成都平原不同种植方式折耳根表面附生细菌群落结构与抗生素抗性基因分析
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摘要
【目的】分析有机、化肥和野生折耳根表面的附生细菌群落结构和抗生素抗性基因(ARGs),揭示细菌群落结构与ARGs相互关系。【方法】高通量测定16SrRNAV3-V4可变区序列分析样品表面附生细菌群落结构;PCR和qPCR扩增29种ARGs基因分析样品表面ARGs污染情况;冗余分析(RDA)探讨细菌群落结构与ARGs的相互关系。【结果】折耳根表面检测到35个属的细菌,其中有机折耳根表面附生细菌多样性低于化肥和野生折耳根(P<0.05);29种被检的ARGs中,有14种在折耳根中被检出,其中有机折耳根含有全部被检出的ARGs,化肥和野生折耳根则含有部分被检出的ARGs。折耳根表面ARGs污染的多样性和丰度显著受到样品表面的菌群结构影响,其中Lactococcus、 Escherichia、Fluviicola、Enterococcus、Sanguibacter和Acidovorax是影响ARGs最主要的菌群。【结论】有机种植极大地改变了折耳根表面附生细菌的群落结构,增加了ARGs的多样性和丰度,对有机折耳根的食品安全带来了潜在威胁。因此,有必要将ARGs污染监测纳入到有机折耳根的食品安全考核范围内。
[Objective] The aim of this study is to analyze bacterial community and antibiotic resistant genes(ARGs) in organic, fertilized and wild Houttuynia cordata Thunb, and to reveal the relationship between bacterial community and ARGs. [Methods] The bacterial community structure was investigated by high-throughput 16S rRNA V3-V4 variable region sequencing. The qualitative and quantitative analysis of 29 ARGs was determined by PCR and qPCR. The redundancy analysis was used to reveal the relationship between bacterial community and ARGs. [Results] A total of 35 genera of bacteria were detected. The bacterial diversity in organic H. cordata Thunb was lower than that in fertilized and wild H. cordata Thunb. Fourteen ARGs were detected on H. cordata Thunb.All detected ARGs were found in the organic H. cordata Thunb, while only part was found in the fertilized and wild H. cordata Thunb. Redundancy analysis showed ARGs were significantly affected by bacterial diversity and abundance, and Lactococcus, Escherichia, Fluviicola, Enterococcus, Sanguibacter and Acidovorax were main bacteria which affected on the diversity and abundance of ARGs on H. cordata Thunb. [Conclusion] Organic planting affects bacterial community on H. Cordata Thunb, and increases diversity and abundance of ARGs,suggesting a potential food safety risk. Therefore, it is necessary to bring ARGs contamination into the scope of food safety assessment for organic H. cordata Thunb.
[Objective] The aim of this study is to analyze bacterial community and antibiotic resistant genes(ARGs) in organic, fertilized and wild Houttuynia cordata Thunb, and to reveal the relationship between bacterial community and ARGs. [Methods] The bacterial community structure was investigated by high-throughput 16S rRNA V3-V4 variable region sequencing. The qualitative and quantitative analysis of 29 ARGs was determined by PCR and qPCR. The redundancy analysis was used to reveal the relationship between bacterial community and ARGs. [Results] A total of 35 genera of bacteria were detected. The bacterial diversity in organic H. cordata Thunb was lower than that in fertilized and wild H. cordata Thunb. Fourteen ARGs were detected on H. cordata Thunb.All detected ARGs were found in the organic H. cordata Thunb, while only part was found in the fertilized and wild H. cordata Thunb. Redundancy analysis showed ARGs were significantly affected by bacterial diversity and abundance, and Lactococcus, Escherichia, Fluviicola, Enterococcus, Sanguibacter and Acidovorax were main bacteria which affected on the diversity and abundance of ARGs on H. cordata Thunb. [Conclusion] Organic planting affects bacterial community on H. Cordata Thunb, and increases diversity and abundance of ARGs,suggesting a potential food safety risk. Therefore, it is necessary to bring ARGs contamination into the scope of food safety assessment for organic H. cordata Thunb.
引文
[1]Oh SY.An effective quality control of pharmacologically active volatiles of Houttuynia cordata Thunb by fast gas chromatography-surface acoustic wave sensor.Molecules,2015,20(6):10298-10312.
[2]Sekita Y,Murakami K,Yumoto H,Mizuguchi H,Amoh T,Ogino S,Matsuo T,Miyake Y,Fukui H,Kashiwada Y.Anti-bacterial and anti-inflammatory effects of ethanol extract from Houttuynia cordata poultice.Bioscience,Biotechnology,and Biochemistry,2016,80(6):1205-1213.
[3]Li J,Zhao FT.Anti-inflammatory functions of Houttuynia cordata Thunb.and its compounds:a perspective on its potential role in rheumatoid arthritis.Experimental and Therapeutic Medicine,2015,10(1):3-6.
[4]Huang CH,Renew JE,Smeby KL,Pinkston K,Sedlak DL.Assessment of potential antibiotic contaminants in water and preliminary occurrence analysis.Journal of Contemporary Water Research and Education,2001,120(1):30-40.
[5]Abriouel H,Omar NB,Molinos AC,López RL,Grande MJ,Martínez-Viedma P,Ortega E,Ca?amero MM,Galvez A.Comparative analysis of genetic diversity and incidence of virulence factors and antibiotic resistance among enterococcal populations from raw fruit and vegetable foods,water and soil,and clinical samples.International Journal of Food Microbiology,2008,123(1/2):38-49.
[6]Boehme S,Werner G,Klare I,Reissbrot R,Witte W.Occurrence of antibiotic-resistant enterobacteria in agricultural foodstuffs.Molecular Nutrition and Food Research,2004,48(7):522-531.
[7]Durso LM,Miller DN,Wienhold BJ.Distribution and quantification of antibiotic resistant genes and bacteria across agricultural and non-agricultural metagenomes.PLoS One,2012,7(11):e48325.
[8]Rodríguez C,Lang L,Wang A,Altendorf K,García F,LipskíA.Lettuce for human consumption collected in Costa Rica contains complex communities of culturable oxytetracyclineand gentamicin-resistant bacteria.Applied and Environmental Microbiology,2006,72(9):5870-5876.
[9]Zhu BK,Chen QL,Chen SC,Zhu YG.Does organically produced lettuce harbor higher abundance of antibiotic resistance genes than conventionally produced?Environment International,2017,98:152-159.
[10]Tang JY,Bu YQ,Zhang XX,Huang KL,He XW,Ye L,Shan ZJ,Ren HQ.Metagenomic analysis of bacterial community composition and antibiotic resistance genes in a wastewater treatment plant and its receiving surface water.Ecotoxicology and Environmental Safety,2016,132:260-269.
[11]Caporaso JG,Kuczynski J,Stombaugh J,Bittinger K,Bushman FD,Costello EK,Fierer N,Pe?a AG,Goodrich JK,Gordon JI,Huttley GA,Kelley ST,Knights D,Koenig JE,Ley RE,Lozupone CA,McDonald D,Muegge BD,Pirrung M,Reeder J,Sevinsky JR,Turnbaugh PJ,Walters WA,Widmann J,Yatsunenko T,Zaneveld J,Knight R.QIIME allows analysis of high-throughput community sequencing data.Nature Methods,2010,7(5):335-336.
[12]Mago?T,Salzberg SL.FLASH:fast length adjustment of short reads to improve genome assemblies.Bioinformatics,2011,27(21):2957-2963.
[13]Edgar RC.Search and clustering orders of magnitude faster than BLAST.Bioinformatics,2010,26(19):2460-2461.
[14]Wang Q,Garrity MG,Tiedje MJ,Cole RJ.Na?ve Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy.Applied and Environmental Microbiology,2007,73(16):5261-5267.
[15]Mao DQ,Yu S,Rysz M,Luo YX,Yang FX,Li FX,Hou J,Mu QH,Alvarez PJJ.Prevalence and proliferation of antibiotic resistance genes in two municipal wastewater treatment plants.Water Research,2015,85:458-466.
[16]Ouoba LII,Lei V,Jensen LB.Resistance of potential probiotic lactic acid bacteria and bifidobacteria of African and European origin to antimicrobials:determination and transferability of the resistance genes to other bacteria.International Journal of Food Microbiology,2008,121(2):217-224.
[17]Zhang J,Yang XH,Ge F,Wang N,Jiao SJ,Ye BP.Effects of long-term application of pig manure containing residual tetracycline on the formation of drug-resistant bacteria and resistance genes.Environmental Science,2014,35(6):2374-2380.(in Chinese)张俊,杨晓洪,葛峰,王娜,焦少俊,叶波平.长期施用四环素残留猪粪对土壤中耐药菌及抗性基因形成的影响.环境科学,2014,35(6):2374-2380.
[18]Jia SY,He XW,Bu YQ,Shi P,Miao Y,Zhou HP,Shan ZJ,Zhang XX.Environmental fate of tetracycline resistance genes originating from swine feedlots in river water.Journal of Environmental Science and Health Part B-Pesticides,Food Contaminants,and Agricultural Wastes,2014,49(8):624-631.
[19]Jiang L,Hu XL,Xu T,Zhang HC,Sheng D,Yin DQ.Prevalence of antibiotic resistance genes and their relationship with antibiotics in the Huangpu River and the drinking water sources,Shanghai,China.Science of the Total Environment,2013,458-460:267-272.
[20]Zhang AY,Wang HN,Tian GB,Zhang Y,Yang X,Xia QQ,Tang JN,Zou LK.Phenotypic and genotypic characterisation of antimicrobial resistance in faecal bacteria from 30 Giant pandas.International Journal of Antimicrobial Agents,2009,33(5):456-460.
[21]Aminov RI,Garrigues-Jeanjean N,Mackie RI.Molecular ecology of tetracycline resistance:development and validation of primers for detection of tetracycline resistance genes encoding ribosomal protection proteins.Applied and Environmental Microbiology,2001,67(1):22-32.
[22]Zhang JY,Chen MX,Sui QW,Wang R,Tong J,Wei YS.Fate of antibiotic resistance genes and its drivers during anaerobic co-digestion of food waste and sewage sludge based on microwave pretreatment.Bioresource Technology,2016,217:28-36.
[23]Casado Mu?oz MdelC,Benomar N,Lavilla LL,Gálvez A,Abriouel H.Antibiotic resistance of Lactobacillus pentosus and Leuconostoc pseudomesenteroides isolated from naturally-fermented Alore?a table olives throughout fermentation process.International Journal of Food Microbiology,2014,172:110-118.
[24]Pruden A,Pei RT,Storteboom H,Carlson KH.Antibiotic resistance genes as emerging contaminants:studies in northern Colorado.Environmental Science&Technology,2006,40(23):7445-7450.
[25]Tian Z,Zhang Y,Yu B,Yang M.Changes of resistome,mobilome and potential hosts of antibiotic resistance genes during the transformation of anaerobic digestion from mesophilic to thermophilic.Water Research,2016,98:261-269.
[26]Martínez JL.Antibiotics and antibiotic resistance genes in natural environments.Science,2008,321(5887):365-367.
[27]van Hoek AHAM,Mevius D,Guerra B,Mullany P,Roberts AP,Aarts HJM.Acquired antibiotic resistance genes:an overview.Frontiers in Microbiology,2011,2:203.
[28]Wang FH,Qiao M,Chen Z,Su JQ,Zhu YG.Antibiotic resistance genes in manure-amended soil and vegetables at harvest.Journal of Hazardous Materials,2015,299:215-221.
[29]Chen QL,An XL,Li H,Su JQ,Ma YB,Zhu YG.Long-term field application of sewage sludge increases the abundance of antibiotic resistance genes in soil.Environment International,2016,92-93:1-10.
[30]Marti R,Scott A,Tien YC,Murray R,Sabourin L,Zhang Y,Topp E.Impact of manure fertilization on the abundance of antibiotic-resistant bacteria and frequency of detection of antibiotic resistance genes in soil and on vegetables at harvest.Applied and Environmental Microbiology,2013,79(18):5701-5709.
[31]Ross J,Topp E.Abundance of antibiotic resistance genes in bacteriophage following soil fertilization with dairy manure or municipal biosolids,and evidence for potential transduction.Applied and Environmental Microbiology,2015,81(22):7905-7913.
[32]Bai Y,Müller DB,Srinivas G,Garrido-Oter R,Potthoff E,Rott M,Dombrowski N,Münch PC,Spaepen S,Remus-Emsermann M,Hüttel B,McHardy AC,Vorholt JA,Schulze-Lefert P.Functional overlap of the Arabidopsis leaf and root microbiota.Nature,2015,528(7582):364-369.
[33]Ruiz-Pérez CA,Restrepo S,Zambrano MM.Microbial and functional diversity within the phyllosphere of Espeletia species in an andean high-mountain ecosystem.Applied and Environmental Microbiology,2016,82(6):1807-1817.
[34]Zarraonaindia I,Owens SM,Weisenhorn P,West K,Hampton-Marcell J,Lax S,Bokulich NA,Mills DA,Martin G,Taghavi S,van der Lelie D,Gilbert JA.The soil microbiome influences grapevine-associated microbiota.mBio,2015,6(2):e02527-14.
[35]Bulgarelli D,Rott M,Schlaeppi K,Ver Loren van Themaat E,Ahmadinejad N,Assenza F,Rauf P,Huettel B,Reinhardt R,Schmelzer E,Peplies J,Gloeckner FO,Amann R,Eickhorst T,Schulze-Lefert P.Revealing structure and assembly cues for Arabidopsis root-inhabiting bacterial microbiota.Nature,2012,488(7409):91-95.
[36]Duan ML,Li HC,Gu J,Tuo XX,Sun W,Qian X,Wang XJ.Effects of biochar on reducing the abundance of oxytetracycline,antibiotic resistance genes,and human pathogenic bacteria in soil and lettuce.Environmental Pollution,2017,224:787-795.
[2]Sekita Y,Murakami K,Yumoto H,Mizuguchi H,Amoh T,Ogino S,Matsuo T,Miyake Y,Fukui H,Kashiwada Y.Anti-bacterial and anti-inflammatory effects of ethanol extract from Houttuynia cordata poultice.Bioscience,Biotechnology,and Biochemistry,2016,80(6):1205-1213.
[3]Li J,Zhao FT.Anti-inflammatory functions of Houttuynia cordata Thunb.and its compounds:a perspective on its potential role in rheumatoid arthritis.Experimental and Therapeutic Medicine,2015,10(1):3-6.
[4]Huang CH,Renew JE,Smeby KL,Pinkston K,Sedlak DL.Assessment of potential antibiotic contaminants in water and preliminary occurrence analysis.Journal of Contemporary Water Research and Education,2001,120(1):30-40.
[5]Abriouel H,Omar NB,Molinos AC,López RL,Grande MJ,Martínez-Viedma P,Ortega E,Ca?amero MM,Galvez A.Comparative analysis of genetic diversity and incidence of virulence factors and antibiotic resistance among enterococcal populations from raw fruit and vegetable foods,water and soil,and clinical samples.International Journal of Food Microbiology,2008,123(1/2):38-49.
[6]Boehme S,Werner G,Klare I,Reissbrot R,Witte W.Occurrence of antibiotic-resistant enterobacteria in agricultural foodstuffs.Molecular Nutrition and Food Research,2004,48(7):522-531.
[7]Durso LM,Miller DN,Wienhold BJ.Distribution and quantification of antibiotic resistant genes and bacteria across agricultural and non-agricultural metagenomes.PLoS One,2012,7(11):e48325.
[8]Rodríguez C,Lang L,Wang A,Altendorf K,García F,LipskíA.Lettuce for human consumption collected in Costa Rica contains complex communities of culturable oxytetracyclineand gentamicin-resistant bacteria.Applied and Environmental Microbiology,2006,72(9):5870-5876.
[9]Zhu BK,Chen QL,Chen SC,Zhu YG.Does organically produced lettuce harbor higher abundance of antibiotic resistance genes than conventionally produced?Environment International,2017,98:152-159.
[10]Tang JY,Bu YQ,Zhang XX,Huang KL,He XW,Ye L,Shan ZJ,Ren HQ.Metagenomic analysis of bacterial community composition and antibiotic resistance genes in a wastewater treatment plant and its receiving surface water.Ecotoxicology and Environmental Safety,2016,132:260-269.
[11]Caporaso JG,Kuczynski J,Stombaugh J,Bittinger K,Bushman FD,Costello EK,Fierer N,Pe?a AG,Goodrich JK,Gordon JI,Huttley GA,Kelley ST,Knights D,Koenig JE,Ley RE,Lozupone CA,McDonald D,Muegge BD,Pirrung M,Reeder J,Sevinsky JR,Turnbaugh PJ,Walters WA,Widmann J,Yatsunenko T,Zaneveld J,Knight R.QIIME allows analysis of high-throughput community sequencing data.Nature Methods,2010,7(5):335-336.
[12]Mago?T,Salzberg SL.FLASH:fast length adjustment of short reads to improve genome assemblies.Bioinformatics,2011,27(21):2957-2963.
[13]Edgar RC.Search and clustering orders of magnitude faster than BLAST.Bioinformatics,2010,26(19):2460-2461.
[14]Wang Q,Garrity MG,Tiedje MJ,Cole RJ.Na?ve Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy.Applied and Environmental Microbiology,2007,73(16):5261-5267.
[15]Mao DQ,Yu S,Rysz M,Luo YX,Yang FX,Li FX,Hou J,Mu QH,Alvarez PJJ.Prevalence and proliferation of antibiotic resistance genes in two municipal wastewater treatment plants.Water Research,2015,85:458-466.
[16]Ouoba LII,Lei V,Jensen LB.Resistance of potential probiotic lactic acid bacteria and bifidobacteria of African and European origin to antimicrobials:determination and transferability of the resistance genes to other bacteria.International Journal of Food Microbiology,2008,121(2):217-224.
[17]Zhang J,Yang XH,Ge F,Wang N,Jiao SJ,Ye BP.Effects of long-term application of pig manure containing residual tetracycline on the formation of drug-resistant bacteria and resistance genes.Environmental Science,2014,35(6):2374-2380.(in Chinese)张俊,杨晓洪,葛峰,王娜,焦少俊,叶波平.长期施用四环素残留猪粪对土壤中耐药菌及抗性基因形成的影响.环境科学,2014,35(6):2374-2380.
[18]Jia SY,He XW,Bu YQ,Shi P,Miao Y,Zhou HP,Shan ZJ,Zhang XX.Environmental fate of tetracycline resistance genes originating from swine feedlots in river water.Journal of Environmental Science and Health Part B-Pesticides,Food Contaminants,and Agricultural Wastes,2014,49(8):624-631.
[19]Jiang L,Hu XL,Xu T,Zhang HC,Sheng D,Yin DQ.Prevalence of antibiotic resistance genes and their relationship with antibiotics in the Huangpu River and the drinking water sources,Shanghai,China.Science of the Total Environment,2013,458-460:267-272.
[20]Zhang AY,Wang HN,Tian GB,Zhang Y,Yang X,Xia QQ,Tang JN,Zou LK.Phenotypic and genotypic characterisation of antimicrobial resistance in faecal bacteria from 30 Giant pandas.International Journal of Antimicrobial Agents,2009,33(5):456-460.
[21]Aminov RI,Garrigues-Jeanjean N,Mackie RI.Molecular ecology of tetracycline resistance:development and validation of primers for detection of tetracycline resistance genes encoding ribosomal protection proteins.Applied and Environmental Microbiology,2001,67(1):22-32.
[22]Zhang JY,Chen MX,Sui QW,Wang R,Tong J,Wei YS.Fate of antibiotic resistance genes and its drivers during anaerobic co-digestion of food waste and sewage sludge based on microwave pretreatment.Bioresource Technology,2016,217:28-36.
[23]Casado Mu?oz MdelC,Benomar N,Lavilla LL,Gálvez A,Abriouel H.Antibiotic resistance of Lactobacillus pentosus and Leuconostoc pseudomesenteroides isolated from naturally-fermented Alore?a table olives throughout fermentation process.International Journal of Food Microbiology,2014,172:110-118.
[24]Pruden A,Pei RT,Storteboom H,Carlson KH.Antibiotic resistance genes as emerging contaminants:studies in northern Colorado.Environmental Science&Technology,2006,40(23):7445-7450.
[25]Tian Z,Zhang Y,Yu B,Yang M.Changes of resistome,mobilome and potential hosts of antibiotic resistance genes during the transformation of anaerobic digestion from mesophilic to thermophilic.Water Research,2016,98:261-269.
[26]Martínez JL.Antibiotics and antibiotic resistance genes in natural environments.Science,2008,321(5887):365-367.
[27]van Hoek AHAM,Mevius D,Guerra B,Mullany P,Roberts AP,Aarts HJM.Acquired antibiotic resistance genes:an overview.Frontiers in Microbiology,2011,2:203.
[28]Wang FH,Qiao M,Chen Z,Su JQ,Zhu YG.Antibiotic resistance genes in manure-amended soil and vegetables at harvest.Journal of Hazardous Materials,2015,299:215-221.
[29]Chen QL,An XL,Li H,Su JQ,Ma YB,Zhu YG.Long-term field application of sewage sludge increases the abundance of antibiotic resistance genes in soil.Environment International,2016,92-93:1-10.
[30]Marti R,Scott A,Tien YC,Murray R,Sabourin L,Zhang Y,Topp E.Impact of manure fertilization on the abundance of antibiotic-resistant bacteria and frequency of detection of antibiotic resistance genes in soil and on vegetables at harvest.Applied and Environmental Microbiology,2013,79(18):5701-5709.
[31]Ross J,Topp E.Abundance of antibiotic resistance genes in bacteriophage following soil fertilization with dairy manure or municipal biosolids,and evidence for potential transduction.Applied and Environmental Microbiology,2015,81(22):7905-7913.
[32]Bai Y,Müller DB,Srinivas G,Garrido-Oter R,Potthoff E,Rott M,Dombrowski N,Münch PC,Spaepen S,Remus-Emsermann M,Hüttel B,McHardy AC,Vorholt JA,Schulze-Lefert P.Functional overlap of the Arabidopsis leaf and root microbiota.Nature,2015,528(7582):364-369.
[33]Ruiz-Pérez CA,Restrepo S,Zambrano MM.Microbial and functional diversity within the phyllosphere of Espeletia species in an andean high-mountain ecosystem.Applied and Environmental Microbiology,2016,82(6):1807-1817.
[34]Zarraonaindia I,Owens SM,Weisenhorn P,West K,Hampton-Marcell J,Lax S,Bokulich NA,Mills DA,Martin G,Taghavi S,van der Lelie D,Gilbert JA.The soil microbiome influences grapevine-associated microbiota.mBio,2015,6(2):e02527-14.
[35]Bulgarelli D,Rott M,Schlaeppi K,Ver Loren van Themaat E,Ahmadinejad N,Assenza F,Rauf P,Huettel B,Reinhardt R,Schmelzer E,Peplies J,Gloeckner FO,Amann R,Eickhorst T,Schulze-Lefert P.Revealing structure and assembly cues for Arabidopsis root-inhabiting bacterial microbiota.Nature,2012,488(7409):91-95.
[36]Duan ML,Li HC,Gu J,Tuo XX,Sun W,Qian X,Wang XJ.Effects of biochar on reducing the abundance of oxytetracycline,antibiotic resistance genes,and human pathogenic bacteria in soil and lettuce.Environmental Pollution,2017,224:787-795.