海南东寨港海水和沉积物中抗生素抗性基因污染特征研究
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摘要
抗生素滥用及其诱导产生的抗生素抗性基因(Antibioticresistancegenes,ARGs)污染已经引起各界的广泛关注。东寨港是海南省重要的滩涂养殖基地,其ARGs的产生、传播和富集都可能对人类及生态健康产生危害。采集该区域海水和沉积物样本各10个,利用LC-MS/MS对海水和沉积物中磺胺类、四环素类、氯霉素类和喹诺酮类等4类15种抗生素进行测定,同时采用RT-PCR阐明了相应的12种ARGs(sul1、sul2、dfr A1;tet A、tet C、tet G、tet M;cata1、cata2、cmle1、cmle3;qnr S)及16S rRNA的分布特征,并对抗生素和ARGs的相关性进行分析。结果显示,所测ARGs在各沉积物样品中的检出率均为100%,海水样品中ARGs的检出率在80%-100%之间。其中,qnr S的检出率最高,存在于所有海水和沉积物样品中,而sul2的检出丰度最高。比较海水和沉积物中ARGs的相对丰度,发现沉积物中ARGs的污染程度高于海水。样品中抗生素总体检出率较低,其中沉积物仅有喹诺酮及磺胺类抗生素被检出,海水中除磺胺类抗生素外其他均未检出。与其他研究相比,该区域ARGs检出水平较高,而抗生素检出水平较低,ARGs含量与抗生素浓度相关性较弱(P>0.05),这说明由历史背景诱导产生的ARGs可进行自我扩增而持续存在于环境中。该研究结果可为当地抗生素的安全使用和ARGs的环境风险评价提供重要依据。
The abuse of antibiotics and the contamination of antibiotic resistance genes(ARGs) induced by antibiotics have attracted extensive attentions. Dongzhai Harbor, as an important tidal flat aquaculture base in Hainan Province, the presence, spreading and accumulating of ARGs may pose potential public health risks on human and environments. In this study, we collected 10 sediment samples and 10 seawater samples from Dongzhai Harbor and quantified 15 kinds of antibiotic belonging to 4 common classes, which were sulfonamides, tetracyclines, chloramphenicol and quinolones using LC-MS/MS. Meanwhile, we also analyzed the distribution patterns of corresponding 12 ARGs(sul1, sul2, dfrA1; tetA, tetC, tetG, tetM; cata1, cata2, cmle1, cmle3; qnrS) as well as the 16S rRNA elucidating by RT-PCR. The correlations between these ARGs and antibiotics were additionally analyzed. The results showed that in sediment samples, the detection rate of measured ARGs was 100%, while in seawater samples, the detection rate of single ARG was varied from 80% to 100%. Among these 12 ARGs, qnrS existed in all the samples with highest detection rate, while sul2 had highest abundance. A comparative analysis of the relative abundance of ARGs in sediments and seawater revealed that the sediment was affected more by ARGs than seawater. The overall detection rate of antibiotics in the samples was low, with only two types of antibiotics(quinolones and sulfa) detected in the sediments, and one(sulfa antibiotics) in the seawater. The correlations between antibiotics(low concentration) and ARGs(high abundance) in the samples were weak(P>0.05), which indicated that ARGs generated before could persist in the environment, likely caused by self-amplification ability of ARGs. The results of this study will provide information and insight for understanding the safe use of antibiotics, as well as environmental risk assessment of ARGs,which should be useful for their management.
The abuse of antibiotics and the contamination of antibiotic resistance genes(ARGs) induced by antibiotics have attracted extensive attentions. Dongzhai Harbor, as an important tidal flat aquaculture base in Hainan Province, the presence, spreading and accumulating of ARGs may pose potential public health risks on human and environments. In this study, we collected 10 sediment samples and 10 seawater samples from Dongzhai Harbor and quantified 15 kinds of antibiotic belonging to 4 common classes, which were sulfonamides, tetracyclines, chloramphenicol and quinolones using LC-MS/MS. Meanwhile, we also analyzed the distribution patterns of corresponding 12 ARGs(sul1, sul2, dfrA1; tetA, tetC, tetG, tetM; cata1, cata2, cmle1, cmle3; qnrS) as well as the 16S rRNA elucidating by RT-PCR. The correlations between these ARGs and antibiotics were additionally analyzed. The results showed that in sediment samples, the detection rate of measured ARGs was 100%, while in seawater samples, the detection rate of single ARG was varied from 80% to 100%. Among these 12 ARGs, qnrS existed in all the samples with highest detection rate, while sul2 had highest abundance. A comparative analysis of the relative abundance of ARGs in sediments and seawater revealed that the sediment was affected more by ARGs than seawater. The overall detection rate of antibiotics in the samples was low, with only two types of antibiotics(quinolones and sulfa) detected in the sediments, and one(sulfa antibiotics) in the seawater. The correlations between antibiotics(low concentration) and ARGs(high abundance) in the samples were weak(P>0.05), which indicated that ARGs generated before could persist in the environment, likely caused by self-amplification ability of ARGs. The results of this study will provide information and insight for understanding the safe use of antibiotics, as well as environmental risk assessment of ARGs,which should be useful for their management.
引文
CESARE A D,ECKERT E M,ROGORA M,et al.,2017.Rainfall increases the abundance of antibiotic resistance genes within a riverine microbial community[J].Environmental Pollution,226:473-478.
CZEKALSKI N,DIEZ E G,BURGMANN N,2014.Wastewater as a point source of antibiotic-resistance genes in the sediment of a freshwater lake[J].The ISME Journal,8(7):1381-1390.
GOGARTEN J P,TOWNSEND J P,2005.Horizontal gene transfer,genome innovation and evolution[J].Nature Reviews Microbiology,3(9):679-687.
GUO X P,LIU X R,NIU Z S,et al.,2018.Seasonal and spatial distribution of antibiotic resistance genes in the sediments along the Yangtze Estuary,China[J].Environmental Pollution,242(Part A):576-584.
HARNISZ M,KORZENIEWSKA E,GOLAS I,2015.The impact of a freshwater fish farm on the community of tetracycline-resistant bacteria and the structure of tetracycline resistance genes in river water[J].Chemosphere,128:134-141.
HARTMANN A,ALDER A C,KOLLER T,et al.,1998.Identification of fluoroquinolone antibiotics as the main source of umuC genotoxicity in native hospital wastewater[J].Environmental Toxicology&Chemistry,17(3):377-382.
HE X T,WANG Z H,NIE X P,et al.,2012.Residues of fluoroquinolones in marine aquaculture environment of the Pearl River Delta,South China[J].Environmental Geochemistry&Health,34(3):323-335.
HU J Y,SHI J C,CHANG H,et al.,2008.Phenotyping and genotyping of antibiotic-resistant Escherichia coli isolated from a natural river basin[J].Environmental Science&Technology,42(9):3415-3420.
HU Y,YAN X,SHEN Y,et al.,2018.Antibiotics in surface water and sediments from Hanjiang River,Central China:Occurrence,behavior and risk assessment[J].Ecotoxicology&Environmental Safety,157:150-158.
JIA J,GUAN Y J,CHENG M Q,et al.,2018.Occurrence and distribution of antibiotics and antibiotic resistance genes in Ba River,China[J].Science of the Total Environment,642:1136-1144.
JIANG L,HU X,XU T,et al.,2013.Prevalence of antibiotic resistance genes and their relationship with antibiotics in the Huangpu River and the drinking water sources,Shanghai,China[J].Science of the Total Environment,458-460(3):267-272.
LI Y,LI Q,ZHOU K,et al.,2016.Occurrence and distribution of the environmental pollutant antibiotics in Gaoqiao mangrove area,China[J].Chemosphere,147:25-35.
LING Z H,YANG Y,HUANG Y L,et al.,2013.A preliminary investigation on the occurrence and distribution of antibiotic resistance genes in the Beijiang River,South China[J].Journal of Environmental Sciences,25(8):1656-1661.
LUO Y,MAO D Q,RYSZ M,et al.,2010.Trends in antibiotic resistance genes occurrence in the Haihe River,China[J].Environmental Science&Technology,44(19):7220-7225.
MACKIE R I,KOIKE S,KRAPAC I,et al.,2006.Tetracycline Residues and Tetracycline Resistance Genes in Groundwater Impacted by Swine Production Facilities[J].Animal Biotechnology,17(2):157-176.
MARTI E,HUERTA B,RODRIGUEZMOZAZ S,et al.,2018.Abundance of antibiotic resistance genes and bacterial community composition in wild freshwater fish species[J].Chemosphere,196:115-119.
MCKINNEY C W,LOFTIN K A,MEYER M T,et al.,2010.Tet and sul antibiotic resistance genes in livestock lagoons of various operation type,configuration,and antibiotic occurrence[J].Environmental Science&Technology,44(16):6102-6109.
MUZIASARI W I,PARNANEN K,JOHNSON T A,et al.,2016.Aquaculture changes the profile of antibiotic resistance and mobile genetic element associated genes in Baltic Sea sediments[J].Fems Microbiology Ecology,92(4):fiw052.
PARK H R,KIM T H,BARK K M,2002.Physicochemical properties of quinolone antibiotics in various environments[J].European Journal of Medicinal Chemistry,37(6):443-460.
PEAK N,KNAPP C W,YANG R K,et al.,2007.Abundance of six tetracycline resistance genes in wastewater lagoons at cattle feedlots with different antibiotic use strategies[J].Environmental Microbiology,9(1):143-151.
PRUDEN A,PEI R,STORTEBOOM H,et al.,2006.Antibiotic Resistance Genes as Emerging Contaminants:?Studies in Northern Colorado[J].Environmental Science&Technology,40(23):7445-7450.
QIAN X,GU J,SUN W,et al.,2017.Diversity,abundance,and persistence of antibiotic resistance genes in various types of animal manure following industrial composting[J].Journal of Hazardous Materials,344:716-722.
SCHLüTER A,SZCZEPANOWSKI R,PUHLER A,et al.,2007.Genomics of IncP-1 antibiotic resistance plasmids isolated from wastewater treatment plants provides evidence for a widely accessible drug resistance gene pool[J].Fems Microbiology Reviews,31(4):449-477.
SK?LD O,2000.Sulfonamide resistance:mechanisms and trends[J].Drug Resistance Updates Reviews&Commentaries in Antimicrobial&Anticancer Chemotherapy,3(3):155-160.
STALIN N,SRINIVASAN P,2016.Molecular characterization of antibiotic resistant Vibrio harveyi isolated from shrimp aquaculture environment in the south east coast of India[J].Microb Pathog,97:110-118.
TENENBAUM I,CHEFETZ B,AVISAR D,et al.,2014.Physicochemical behavior of tetracycline and 17α-ethinylestradiol with wastewater sludge-derived humic substances[J].Water Air&Soil Pollution,225(10):1-11.
THIELE-BRUHN S,2003.Pharmaceutical antibiotic compounds in soils-a review[J].Journal of Plant Nutrition and Soil Science,166(2):145-167.
TURTON J A,HAVARD A C,ROBINSON S,et al.,2000.An assessment of chloramphenicol and thiamphenicol in the induction of aplastic anaemia in the BALB/c mouse[J].Food&Chemical Toxicology,38(10):925-938.
WANG N,GUO X Y,YAN Z,et al.,2016.A Comprehensive Analysis on Spread and Distribution Characteristic of Antibiotic Resistance Genes in Livestock Farms of Southeastern China[J].PLoS ONE 11(7):e0156889.
ZHANG Q Q,YING G G,PAN C G,et al.,2015.Comprehensive evaluation of antibiotics emission and fate in the river basins of China:source analysis,multimedia modeling,and linkage to bacterial resistance[J].Environmental Science&Technology,49(11):6772-6782.
ZOU S C,XU W H,ZHANG R J,et al.,2011.Occurrence and distribution of antibiotics in coastal water of the Bohai Bay,China:impacts of river discharge and aquaculture activities[J].Environmental Pollution,159(10):2913-2920.
冀秀玲,刘芳,沈群辉,等,2011.养殖场废水中磺胺类和四环素抗生素及其抗性基因的定量检测[J].生态环境学报,20(5):927-933.JI X L,LIU F,SHEN Q H,et al.,2011.Quantitative detection of sulfonamides and tetracycline antibiotics and resistence genes in sewage farms[J].Ecology and Evironmetal Sciences.20(5):927-933.
李仕平,李仙,张光磊,等,2017.东寨港海水增养殖区水质状况及变化趋势研究[J].海南师范大学学报(自然科学版),30(4):430-435.LI S P,L X,ZHANG G L,et al.,2017.Study on Water Quality and Change Trends in the Mariculture Zone of Dongzhaigang[J].Journal of Hainan Normal University(Natural Sciences),30(4):430-435.
李壹,曲凌云,朱鹏飞,等,2016.山东地区海水养殖区常见抗生素耐药菌及耐药基因分布特征[J].海洋环境科学,35(1):55-62.LI Y,QU L Y,ZHU P F,et al.,2016.Distribution characteristics of antibiotic resistance bacteria and related resistance genes in mariculture area of Shandong[J].Marine Environmental Science,35(1):55-62.
梁惜梅,聂湘平,施震,2013.珠江口典型水产养殖区抗生素抗性基因污染的初步研究[J].环境科学,34(10):4073-4080.LANG X M,NIE X P,SHI Z.,2013.Preliminary Studies on the Occurrence of Antibiotic Resistance Genes in Typical Aquaculture Area of the Pearl River Estuary[J].Environmental Science,34(10):4073-4080.
罗方园,潘根兴,李恋卿,等,2017.洪泽湖沉积物中四环素土霉素及相关抗性基因的分布特征及潜在风险分析[J].农业环境科学学报,36(2):369-375.LUO F Y,PAN G X,LI L Q,et al.,2017.The distribution characteristics and potential risk of tetracycline and their corresponding genes pollution in sediment of Hongze Lake[J].Journal of Agro-Environment Science,36(2):369-375.
王丽梅,罗义,毛大庆,等,2010.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,21(4):1063-1069.WANG L M,LUO Y,MAO D Q,et al.,2010.Transport of antibiotic resistance genes in environment and detection methods of antibiotic resistance[J].Chinese Journal of Applied Ecology,21(4):1063-1069.
CZEKALSKI N,DIEZ E G,BURGMANN N,2014.Wastewater as a point source of antibiotic-resistance genes in the sediment of a freshwater lake[J].The ISME Journal,8(7):1381-1390.
GOGARTEN J P,TOWNSEND J P,2005.Horizontal gene transfer,genome innovation and evolution[J].Nature Reviews Microbiology,3(9):679-687.
GUO X P,LIU X R,NIU Z S,et al.,2018.Seasonal and spatial distribution of antibiotic resistance genes in the sediments along the Yangtze Estuary,China[J].Environmental Pollution,242(Part A):576-584.
HARNISZ M,KORZENIEWSKA E,GOLAS I,2015.The impact of a freshwater fish farm on the community of tetracycline-resistant bacteria and the structure of tetracycline resistance genes in river water[J].Chemosphere,128:134-141.
HARTMANN A,ALDER A C,KOLLER T,et al.,1998.Identification of fluoroquinolone antibiotics as the main source of umuC genotoxicity in native hospital wastewater[J].Environmental Toxicology&Chemistry,17(3):377-382.
HE X T,WANG Z H,NIE X P,et al.,2012.Residues of fluoroquinolones in marine aquaculture environment of the Pearl River Delta,South China[J].Environmental Geochemistry&Health,34(3):323-335.
HU J Y,SHI J C,CHANG H,et al.,2008.Phenotyping and genotyping of antibiotic-resistant Escherichia coli isolated from a natural river basin[J].Environmental Science&Technology,42(9):3415-3420.
HU Y,YAN X,SHEN Y,et al.,2018.Antibiotics in surface water and sediments from Hanjiang River,Central China:Occurrence,behavior and risk assessment[J].Ecotoxicology&Environmental Safety,157:150-158.
JIA J,GUAN Y J,CHENG M Q,et al.,2018.Occurrence and distribution of antibiotics and antibiotic resistance genes in Ba River,China[J].Science of the Total Environment,642:1136-1144.
JIANG L,HU X,XU T,et al.,2013.Prevalence of antibiotic resistance genes and their relationship with antibiotics in the Huangpu River and the drinking water sources,Shanghai,China[J].Science of the Total Environment,458-460(3):267-272.
LI Y,LI Q,ZHOU K,et al.,2016.Occurrence and distribution of the environmental pollutant antibiotics in Gaoqiao mangrove area,China[J].Chemosphere,147:25-35.
LING Z H,YANG Y,HUANG Y L,et al.,2013.A preliminary investigation on the occurrence and distribution of antibiotic resistance genes in the Beijiang River,South China[J].Journal of Environmental Sciences,25(8):1656-1661.
LUO Y,MAO D Q,RYSZ M,et al.,2010.Trends in antibiotic resistance genes occurrence in the Haihe River,China[J].Environmental Science&Technology,44(19):7220-7225.
MACKIE R I,KOIKE S,KRAPAC I,et al.,2006.Tetracycline Residues and Tetracycline Resistance Genes in Groundwater Impacted by Swine Production Facilities[J].Animal Biotechnology,17(2):157-176.
MARTI E,HUERTA B,RODRIGUEZMOZAZ S,et al.,2018.Abundance of antibiotic resistance genes and bacterial community composition in wild freshwater fish species[J].Chemosphere,196:115-119.
MCKINNEY C W,LOFTIN K A,MEYER M T,et al.,2010.Tet and sul antibiotic resistance genes in livestock lagoons of various operation type,configuration,and antibiotic occurrence[J].Environmental Science&Technology,44(16):6102-6109.
MUZIASARI W I,PARNANEN K,JOHNSON T A,et al.,2016.Aquaculture changes the profile of antibiotic resistance and mobile genetic element associated genes in Baltic Sea sediments[J].Fems Microbiology Ecology,92(4):fiw052.
PARK H R,KIM T H,BARK K M,2002.Physicochemical properties of quinolone antibiotics in various environments[J].European Journal of Medicinal Chemistry,37(6):443-460.
PEAK N,KNAPP C W,YANG R K,et al.,2007.Abundance of six tetracycline resistance genes in wastewater lagoons at cattle feedlots with different antibiotic use strategies[J].Environmental Microbiology,9(1):143-151.
PRUDEN A,PEI R,STORTEBOOM H,et al.,2006.Antibiotic Resistance Genes as Emerging Contaminants:?Studies in Northern Colorado[J].Environmental Science&Technology,40(23):7445-7450.
QIAN X,GU J,SUN W,et al.,2017.Diversity,abundance,and persistence of antibiotic resistance genes in various types of animal manure following industrial composting[J].Journal of Hazardous Materials,344:716-722.
SCHLüTER A,SZCZEPANOWSKI R,PUHLER A,et al.,2007.Genomics of IncP-1 antibiotic resistance plasmids isolated from wastewater treatment plants provides evidence for a widely accessible drug resistance gene pool[J].Fems Microbiology Reviews,31(4):449-477.
SK?LD O,2000.Sulfonamide resistance:mechanisms and trends[J].Drug Resistance Updates Reviews&Commentaries in Antimicrobial&Anticancer Chemotherapy,3(3):155-160.
STALIN N,SRINIVASAN P,2016.Molecular characterization of antibiotic resistant Vibrio harveyi isolated from shrimp aquaculture environment in the south east coast of India[J].Microb Pathog,97:110-118.
TENENBAUM I,CHEFETZ B,AVISAR D,et al.,2014.Physicochemical behavior of tetracycline and 17α-ethinylestradiol with wastewater sludge-derived humic substances[J].Water Air&Soil Pollution,225(10):1-11.
THIELE-BRUHN S,2003.Pharmaceutical antibiotic compounds in soils-a review[J].Journal of Plant Nutrition and Soil Science,166(2):145-167.
TURTON J A,HAVARD A C,ROBINSON S,et al.,2000.An assessment of chloramphenicol and thiamphenicol in the induction of aplastic anaemia in the BALB/c mouse[J].Food&Chemical Toxicology,38(10):925-938.
WANG N,GUO X Y,YAN Z,et al.,2016.A Comprehensive Analysis on Spread and Distribution Characteristic of Antibiotic Resistance Genes in Livestock Farms of Southeastern China[J].PLoS ONE 11(7):e0156889.
ZHANG Q Q,YING G G,PAN C G,et al.,2015.Comprehensive evaluation of antibiotics emission and fate in the river basins of China:source analysis,multimedia modeling,and linkage to bacterial resistance[J].Environmental Science&Technology,49(11):6772-6782.
ZOU S C,XU W H,ZHANG R J,et al.,2011.Occurrence and distribution of antibiotics in coastal water of the Bohai Bay,China:impacts of river discharge and aquaculture activities[J].Environmental Pollution,159(10):2913-2920.
冀秀玲,刘芳,沈群辉,等,2011.养殖场废水中磺胺类和四环素抗生素及其抗性基因的定量检测[J].生态环境学报,20(5):927-933.JI X L,LIU F,SHEN Q H,et al.,2011.Quantitative detection of sulfonamides and tetracycline antibiotics and resistence genes in sewage farms[J].Ecology and Evironmetal Sciences.20(5):927-933.
李仕平,李仙,张光磊,等,2017.东寨港海水增养殖区水质状况及变化趋势研究[J].海南师范大学学报(自然科学版),30(4):430-435.LI S P,L X,ZHANG G L,et al.,2017.Study on Water Quality and Change Trends in the Mariculture Zone of Dongzhaigang[J].Journal of Hainan Normal University(Natural Sciences),30(4):430-435.
李壹,曲凌云,朱鹏飞,等,2016.山东地区海水养殖区常见抗生素耐药菌及耐药基因分布特征[J].海洋环境科学,35(1):55-62.LI Y,QU L Y,ZHU P F,et al.,2016.Distribution characteristics of antibiotic resistance bacteria and related resistance genes in mariculture area of Shandong[J].Marine Environmental Science,35(1):55-62.
梁惜梅,聂湘平,施震,2013.珠江口典型水产养殖区抗生素抗性基因污染的初步研究[J].环境科学,34(10):4073-4080.LANG X M,NIE X P,SHI Z.,2013.Preliminary Studies on the Occurrence of Antibiotic Resistance Genes in Typical Aquaculture Area of the Pearl River Estuary[J].Environmental Science,34(10):4073-4080.
罗方园,潘根兴,李恋卿,等,2017.洪泽湖沉积物中四环素土霉素及相关抗性基因的分布特征及潜在风险分析[J].农业环境科学学报,36(2):369-375.LUO F Y,PAN G X,LI L Q,et al.,2017.The distribution characteristics and potential risk of tetracycline and their corresponding genes pollution in sediment of Hongze Lake[J].Journal of Agro-Environment Science,36(2):369-375.
王丽梅,罗义,毛大庆,等,2010.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,21(4):1063-1069.WANG L M,LUO Y,MAO D Q,et al.,2010.Transport of antibiotic resistance genes in environment and detection methods of antibiotic resistance[J].Chinese Journal of Applied Ecology,21(4):1063-1069.