制药废水厂微生物群落和多种抗性基因相关性分析
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摘要
为了研究多种生物处理工艺制药废水中微生物群落结构和抗性基因的分布特征、扩增情况及其相关性,采用Miseq高通量测序分析技术和荧光定量PCR技术对制药废水厂中活性污泥进行检测。荧光定量结果表明:sul1,sul2,tetO,tetQ,tetW,OXA-1和可移动遗传元件int1在制药废水厂中各个阶段均能被广泛地检测到,总抗性基因浓度范围为3.09×10~8～2.26×10~9 copies/g(干重),基因总浓度上升了7.3倍。Miseq测序结果表明:制药废水中主要优势菌门为Proteobacteria,Bacteroidetes,Firmicutes,Thermus和Gemmatimonadetes等门,其平均总相对丰度比例占到81.05%;冗余分析显示,Aeromicrobium与sul2呈较高程度的正相关性,可能是sul2在微生物群落中存在的可能的主要菌群;Rhodovulum和Rhodospirillaceae菌属与OXA-1和tetQ呈较高程度的正相关性,这些菌属是这些ARGs分布的主要菌属;Acinetobacter与tetO和tetW呈现较高程度的正相关性,可能是tetO和tetW在微生物群落中存在的可能的主要菌属。因此,相关抗性基因的增值和分布与相关特定菌属有关,可以通过控制相关菌属的丰度来消减工业废水厂中的抗性基因。
In order to study the distribution, amplification and correlation of microbial community structure and resistance genes in pharmaceutical wastewater, a variety of biological treatment processes are used to treat pharmaceutical wastewater. Combining Miseq high throughput sequencing analysis technology and fluorescence quantitative PCR technology, activated sludge in pharmaceutical wastewater plants is detected. The results show that sul1, sul2, tetO, tetQ, tetW, OXA-1 and INT1 are widely detected at all stages of pharmaceutical wastewater treatment plant. The total resistance gene concentration ranges from 3.09×10~8 to 2.26×10~9 copies/g(dry weight), and the total gene concentration increases by 7.3 times. Miseq sequencing results show that the dominant bacteria in pharmaceutical wastewater are Proteobacteria, Bacteroidetes, Firmicutes, Thermus and Gemmatimonadetes, with an average relative abundance ratio of 81.05%. Redundancy analysis shows that Aeromicrobium is positively correlated with sul2 to a high degree, which might be sul2 existing in microbial communities. Among the possible major microflora, Rhodovulum and Rhodospirillaceae are highly positively correlated with OXA-1 and tetQ, which are the main microflora of ARGs distribution. Acinetobacter has a high degree of positive correlation with tetO and tetW, which may be the main possible microflora of tetO and tetW in microbial community. The results show that appreciation and distribution of antibiotic resistance gens are connected with concerned specific bacterial genus. The controlled abundance of concerned bacterial genus could subduce resistance gene in industrial wastewater factory.
In order to study the distribution, amplification and correlation of microbial community structure and resistance genes in pharmaceutical wastewater, a variety of biological treatment processes are used to treat pharmaceutical wastewater. Combining Miseq high throughput sequencing analysis technology and fluorescence quantitative PCR technology, activated sludge in pharmaceutical wastewater plants is detected. The results show that sul1, sul2, tetO, tetQ, tetW, OXA-1 and INT1 are widely detected at all stages of pharmaceutical wastewater treatment plant. The total resistance gene concentration ranges from 3.09×10~8 to 2.26×10~9 copies/g(dry weight), and the total gene concentration increases by 7.3 times. Miseq sequencing results show that the dominant bacteria in pharmaceutical wastewater are Proteobacteria, Bacteroidetes, Firmicutes, Thermus and Gemmatimonadetes, with an average relative abundance ratio of 81.05%. Redundancy analysis shows that Aeromicrobium is positively correlated with sul2 to a high degree, which might be sul2 existing in microbial communities. Among the possible major microflora, Rhodovulum and Rhodospirillaceae are highly positively correlated with OXA-1 and tetQ, which are the main microflora of ARGs distribution. Acinetobacter has a high degree of positive correlation with tetO and tetW, which may be the main possible microflora of tetO and tetW in microbial community. The results show that appreciation and distribution of antibiotic resistance gens are connected with concerned specific bacterial genus. The controlled abundance of concerned bacterial genus could subduce resistance gene in industrial wastewater factory.
引文
[1] 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: 210.
[2] RODRIGUEZ-MOZAZ S, CHAMORRO S, MARTI E, et al. Occurrence of antibiotics and antibiotic resistance genes in hospital and urban wastewaters and their impact on the receiving river [J]. Water Research, 2015, 69: 234-242.
[3] 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.
[4] 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.
[5] FORSBERG K J, REYES A, WANG B, et al. The shared antibiotic resistome of soil bacteria and human pathogens [J]. Science, 2012, 337(6098): 1107-1111.
[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.
[7] 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.
[8] 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.
[9] 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.
[10] 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.
[11] 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.
[12] WANG F H, QIAO M, LYU Z E, et al. Impact of reclaimed water irrigation on antibiotic resistance in public parks, Beijing, China [J]. Environmental Pollution, 2014, 184(1): 247-253.
[13] XU Z, LI L, SHIRTLIFF M E, et al. Resistance class 1 integron in clinical methicillin‐resistant Staphylococcus aureus strains in southern China, 2001-2006 [J]. Clinical Microbiology & Infection, 2011, 17(5): 714-718.
[14] BEN-SHAHAR O, OBARA I, ARY A W, et al. Antimicrobial resistance of integron-harboring Escherichia coli isolates from clinical samples, wastewater treatment plant and river water [J]. Science of the Total Environment, 2012, 414(414): 680-685.
[15] LING A L, PACE N R, HERNANDEZ M T, et al. Tetracycline resistance and Class 1 integron genes associated with indoor and outdoor aerosols [J]. Environmental Science & Technology, 2013, 47(9): 4046-4052.
[16] 国家环境保护总局. 水和废水监测分析方法[M].第4版. 北京:中国环境科学出版社, 2002.
[17] GAZE W H, ZHANG L, ABDOUSLAM N A, et al. Impacts of anthropogenic activity on the ecology of class 1 integrons and integron-associated genes in the environment [J]. Isme Journal, 2011, 5(8): 1253.
[18] CHEN H, ZHANG M. Occurrence and removal of antibiotic resistance genes in municipal wastewater and rural domestic sewage treatment systems in eastern China [J]. Environment International, 2013, 55(4): 9.
[19] 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 Wenchao. The Fate and Control Principle of Antibiotic Resistance Genes in Pharmaceutical Wastewater Treatment Systems[D].Tianjin: Nankai University, 2014.
[20] LUO Y, MAO D, RYSZ M, et al. Trends in antibiotic resistance genes occurrence in the Haihe River, China [J]. Environmental Science & Technology, 2010, 44(19): 7220.
[21] AMINOV R I, GARRIGUESJEANJEAN N, MACKIE R I. Molecular ecology of tetracycline resistance: development and validation of primers for detection of tetracycline resistance genes encoding ribosomal protection proteins [J]. Applied & Environmental Microbiology, 2001, 67(1): 22.
[22] 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.
[23] PEAK N, KNAPP C W, YANG R K, et al. Abundance of six tetracycline resistance genes in wastewater lagoons at cattle feedlots with different antibiotic use strategies [J]. Environmental Microbiology, 2007, 9(1): 143-151.
[24] GUO X, XIA R, HAN N, et al. Genetic diversity analyses of class 1 integrons 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.
[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] HOA P T P, NONAKA L, VIET P H, et al. Detection of the sul1, sul2, and sul3 genes in sulfonamide-resistant bacteria from wastewater and shrimp ponds of north Vietnam [J]. Science of the Total Environment, 2008, 405(1/2/3): 377-384.
[27] MOURA A, PEREIRA C, HENRIQUES I, et al. Novel gene cassettes and integrons in antibiotic-resistant bacteria isolated from urban wastewaters [J]. Research in Microbiology, 2012, 163(2): 92-100.
[28] TENNSTEDT T, SZCZEPANOWSKI R, BRAUN S, et al. Occurrence of integron-associated resistance gene cassettes located on antibiotic resistance plasmids isolated from a wastewater treatment plant [J]. Fems Microbiology Ecology, 2010, 45(3): 239-252.
[29] DU J, REN H, GENG J, et al. Occurrence and abundance of tetracycline, sulfonamide resistance genes, and class 1 integron in five wastewater treatment plants [J]. Environmental Science & Pollution Research, 2014, 21(12): 7276-7284.
[30] 王丽梅, 罗义, 毛大庆, 等. 抗生素抗性基因在环境中的传播扩散及抗性研究方法 [J]. 应用生态学报, 2010, 21(4): 1063-1069.WANG Limei, LUO Yi, MAO Daqing, et al. Transport of antibiotic resistance genes in environment and detection methods of antibiotic resistance, 2010, 21(4): 1063-1069.
[31] 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 Xiaorong. Study on Hydrolysis and Acidification of Activated Sludge Enhanced by Ultrasound Combined with Alkaline [D]. Harbin:Harbin Institute of Technology, 2013.
[32] 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.
[2] RODRIGUEZ-MOZAZ S, CHAMORRO S, MARTI E, et al. Occurrence of antibiotics and antibiotic resistance genes in hospital and urban wastewaters and their impact on the receiving river [J]. Water Research, 2015, 69: 234-242.
[3] 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.
[4] 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.
[5] FORSBERG K J, REYES A, WANG B, et al. The shared antibiotic resistome of soil bacteria and human pathogens [J]. Science, 2012, 337(6098): 1107-1111.
[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.
[7] 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.
[8] 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.
[9] 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.
[10] 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.
[11] 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.
[12] WANG F H, QIAO M, LYU Z E, et al. Impact of reclaimed water irrigation on antibiotic resistance in public parks, Beijing, China [J]. Environmental Pollution, 2014, 184(1): 247-253.
[13] XU Z, LI L, SHIRTLIFF M E, et al. Resistance class 1 integron in clinical methicillin‐resistant Staphylococcus aureus strains in southern China, 2001-2006 [J]. Clinical Microbiology & Infection, 2011, 17(5): 714-718.
[14] BEN-SHAHAR O, OBARA I, ARY A W, et al. Antimicrobial resistance of integron-harboring Escherichia coli isolates from clinical samples, wastewater treatment plant and river water [J]. Science of the Total Environment, 2012, 414(414): 680-685.
[15] LING A L, PACE N R, HERNANDEZ M T, et al. Tetracycline resistance and Class 1 integron genes associated with indoor and outdoor aerosols [J]. Environmental Science & Technology, 2013, 47(9): 4046-4052.
[16] 国家环境保护总局. 水和废水监测分析方法[M].第4版. 北京:中国环境科学出版社, 2002.
[17] GAZE W H, ZHANG L, ABDOUSLAM N A, et al. Impacts of anthropogenic activity on the ecology of class 1 integrons and integron-associated genes in the environment [J]. Isme Journal, 2011, 5(8): 1253.
[18] CHEN H, ZHANG M. Occurrence and removal of antibiotic resistance genes in municipal wastewater and rural domestic sewage treatment systems in eastern China [J]. Environment International, 2013, 55(4): 9.
[19] 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 Wenchao. The Fate and Control Principle of Antibiotic Resistance Genes in Pharmaceutical Wastewater Treatment Systems[D].Tianjin: Nankai University, 2014.
[20] LUO Y, MAO D, RYSZ M, et al. Trends in antibiotic resistance genes occurrence in the Haihe River, China [J]. Environmental Science & Technology, 2010, 44(19): 7220.
[21] AMINOV R I, GARRIGUESJEANJEAN N, MACKIE R I. Molecular ecology of tetracycline resistance: development and validation of primers for detection of tetracycline resistance genes encoding ribosomal protection proteins [J]. Applied & Environmental Microbiology, 2001, 67(1): 22.
[22] 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.
[23] PEAK N, KNAPP C W, YANG R K, et al. Abundance of six tetracycline resistance genes in wastewater lagoons at cattle feedlots with different antibiotic use strategies [J]. Environmental Microbiology, 2007, 9(1): 143-151.
[24] GUO X, XIA R, HAN N, et al. Genetic diversity analyses of class 1 integrons 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.
[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] HOA P T P, NONAKA L, VIET P H, et al. Detection of the sul1, sul2, and sul3 genes in sulfonamide-resistant bacteria from wastewater and shrimp ponds of north Vietnam [J]. Science of the Total Environment, 2008, 405(1/2/3): 377-384.
[27] MOURA A, PEREIRA C, HENRIQUES I, et al. Novel gene cassettes and integrons in antibiotic-resistant bacteria isolated from urban wastewaters [J]. Research in Microbiology, 2012, 163(2): 92-100.
[28] TENNSTEDT T, SZCZEPANOWSKI R, BRAUN S, et al. Occurrence of integron-associated resistance gene cassettes located on antibiotic resistance plasmids isolated from a wastewater treatment plant [J]. Fems Microbiology Ecology, 2010, 45(3): 239-252.
[29] DU J, REN H, GENG J, et al. Occurrence and abundance of tetracycline, sulfonamide resistance genes, and class 1 integron in five wastewater treatment plants [J]. Environmental Science & Pollution Research, 2014, 21(12): 7276-7284.
[30] 王丽梅, 罗义, 毛大庆, 等. 抗生素抗性基因在环境中的传播扩散及抗性研究方法 [J]. 应用生态学报, 2010, 21(4): 1063-1069.WANG Limei, LUO Yi, MAO Daqing, et al. Transport of antibiotic resistance genes in environment and detection methods of antibiotic resistance, 2010, 21(4): 1063-1069.
[31] 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 Xiaorong. Study on Hydrolysis and Acidification of Activated Sludge Enhanced by Ultrasound Combined with Alkaline [D]. Harbin:Harbin Institute of Technology, 2013.
[32] 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.