滥用饲用抗生素现状及对生态环境的影响[J].现代畜牧兽医,2009(10):35-36.
[10]狄静波.环境残留抗生素降解产物的生物活性及毒性影响研究[A].中国环境科学学会.2017中国环境科学学会科学与技术年会论文集(第四卷)[C].中国环境科学学会:,2017:6.
[11]乔栋,刘隆勇,黄璐.兽用抗生素残留对微生物的影响及防治对策[J].中国畜牧兽医文摘,2013(1):224-226.
[12]刘开永,李英伦.二氟沙星的应用研究概况[J].兽药与饲料添加剂,2004(03):12-14.
[13]王加龙,刘坚真,陈杖榴,等.恩诺沙星残留对土壤微生物数量及群落功能多样性的影响[J].应用与环境生物学报,2005,11(1):86-89.
[14]王敏,唐景春.土壤中的抗生素污染及其生态毒性研究进展[J].农业环境科学学报,2010,29(b03):261-266.
[15]谢全喜,张建梅,李晓颖,等.复合微生态制剂与饲用抗生素对肉雏鸡免疫性能和肠道微生物菌群的影响[J].家畜生态学报,2011,32(5):77-80.
[16]王冲.高通量16SrRNA标签测序法比较抗生素使用人群与正常人群肠道微生物组多样性[C]//第四届国际人体微生态大会、第十三届中华预防医学会微生态学学术会议、第五届中国医师协会感染科医师大会暨传染病诊治高峰论坛、2013医学前沿论坛暨我国感染病学发展战略研究研讨会论文集.2013:317-317.
[17]Kim M,Morrison M,Yu Z.Status of the phylogenetic diversity census of ruminal microbiomes[J].Fems Microbiol Ecol,2015,76(1):49-63.
[18]吴森,张莺莺,昝林森.基于高通量测序的宏基因组学技术在动物胃肠道微生物方面的研究进展[J].生物技术进展,2015(2):77-84.
[19]王继文,王立志,闫天海,等.山羊瘤胃与粪便微生物多样性[J].动物营养学报,2015,27(8):2559-2571.
[20]Jami E,Mizrahi I.Composition and similarity of bovine rumen microbiota across individual animals[J].Plos One,2012,7(3):e33306.
[21]Sommer M O A,Church G M.Functional characterization of the antibiotic resistance reservoir in the human microflora[J].Science,2009,325(5944):1128-31.
[22]Singh K M,Jakhesara S J,Koringa P G,et al.Metagenomic analysis of virulence-associated and antibiotic resistance genes of microbes in rumen of Indian buffalo(Bubalus bubalis)[J].Gene,2012,507(2):146-151.
[23]Santi C D,Altermark B,Pierechod M M,et al.Characterization of a cold-active and salt tolerant esterase identified by functional screening of Arctic metagenomic libraries[J].Bmc Biochemistry,2016,17(1):1.
[24]Pandey S,Gulati S,Goyal E,et al.Construction and screening of metagenomic library derived from soil forβ-1,4-endoglucanase gene[J].Biocatal Biotransfor,2016,5:186-192.
[25]Xue D,Chen H,Chen F,et al.Analysis of the rumen bacteria and methanogenic archaea of yak(Bos grunniens)steers grazing on the Qinghai-Tibetan Plateau[J].LIVEST SCI,2016,188:61-71.
[26]Singh K M,Reddy B,Patel D,et al.High Potential Source for Biomass Degradation Enzyme Discovery and Environmental Aspects Revealed through Metagenomics of Indian Buffalo Rumen[J].Biomed Res Int,2014,2014(5938):267189.
[27]Parmar N R,Solanki J V,Patel A B,et al.Metagenome of Mehsani buffalo rumen microbiota:an assessment of variation in feed-dependent phylogenetic and functional classification[J].J Ind Microbiol Biot,2014,24(4):249-261.
[28]Yang L Y,Chen J,Cheng X L,et al.Phylogenetic analysis of 16SrRNA gene sequences reveals rumen bacterial diversity in Yaks(Bos grunniens)[J].Mol Biol Rep,2010,37(1):553-562.
[29]Leng J,Zhu R,Yang S,et al.Dominant bacterial communities in the rumen of Gayals(Bos frontalis),Yaks(Bos grunniens)and Yunnan Yellow Cattle(Bos taurs)revealed by denaturing gradient gel electrophoresis[J].Mol Biol Rep,2011,38(8):4863-4872.
[30]Henderson G,Cox F,Ganesh S,et al.Rumen microbial community composition varies with diet and host,but a core microbiome is found across a wide geographical range[J].Sci Rep-Uk,2015,5:14567.
[31]Wongwilaiwalin S,Laothanachareon T,Mhuantong W,et al.Comparative metagenomic analysis of microcosm structures and lignocellulolytic enzyme systems of symbiotic biomass-degrading consortia[J].Appl Microbiol Biot,2013,97(20):8941-8954.
[32]Saro C,Ranilla M J,Carro M D.Postprandial changes of fiber-degrading microbes in the rumen of sheep fed diets varying in type of forage as monitored by real-time PCR and automated ribosomal intergenic spacer analysis[J].J Anim Sci,2012,90(12):4487-4494.
[33]Fernando S C,Nd P H,Najar F Z,et al.Rumen Microbial Population Dynamics during Adaptation to a High-Grain Diet[J].Appl Environ Microb,2010,76(22):7482-7490.
[34]Leser T D,Amenuvor J Z,Jensen T K,et al.Culture-independent analysis of gut bacteria:the pig gastrointestinal tract microbiota revisited[J].Appl Mircrobiol Biot,2002,68(2):673-690.
[35]Huo W,Zhu W,Mao S.Impact of subacute ruminal acidosis on the diversity of liquid and solid-associated bacteria in the rumen of goats[J].World J Microb Biot,2014,30(2):669-680.
[36]Bekele A Z,Koike S,Kobayashi Y.Phylogenetic diversity and dietary association of rumen Treponema revealed using group-specific16S rRNA gene-based analysis[J].Fems Microbiol Lett,2011,316(1):51-60.
[37]田彦.基于高通量测序的中国荷斯坦奶牛瘤胃宏转录组研究[D].北京:中国科学院北京基因组研究所,2015.
[38]Rodríguezsanoja R,Oviedo N,Sánchez S.Microbial starch-binding domain[J].Curr Opin Microbiol,2005,8(3):260-267.
[39]M.Machovic,.Janecek.Starch-binding domains in the post-genome era[J].Cell Mol Life Sci,2006,63(23):2710.
[40]Macgregor E A,Janecek S,Svensson B.Relationship of sequence and structure to specificity in the alpha-amylase family of enzymes[J].Biochim Biophys Acta,2001,1546(1):1-20.
[41]Lynd L R,Weimer P J,van Zyl W H,et al.Microbial cellulose utilization:fundamentals and biotechnology[J].Microbiol Mol Biol R,2002,66(3):506.
[42]Hinz S W,Pastink M I,La V D B,et al.Bifidobacterium longum endogalactanase liberates galactotriose from type I galactans[J].Appl Environ Microb,2005,71(9):5501-5510.
[43]Hess M,Sczyrba A,Egan R,et al.Metagenomic discovery of biomass-degrading genes and genomes from cow rumen[J].Science,2011,331(6016):463-467.
[44]Hyeon J E,Jeon S D,Han S O.Cellulosome-based,Clostridiumderived multi-functional enzyme complexes for advanced biotechnology tool development:advances and applications[J].Biotechnol Adv,2013,31(6):936-944.
[2]王云鹏,马越.养殖业抗生素的使用及其潜在危害[J].中国抗生素杂志,2008,33(9):519-523.
[3]Vaccaro E,Giorgi M,Longo V,et al.Inhibition of cytochrome p450enzymes byenrofloxacin in the sea bass(Dicentrarchus labrax)[J].Aquat Toxicol,2003,62(1):27-33.
[4]Jianying Hu,Wanfeng Wang,Zhu Z,et al.Quantitative Structureactivity Relationship Model for Prediction of Genotoxic Potential for Quinolone Antibacterials[J].Environ Sci Technol,2007,41(13):4806-4812.
[5]Kemper N.Veterinary antibiotics in the aquatic and terrestrial environment[J].Ecol Indic,2008,8(1):1-13.
[6]Burgess C.Livestock Drugs Infiltrate Dust:Another Hazard for Farmers[J].ENVIRON HEALTH PERSP,2003,111(13):a717-a717.
[7]Moreno-Bondi M C,Marazuela M D,Herranz S,et al.An overview of sample preparation procedures for LC-MS multiclass antibiotic determination in environmental and food samples[J].Anal Bioanal Chem,2009,395(4):921-46.
[8]Sarmah A K,Meyer M T,Boxall A B.A global perspective on the use,sales,exposure pathways,occurrence,fate and effects of veterinary antibiotics(VAs)in the environment[J].Chemosphere,2006,65(5):725-759.
[9]邓玉英,陈桂先,曾年英.滥用饲用抗生素现状及对生态环境的影响[J].现代畜牧兽医,2009(10):35-36.
[10]狄静波.环境残留抗生素降解产物的生物活性及毒性影响研究[A].中国环境科学学会.2017中国环境科学学会科学与技术年会论文集(第四卷)[C].中国环境科学学会:,2017:6.
[11]乔栋,刘隆勇,黄璐.兽用抗生素残留对微生物的影响及防治对策[J].中国畜牧兽医文摘,2013(1):224-226.
[12]刘开永,李英伦.二氟沙星的应用研究概况[J].兽药与饲料添加剂,2004(03):12-14.
[13]王加龙,刘坚真,陈杖榴,等.恩诺沙星残留对土壤微生物数量及群落功能多样性的影响[J].应用与环境生物学报,2005,11(1):86-89.
[14]王敏,唐景春.土壤中的抗生素污染及其生态毒性研究进展[J].农业环境科学学报,2010,29(b03):261-266.
[15]谢全喜,张建梅,李晓颖,等.复合微生态制剂与饲用抗生素对肉雏鸡免疫性能和肠道微生物菌群的影响[J].家畜生态学报,2011,32(5):77-80.
[16]王冲.高通量16SrRNA标签测序法比较抗生素使用人群与正常人群肠道微生物组多样性[C]//第四届国际人体微生态大会、第十三届中华预防医学会微生态学学术会议、第五届中国医师协会感染科医师大会暨传染病诊治高峰论坛、2013医学前沿论坛暨我国感染病学发展战略研究研讨会论文集.2013:317-317.
[17]Kim M,Morrison M,Yu Z.Status of the phylogenetic diversity census of ruminal microbiomes[J].Fems Microbiol Ecol,2015,76(1):49-63.
[18]吴森,张莺莺,昝林森.基于高通量测序的宏基因组学技术在动物胃肠道微生物方面的研究进展[J].生物技术进展,2015(2):77-84.
[19]王继文,王立志,闫天海,等.山羊瘤胃与粪便微生物多样性[J].动物营养学报,2015,27(8):2559-2571.
[20]Jami E,Mizrahi I.Composition and similarity of bovine rumen microbiota across individual animals[J].Plos One,2012,7(3):e33306.
[21]Sommer M O A,Church G M.Functional characterization of the antibiotic resistance reservoir in the human microflora[J].Science,2009,325(5944):1128-31.
[22]Singh K M,Jakhesara S J,Koringa P G,et al.Metagenomic analysis of virulence-associated and antibiotic resistance genes of microbes in rumen of Indian buffalo(Bubalus bubalis)[J].Gene,2012,507(2):146-151.
[23]Santi C D,Altermark B,Pierechod M M,et al.Characterization of a cold-active and salt tolerant esterase identified by functional screening of Arctic metagenomic libraries[J].Bmc Biochemistry,2016,17(1):1.
[24]Pandey S,Gulati S,Goyal E,et al.Construction and screening of metagenomic library derived from soil forβ-1,4-endoglucanase gene[J].Biocatal Biotransfor,2016,5:186-192.
[25]Xue D,Chen H,Chen F,et al.Analysis of the rumen bacteria and methanogenic archaea of yak(Bos grunniens)steers grazing on the Qinghai-Tibetan Plateau[J].LIVEST SCI,2016,188:61-71.
[26]Singh K M,Reddy B,Patel D,et al.High Potential Source for Biomass Degradation Enzyme Discovery and Environmental Aspects Revealed through Metagenomics of Indian Buffalo Rumen[J].Biomed Res Int,2014,2014(5938):267189.
[27]Parmar N R,Solanki J V,Patel A B,et al.Metagenome of Mehsani buffalo rumen microbiota:an assessment of variation in feed-dependent phylogenetic and functional classification[J].J Ind Microbiol Biot,2014,24(4):249-261.
[28]Yang L Y,Chen J,Cheng X L,et al.Phylogenetic analysis of 16SrRNA gene sequences reveals rumen bacterial diversity in Yaks(Bos grunniens)[J].Mol Biol Rep,2010,37(1):553-562.
[29]Leng J,Zhu R,Yang S,et al.Dominant bacterial communities in the rumen of Gayals(Bos frontalis),Yaks(Bos grunniens)and Yunnan Yellow Cattle(Bos taurs)revealed by denaturing gradient gel electrophoresis[J].Mol Biol Rep,2011,38(8):4863-4872.
[30]Henderson G,Cox F,Ganesh S,et al.Rumen microbial community composition varies with diet and host,but a core microbiome is found across a wide geographical range[J].Sci Rep-Uk,2015,5:14567.
[31]Wongwilaiwalin S,Laothanachareon T,Mhuantong W,et al.Comparative metagenomic analysis of microcosm structures and lignocellulolytic enzyme systems of symbiotic biomass-degrading consortia[J].Appl Microbiol Biot,2013,97(20):8941-8954.
[32]Saro C,Ranilla M J,Carro M D.Postprandial changes of fiber-degrading microbes in the rumen of sheep fed diets varying in type of forage as monitored by real-time PCR and automated ribosomal intergenic spacer analysis[J].J Anim Sci,2012,90(12):4487-4494.
[33]Fernando S C,Nd P H,Najar F Z,et al.Rumen Microbial Population Dynamics during Adaptation to a High-Grain Diet[J].Appl Environ Microb,2010,76(22):7482-7490.
[34]Leser T D,Amenuvor J Z,Jensen T K,et al.Culture-independent analysis of gut bacteria:the pig gastrointestinal tract microbiota revisited[J].Appl Mircrobiol Biot,2002,68(2):673-690.
[35]Huo W,Zhu W,Mao S.Impact of subacute ruminal acidosis on the diversity of liquid and solid-associated bacteria in the rumen of goats[J].World J Microb Biot,2014,30(2):669-680.
[36]Bekele A Z,Koike S,Kobayashi Y.Phylogenetic diversity and dietary association of rumen Treponema revealed using group-specific16S rRNA gene-based analysis[J].Fems Microbiol Lett,2011,316(1):51-60.
[37]田彦.基于高通量测序的中国荷斯坦奶牛瘤胃宏转录组研究[D].北京:中国科学院北京基因组研究所,2015.
[38]Rodríguezsanoja R,Oviedo N,Sánchez S.Microbial starch-binding domain[J].Curr Opin Microbiol,2005,8(3):260-267.
[39]M.Machovic,.Janecek.Starch-binding domains in the post-genome era[J].Cell Mol Life Sci,2006,63(23):2710.
[40]Macgregor E A,Janecek S,Svensson B.Relationship of sequence and structure to specificity in the alpha-amylase family of enzymes[J].Biochim Biophys Acta,2001,1546(1):1-20.
[41]Lynd L R,Weimer P J,van Zyl W H,et al.Microbial cellulose utilization:fundamentals and biotechnology[J].Microbiol Mol Biol R,2002,66(3):506.
[42]Hinz S W,Pastink M I,La V D B,et al.Bifidobacterium longum endogalactanase liberates galactotriose from type I galactans[J].Appl Environ Microb,2005,71(9):5501-5510.
[43]Hess M,Sczyrba A,Egan R,et al.Metagenomic discovery of biomass-degrading genes and genomes from cow rumen[J].Science,2011,331(6016):463-467.
[44]Hyeon J E,Jeon S D,Han S O.Cellulosome-based,Clostridiumderived multi-functional enzyme complexes for advanced biotechnology tool development:advances and applications[J].Biotechnol Adv,2013,31(6):936-944.