制药废水厂抗性基因和微生物群落相关性研究
|
摘要
以2座制药废水厂的生物曝气阶段为例,结合Miseq测序分析技术和荧光定量PCR技术研究活性污泥中微生物群落和β-内酰胺类抗性基因的分布特征、扩增情况及其相关性.结果表明:β-内酰胺类抗性基因OXA-1、OXA-2和OXA-10在2个水厂(J厂和K厂)中均能被检出,OXA型基因丰度在K厂中为5.82×10~5～3.94×10~7copies/g(干重),在J厂的丰度范围为4.84×10~7～1.09×10~(10)copies/g,3种基因丰度在曝气处理中显著扩增. Miseq测序结果表明:K厂中主要优势菌门为Proteobacteria,Planctomycetes,Bacteroidetes,Chloroflexi和Acidobacteria等,总平均相对丰度比例为82.13%;J厂中主要优势菌门Proteobacteria,Bacteroidetes,Verrucomicrobia,Gemmatimonadetes和Thermi,总平均相对丰度比例为85.76%.冗余分析显示:生物群落中Bdellovibrio、KD8-87和Paracoccus等菌属可能是OXA-1、OXA-2和OXA-10的主要携带菌属;Hyphomicrobium、Thermomonas和Comamonadaceae可能是OXA-1的主要携带菌属,Caldilineaceae、Myxococcales和Pirellulaceae可能是OXA-10的主要携带菌属.
The distribution characteristics, amplification and correlation of microbial communities and β-lactam resistance genes inactive sludge were studied by Miseq sequencing analysis and fluorescence quantitative PCR. The samples of active sludge wereobtained from the biological aeration stages of two pharmaceutical wastewater treatment plant(Plant J and K). β-lactam resistancegenes OXA-1, OXA-2 and OXA-10 can be detected in both plants. The abundance of OXA gene was 5.82×10~5～3.94×10~7 copies/g(dry weight) in K plant and was 4.84×10~7～1.09×10~(10) copies/g in J plant. The Abundance of three genes in aeration treatment issignificantly amplified. Miseq sequencing showed that the main dominant bacterium were Proteobacteria, Planctomycetes,Bacteroidetes, Chloroflexi and Acidobacteria in K plant and the total average relative abundance ratio was 85.76%. The maindominant bacterium were Proteobacteria, Bacteroidetes, Verrucomicrobia, Gemmatimonadetes and Thermi in J plant, the totalaverage relative abundance ratio was 85.76%. Redundancy analysis display showed that Bdellovibrio、KD8-87 and Paracoccus inbiological community were the major distribution factors of OXA-1、OXA-2 and OXA-10. Hyphomicrobium、Thermomonas andComamonadaceae were the major distribution factor of OXA-1. Caldilineaceae、Myxococcales and Pirellulaceae were the majordistribution factor of OXA-10.
The distribution characteristics, amplification and correlation of microbial communities and β-lactam resistance genes inactive sludge were studied by Miseq sequencing analysis and fluorescence quantitative PCR. The samples of active sludge wereobtained from the biological aeration stages of two pharmaceutical wastewater treatment plant(Plant J and K). β-lactam resistancegenes OXA-1, OXA-2 and OXA-10 can be detected in both plants. The abundance of OXA gene was 5.82×10~5～3.94×10~7 copies/g(dry weight) in K plant and was 4.84×10~7～1.09×10~(10) copies/g in J plant. The Abundance of three genes in aeration treatment issignificantly amplified. Miseq sequencing showed that the main dominant bacterium were Proteobacteria, Planctomycetes,Bacteroidetes, Chloroflexi and Acidobacteria in K plant and the total average relative abundance ratio was 85.76%. The maindominant bacterium were Proteobacteria, Bacteroidetes, Verrucomicrobia, Gemmatimonadetes and Thermi in J plant, the totalaverage relative abundance ratio was 85.76%. Redundancy analysis display showed that Bdellovibrio、KD8-87 and Paracoccus inbiological community were the major distribution factors of OXA-1、OXA-2 and OXA-10. Hyphomicrobium、Thermomonas andComamonadaceae were the major distribution factor of OXA-1. Caldilineaceae、Myxococcales and Pirellulaceae were the majordistribution factor of OXA-10.
引文
[1]Borg M A,Zarb P,Scicluna E A,et al.Antibiotic consumption as a driver for resistance in Staphylococcus aureus and Escherichia coli within a developing region[J].Ajic American Journal of Infection Control,2010,38(3):212-216.
[2]Naquin A,Shrestha A,Sherpa M,et al.Presence of antibiotic resistance genes in a sewage treatment plant in Thibodaux,Louisiana,USA[J].Bioresource Technology,2015,188(11):79-83.
[3]Tong Z,Zhang X X,Lin Y.Plasmid metagenome reveals high levels of antibiotic resistance genes and mobile genetic elements in activated sludge[J].Plos One,2011,6(10):e26041.
[4]Pehrsson E C,Tsukayama P,Patel S,et al.Interconnected microbiomes and resistomes in low-income human habitats[J].Nature,2016,533(7602):212-216.
[5]He L Y,Ying G G,Liu Y S,et al.Discharge of swine wastes risks water quality and food safety:Antibiotics and antibiotic resistance genes from swine sources to the receiving environments[J].Environment International,2016:92-93.
[6]Pruden A,Pei R,Storteboom H,et al.Antibiotic resistance genes as emerging contaminants:studies in northern Colorado[J].Environmental Science&Technology,2006,40(23):7445-7450.
[7]王玉倩,薛秀花.实时荧光定量PCR技术研究进展及其应用[J].生物学通报,2016,51(2):1-6.Wang Y Q,Xue X H.The application and development of real-time fluorescent quantitative PCR[J].Bulletin of Biology,2016,51(2):1-6.
[8]Aydin S,Ince B,Ince O.Development of antibiotic resistance genes in microbial communities during long-term operation of anaerobic reactors in the treatment of pharmaceutical wastewater[J].Water Research,2015,83(4):337-344.
[9]Liu M,Zhang Y,Yang M,et al.Abundance and distribution of tetracycline resistance genes and mobile elements in an oxytetracycline production wastewater treatment system[J].Environmental Science&Technology,2012,46(14):7551-7557.
[10]Guo X,Yan Z,Zhang Y,et al.Behavior of antibiotic resistance genes under extremely high-level antibiotic selection pressures in pharmaceutical wastewater treatment plants[J].Science of the Total Environment,2017,612:119-128.
[11]张佳奇.湘江流域β内酰胺抗性基因污染特征与影响因素研究[D].天津:天津大学,2015.Zhang J Q.Pollution characteristics and influencing factors of beta lactam resistant genes in Xiangjiang Basin[D].Tianjin:Tianjin University,2015.
[12]Li Y,Sun Q L,Shen Y,et al.Rapid increase in the prevalence of carbapenem-resistant Enterobacteriaceae(CRE)and emergence of colistin resistance gene mcr-1in CRE in a hospital in Henan,China[J].Journal of Clinical Microbiology,2018,JCM.01932-01917.
[13]Diwan V,Chandran S P,Tamhankar A J,et al.Identification of extended-spectrumβ-lactamase and quinolone resistance genes in Escherichia coli isolated from hospital wastewater from central India[J].Journal of Antimicrobial Chemotherapy,2012,67(4):857-859.
[14]Yang Y,Li B,Zou S,et al.Fate of antibiotic resistance genes in sewage treatment plant revealed by metagenomic approach[J].Water Research,2014,62(7):97-106.
[15]Zhang T,Li B.Occurrence,transformation,and fate of antibiotics in municipal wastewater treatment plants[J].Critical Reviews in Environmental Science&Technology,2011,41(11):951-998.
[16]Wang J,Mao D,Mu Q,et al.Fate and proliferation of typical antibiotic resistance genes in five full-scale pharmaceutical wastewater treatment plants[J].Science of the Total Environment,2015,526(4):366-373.
[17]国家环境保护总局.水和废水监测分析方法.[M].4版.北京:中国环境科学出版社,2002.The State Environmental Protection Administration.Water and wastewater monitoring and analysis method[M].4th Edition.Beijing:China Environmental Science Press,2002.
[18]翟文超.抗生素抗性基因在抗生素制药废水处理过程中的分布特征及控制原理研究[D].天津:南开大学,2014.Zhai W.The fate and control principle of antibiotic resistance genes in pharmaceutical wastewater treatment systems[D].Tianjin:Nankai University,2014.
[19]余忻.抗生素废水的生物毒性与微生物耐药性及其控制技术研究[D].北京:清华大学,2014.Yu X.Biotoxicity and antimicrobial resistance of antibiotic wastewater and its control technology[D].Beijing:Tsinghua University,2014.
[20]Shu D,He Y,Yue H,et al.Metagenomic insights into the effects of volatile fatty acids on microbial community structures and functional genes in organotrophic anammox process[J].Bioresource Technology,2015,196:621-633.
[21]王智,张志勇,张君倩,等.水葫芦修复富营养化湖泊水体区域内外底栖动物群落特征[J].中国环境科学,2012,32(1):142-149.Wang Z,Zhang Z Y,Zhang J Q,et al.The fauna structure of benthic macro-invertebrates for environmental restoration in a eutrophic lake using water hyacinths[J].China Environmental Science,2012,32(1):142-149.
[22]刁晓君,李一葳,王曙光.水华生消过程对巢湖沉积物微生物群落结构的影响[J].环境科学,2015,(1):107-113.Diao X J,Li Y W,Wang S G.Effects of outbreak and extinction of algal blooms on the microbial community structure in sediments of Chaohu Lake[J].Environmental Science,2015,(1):107-113.
[23]郑向阳,罗晓,袁立霞,等.AO工艺处理淀粉污水效能及微生物群落解析[J/OL][J].环境工程学报,2018,3:804-814.Zheng X,Luo X,Yuan L,et al.Performance and bacterial community analysis of AO process treating starch wastewater[J].Chinese Journal of Environmental Engineering,2018,12(3):804-814.
[24]康晓荣.超声联合碱促进剩余污泥水解酸化及产物研究[D].哈尔滨:哈尔滨工业大学,2013.Kang X R.Study on hydrolysis and acidification of activated sludge enhanced by ultrasound combined with alkaline[D].Harbin:Harbin institute of technology,2013.
[25]Liu Y J,Wang X C,Yuan H L.Characterization of microbial communities in a fluidized-pellet-bed bioreactor for wastewater treatment[J].Desalination,2009,249(1):445-452.
[26]Nielsen P H,Mielczarek A T,Kragelund C,et al.A conceptual ecosystem model of microbial communities in enhanced biological phosphorus removal plants[J].Water Research,2010,44(17):5070-5088.
[27]高大文,辛晓东.MBR膜污染过程中微生物群落结构与代谢产物分析[J].哈尔滨工业大学学报,2014,46(2):26-32.Gao D W,Xin X D.Analysis of microbial community structure and metabolites during the MBR membrane fouling process[J].Journal of Harbin Institute of Technology,2014,46(2):26-32.
[28]刘燕,甘莉,黄哲强,等.脱氮副球菌YF1目的反硝化特性研究[J].水处理技术,2010,36(10):61-65.Liu Y,Gan L,Huang Z Q,et al.Study on denitrification characteristics of Paracoccus YF1[J].Technology of Water Treatment,2010,36(10):61-65.
[29]李卫华,孙英杰,刘子梁,等.序批式生物反应器填埋场脱氮微生物多样性分析[J].环境科学,2016,37(1):342-349.Li W,Sun Y,Liu Z,et al.analysis on diversity of denitrifying microorganisms in sequential batch bioreactor landfill[J].Environmental Science,2016,37(1):342-349.
[30]魏京焘,龚雪,李雪,等.不同地区污水厂尾水中微生物群落结构多样性研究[J].安徽农业科学,2016,(11):23-25.Wei J,Gong X,Li X,et al.Research on the Diversity of microbial community structure of tail water from sewage plants in different areas[J].Journal of Anhui Agricultural Sciences,2016,(11):23-25.
[31]罗晓,郑向阳,赵丛丛,等.A/O工艺中污泥浓度对微生物群落结构的影响[J].中国环境科学,2018,38(1):275-283.Luo X,Zheng X,Zhao C,et al.Effects of sludge concentration on microbial community structure in A/O process[J].China Environmental Science,2018,38(1):275-283.
[32]孙涛.水的深度处理生物安全性及自养反硝化研究[D].北京:中国地质大学,2009.Sun T.Study on biological safety and autotrophic denitrification of advanced treatment of water[D].Beijing:China University of Geosciences,2009.
[33]Guo X,Xia R,Han N,et al.Genetic diversity analyses of class1integrons and their associated antimicrobial resistance genes in Enterobacteriaceae strains recovered from aquatic habitats in China[J].Letters in Applied Microbiology,2011,52(6):667-675.
[34]Laviad S,Lapidus A,Han J,et al.High quality draft genome sequence of Brachymonas chironomi AIMA4(T)(DSM 19884(T))isolated from a Chironomus sp.egg mass.[J].Standards in Genomic Sciences,2015,10(1):29.
[35]Amiri J M,Poehlein A,Fisch K,et al.Draft genome sequences of the obligatory marine myxobacterial strains enhygromyxa salina SWB005and SWB007[J].Genome Announcements,2018,6(17):e00324-18.
[2]Naquin A,Shrestha A,Sherpa M,et al.Presence of antibiotic resistance genes in a sewage treatment plant in Thibodaux,Louisiana,USA[J].Bioresource Technology,2015,188(11):79-83.
[3]Tong Z,Zhang X X,Lin Y.Plasmid metagenome reveals high levels of antibiotic resistance genes and mobile genetic elements in activated sludge[J].Plos One,2011,6(10):e26041.
[4]Pehrsson E C,Tsukayama P,Patel S,et al.Interconnected microbiomes and resistomes in low-income human habitats[J].Nature,2016,533(7602):212-216.
[5]He L Y,Ying G G,Liu Y S,et al.Discharge of swine wastes risks water quality and food safety:Antibiotics and antibiotic resistance genes from swine sources to the receiving environments[J].Environment International,2016:92-93.
[6]Pruden A,Pei R,Storteboom H,et al.Antibiotic resistance genes as emerging contaminants:studies in northern Colorado[J].Environmental Science&Technology,2006,40(23):7445-7450.
[7]王玉倩,薛秀花.实时荧光定量PCR技术研究进展及其应用[J].生物学通报,2016,51(2):1-6.Wang Y Q,Xue X H.The application and development of real-time fluorescent quantitative PCR[J].Bulletin of Biology,2016,51(2):1-6.
[8]Aydin S,Ince B,Ince O.Development of antibiotic resistance genes in microbial communities during long-term operation of anaerobic reactors in the treatment of pharmaceutical wastewater[J].Water Research,2015,83(4):337-344.
[9]Liu M,Zhang Y,Yang M,et al.Abundance and distribution of tetracycline resistance genes and mobile elements in an oxytetracycline production wastewater treatment system[J].Environmental Science&Technology,2012,46(14):7551-7557.
[10]Guo X,Yan Z,Zhang Y,et al.Behavior of antibiotic resistance genes under extremely high-level antibiotic selection pressures in pharmaceutical wastewater treatment plants[J].Science of the Total Environment,2017,612:119-128.
[11]张佳奇.湘江流域β内酰胺抗性基因污染特征与影响因素研究[D].天津:天津大学,2015.Zhang J Q.Pollution characteristics and influencing factors of beta lactam resistant genes in Xiangjiang Basin[D].Tianjin:Tianjin University,2015.
[12]Li Y,Sun Q L,Shen Y,et al.Rapid increase in the prevalence of carbapenem-resistant Enterobacteriaceae(CRE)and emergence of colistin resistance gene mcr-1in CRE in a hospital in Henan,China[J].Journal of Clinical Microbiology,2018,JCM.01932-01917.
[13]Diwan V,Chandran S P,Tamhankar A J,et al.Identification of extended-spectrumβ-lactamase and quinolone resistance genes in Escherichia coli isolated from hospital wastewater from central India[J].Journal of Antimicrobial Chemotherapy,2012,67(4):857-859.
[14]Yang Y,Li B,Zou S,et al.Fate of antibiotic resistance genes in sewage treatment plant revealed by metagenomic approach[J].Water Research,2014,62(7):97-106.
[15]Zhang T,Li B.Occurrence,transformation,and fate of antibiotics in municipal wastewater treatment plants[J].Critical Reviews in Environmental Science&Technology,2011,41(11):951-998.
[16]Wang J,Mao D,Mu Q,et al.Fate and proliferation of typical antibiotic resistance genes in five full-scale pharmaceutical wastewater treatment plants[J].Science of the Total Environment,2015,526(4):366-373.
[17]国家环境保护总局.水和废水监测分析方法.[M].4版.北京:中国环境科学出版社,2002.The State Environmental Protection Administration.Water and wastewater monitoring and analysis method[M].4th Edition.Beijing:China Environmental Science Press,2002.
[18]翟文超.抗生素抗性基因在抗生素制药废水处理过程中的分布特征及控制原理研究[D].天津:南开大学,2014.Zhai W.The fate and control principle of antibiotic resistance genes in pharmaceutical wastewater treatment systems[D].Tianjin:Nankai University,2014.
[19]余忻.抗生素废水的生物毒性与微生物耐药性及其控制技术研究[D].北京:清华大学,2014.Yu X.Biotoxicity and antimicrobial resistance of antibiotic wastewater and its control technology[D].Beijing:Tsinghua University,2014.
[20]Shu D,He Y,Yue H,et al.Metagenomic insights into the effects of volatile fatty acids on microbial community structures and functional genes in organotrophic anammox process[J].Bioresource Technology,2015,196:621-633.
[21]王智,张志勇,张君倩,等.水葫芦修复富营养化湖泊水体区域内外底栖动物群落特征[J].中国环境科学,2012,32(1):142-149.Wang Z,Zhang Z Y,Zhang J Q,et al.The fauna structure of benthic macro-invertebrates for environmental restoration in a eutrophic lake using water hyacinths[J].China Environmental Science,2012,32(1):142-149.
[22]刁晓君,李一葳,王曙光.水华生消过程对巢湖沉积物微生物群落结构的影响[J].环境科学,2015,(1):107-113.Diao X J,Li Y W,Wang S G.Effects of outbreak and extinction of algal blooms on the microbial community structure in sediments of Chaohu Lake[J].Environmental Science,2015,(1):107-113.
[23]郑向阳,罗晓,袁立霞,等.AO工艺处理淀粉污水效能及微生物群落解析[J/OL][J].环境工程学报,2018,3:804-814.Zheng X,Luo X,Yuan L,et al.Performance and bacterial community analysis of AO process treating starch wastewater[J].Chinese Journal of Environmental Engineering,2018,12(3):804-814.
[24]康晓荣.超声联合碱促进剩余污泥水解酸化及产物研究[D].哈尔滨:哈尔滨工业大学,2013.Kang X R.Study on hydrolysis and acidification of activated sludge enhanced by ultrasound combined with alkaline[D].Harbin:Harbin institute of technology,2013.
[25]Liu Y J,Wang X C,Yuan H L.Characterization of microbial communities in a fluidized-pellet-bed bioreactor for wastewater treatment[J].Desalination,2009,249(1):445-452.
[26]Nielsen P H,Mielczarek A T,Kragelund C,et al.A conceptual ecosystem model of microbial communities in enhanced biological phosphorus removal plants[J].Water Research,2010,44(17):5070-5088.
[27]高大文,辛晓东.MBR膜污染过程中微生物群落结构与代谢产物分析[J].哈尔滨工业大学学报,2014,46(2):26-32.Gao D W,Xin X D.Analysis of microbial community structure and metabolites during the MBR membrane fouling process[J].Journal of Harbin Institute of Technology,2014,46(2):26-32.
[28]刘燕,甘莉,黄哲强,等.脱氮副球菌YF1目的反硝化特性研究[J].水处理技术,2010,36(10):61-65.Liu Y,Gan L,Huang Z Q,et al.Study on denitrification characteristics of Paracoccus YF1[J].Technology of Water Treatment,2010,36(10):61-65.
[29]李卫华,孙英杰,刘子梁,等.序批式生物反应器填埋场脱氮微生物多样性分析[J].环境科学,2016,37(1):342-349.Li W,Sun Y,Liu Z,et al.analysis on diversity of denitrifying microorganisms in sequential batch bioreactor landfill[J].Environmental Science,2016,37(1):342-349.
[30]魏京焘,龚雪,李雪,等.不同地区污水厂尾水中微生物群落结构多样性研究[J].安徽农业科学,2016,(11):23-25.Wei J,Gong X,Li X,et al.Research on the Diversity of microbial community structure of tail water from sewage plants in different areas[J].Journal of Anhui Agricultural Sciences,2016,(11):23-25.
[31]罗晓,郑向阳,赵丛丛,等.A/O工艺中污泥浓度对微生物群落结构的影响[J].中国环境科学,2018,38(1):275-283.Luo X,Zheng X,Zhao C,et al.Effects of sludge concentration on microbial community structure in A/O process[J].China Environmental Science,2018,38(1):275-283.
[32]孙涛.水的深度处理生物安全性及自养反硝化研究[D].北京:中国地质大学,2009.Sun T.Study on biological safety and autotrophic denitrification of advanced treatment of water[D].Beijing:China University of Geosciences,2009.
[33]Guo X,Xia R,Han N,et al.Genetic diversity analyses of class1integrons and their associated antimicrobial resistance genes in Enterobacteriaceae strains recovered from aquatic habitats in China[J].Letters in Applied Microbiology,2011,52(6):667-675.
[34]Laviad S,Lapidus A,Han J,et al.High quality draft genome sequence of Brachymonas chironomi AIMA4(T)(DSM 19884(T))isolated from a Chironomus sp.egg mass.[J].Standards in Genomic Sciences,2015,10(1):29.
[35]Amiri J M,Poehlein A,Fisch K,et al.Draft genome sequences of the obligatory marine myxobacterial strains enhygromyxa salina SWB005and SWB007[J].Genome Announcements,2018,6(17):e00324-18.