城市生活污水和生活垃圾渗滤液抗生素抗性基因污染的比较研究黄福义

3710201610ENVIＲONMENTALSCIENCEVol．37No．10Oct．201611212121*1.3610212．100049PCＲ．39187P＜0.05119P＜0.05tnpA-04blaVEB33381061β-．PCＲX171.5A0250-3301201610-3949-06DOI10．13227/j．hjkx．2016．10．0372016-03-042016-05-162016YFD080020531270153212100081987～E-mailfyhuang@iue．ac．cn*E-mailjqsu@iue．ac．cnComparativeInvestigationofAntiboticＲesistanceGenesBetweenWastewaterandLandfillLeachateHUANGFu-yi1LIHu12ANXin-li12OUYANGWei-ying12SUJian-qiang1*1．KeyLaboratoryofUrbanEnvironmentandHealthInstituteofUrbanEnvironmentChineseAcademyofSciencesXiamen361021China2．UniversityofChineseAcademyofSciencesBeijing100049ChinaAbstractTheincreasingoutputofwastewaterandlandfillleachateduringtherapidurbanizationexacerbatedthehumanhealthrisksinducedbyantibioticresistancegenesAＲGs．ForfurtherinsightintoantibioticresistancegenesAＲGsofwastewateraddingleachatebeforetreatmentandlandfillleachatehigh-throughputqPCＲtechniquewasusedtoinvestigatetheirabundanceanddiversities．Atotalof187uniqueAＲGsweredetectedinwastewaterwhichwassignificantlyhigherthanthatinleachate39uniqueAＲGsweredetectedP＜0.05．Atotalof119antibioticresistancegenesweresignificantlyenrichedinwastewatercomparedwiththelandfillleachateP＜0.05especiallythetnpA-04andblaVEBwhichbelongstomobilegeneelementsMGEsandβ-Lactamaseincreasedby3338-foldand1061-foldrespectively．OurresultsshowedthattheprofileofAＲGswassignificantlydifferentbetweenthewastewaterandleachateandthewastewaterwastheimportantreservoirofAＲGs．Keywordswastewaterlandfillleachateantibioticantibioticresistancegeneshigh-throughputqPCＲ．．、、．12、34、5、6789．Pruden102006．、、、、、1112．、、、．“”．、375a13．14～16．1317．1819、．．、．11.1201571L350%．201571L3．－20℃DNA．1.2DNArawwastewaterＲWWrawleachateＲLC40mL50mL12000r·min－110min1mL．1.5mL18000r·min－1．FastDNASpinKitforSoilMPBiomedicalsPBS978μL．PBSLysingMatrixEtubeDNA1%．DNAQuantiFluordsDNASystemPromegaCorporationDNA．DNA50ng·μL－1．1.3PCＲSmartChipＲeal-TimePCＲSystemsWaferGenInc．．2931．1blaNDM-1201intI1class1integron211CintI1clinicalclass1integon22．PCＲ100nL．1×LightCycler480SYBＲGreenⅠMasterMixＲochenuclease-freePCＲ-gradewater1ng·μL－1BSA5ng·μL－1DNA1μmol·L－1．PCＲ95℃10min95℃30s60℃30s40．PCＲDNA．qPCＲCycler1.8～2.2．SmartChipＲeal-TimePCＲSystemsCT31．333．1.4generelativecopynumberLooft231．Schmittgen24FCfoldchange2．=1031－CT/10/31059310ΔCT=CTAＲG－CT16SΔΔCT=ΔCTTarget－ΔCTＲef2FC=2－ΔΔCTCTPCＲAＲG16S16SrDNATargetＲefFCAＲGs．FC=2－ΔΔCT+2s＞1studentt．22.1296Aminoglycoside、β-β-Lactamase、Chloramphenicol、--BMLSB、Sulfonamide、Tetracycline、Vancomycinother/efflux8．ＲWW187391．8Aminoglycoside、--BMLSB、Tetracyclineother/efflux4．62526．4．deactivation、efflux、protectionunkown．deactivation48.13%51.28%2．39erm3538．P＜0.05．1Fig．1Numberofdetectedantibioticresistancegenes2Fig．2Ｒatiosofmechanismindetectedantibioticresistancegenes2.2PCA198.8%3．．Auerbach27109copies·L－1．4P＜0.05．4AA11593373Fig．3PrinciplecomponentanalysisofAＲGsinwastewaterandlandfillleachateA2．BCDBC．“”285a13PCＲ．2.3、“”1317．．Schmittgen24genecopynumber5．others、、1.4×108copies·L－1、3.6×107copies·L－1．3944Fig．4PollutionprofileofAＲGsinwastewaterandlandfillleachate5Fig．5AbundanceofAＲGsinwastewaterandlandfillleachate1.1×105copies·L－1P＜0.05．13．7119259310P＜0.05tnpA-04、blaVEBblaOXA10-013338、106110016β-．horizontalgenetransferHGTMGEs7．1029．“”．6Fig．6EnrichedAＲGsinwastewaterWang25Wu26．．31．2．1ZhuYGJohnsonTASuJQetal．DiverseandabundantantibioticresistancegenesinChineseswinefarmsJ．ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica201311093435-3440．2．5J．200945705-710．WuBQiaoMZhuYG．QuantificationoffivetetracyclineresistancegenesinsoilfromaswinefeedlotJ．AsianJournalofEcotoxicology200945705-710．3SuJQWeiBOuyangWYetal．AntibioticresistomeanditsassociationwithbacterialcommunitiesduringsewagesludgecompostingJ．EnvirontalScience＆Technology201549127356-7363．4ＲahubeTOMartiＲScottAetal．Impactoffertilizingwithraworanaerobicallydigestedsewagesludgeontheabundanceofantibiotic-resistantcoliformsantibioticresistancegenesandpathogenicbacteriainsoilandonvegetablesatharvestJ．AppliedandEnvironmentalMicrobiology201480226898-6907．5OuyangWYHuangFYZhaoYetal．IncreasedlevelsofantibioticresistanceinurbanstreamofJiulongjiangＲiverChinaJ．AppliedMicrobiologyandBiotechnology201599135697-5707．6．J．201435103869-3873．HuangFYLiHWeiBetal．Long-termmanureapplicationinducedshiftofdiversityandabundanceofantibioticresistancegenesinpaddysoilJ．EnvironmentalScience201435103869-3873．7XuLKOuyangWYQianYYetal．High-throughputprofilingofantibioticresistancegenesindrinkingwatertreatmentplantsanddistributionsystemsJ．EnvironmentalPollution2016213119-126．8XiCWZhangYLMarrsCFetal．PrevalenceofantibioticresistanceindrinkingwatertreatmentanddistributionsystemsJ．AppliedandEnvironmentalMicrobiology200975175714-5718．9．J．2013214481-487．SuJQHuangFYZhuYG．AntibioticresistancegenesintheenvironmentJ．BiodiversityScience2013214481-487．10PrudenAPeiＲTStorteboomHetal．AntibioticresistancegenesasemergingcontaminantsstudiesinnorthernColoradoJ．EnvironmentalScience＆Technology200640237445-7450．11．J．200420136-39．WeiDBHuHY．Safetyindexsystemofreclaimedandreused359337waterqualityJ．ChinaWater＆Wastewater200420136-39．12Mol