鲍曼不动杆菌ATCC19606多重耐性分析及耐药基因的克隆

鲍曼不动杆菌ATCC19606多重耐性分析及耐药基因的克隆 鲍曼不动杆菌ATCC19606多重耐性分析及 耐药基因的克隆 ?688?中国抗生察杂志2006年11月第31卷第l1期 文章编号1001—8689(2006)11-0688-04 MultipleresistanceinAcinetobacterbaumanniiATCC19606 andcloningofgenesresponsiblefortheresistance SuXian—zhong,ZhangXing,ChenYan,andTomofusaTsuchiya. (1DepartmentofBiologicalEngineering,SchoolofChemicalEngineering, ChinaUniversityofMiningandTechnology,Xuzhou221008; 2XuzhouMedicalCollege,Xuzhou221002} 3DepartmentofMicrobiology,FacultyofPharmaceuticalSciences, OkayamaUniversity,Okayama700-8530,Japan) ABSTRACTDrugresistanceofAcinetobacterbaumanniiATCC19606wastestedinthisstudy.Theresult showedfairlyhighresistancetomanyantimicrobialagentstestedincludingstreptomycin,norfloxacin,chloram— phenicol,erythromycin,tetracycline,ampicillin,andantimicrobialdyes.Usingthedrug— hypersensitivestrain ofEscherichiacoliKAM32asthehost,weclonedthegenesresponsibleformultipleresistancefromchromoso- malDNAofA.baumanniiATCC19606.Weobtained9hybridplasmidsthatmadehostcellsresistanttoseveral antimierobialagents.Manyofthetransformantsharboringeachoftheplasmidsshowedmultipleresistance, andoneshowedresistancetospecificdrug.Thehybridplasmidswereclassifiedintoseveralgroupsbasedon theirdrugspecificity.Itappearsthateachclassofplasmidcarriesdifferenttypesofdrugresista ncegenes. AnalysisofsuchgeneswillrevealthevariousmechanismsinvolvedinmultipleresistanceinA .baumannii ATCC19606. KEYWORDSMultipleresistance;Genecloning;Acinetobacterbaumannii 中圈分类号:R378文献标识码:A 鲍曼不动杆菌ATCC19606多重耐性分析及耐药基因的克隆 苏显中张兴陈彦土屋友房 (1中国矿业大学化工学院生物工程系,徐州221008; 2徐州医学院,徐州221002; 3冈山大学药学部微生物系,冈山700-8530日本) 摘要:鲍曼不动杆菌已成为重要的院内感染病菌.我们测定了鲍曼不动杆菌ATCC19606的药物最小抑制浓度(MIC),结果 显示该菌株对多种抗菌药物都有很高的耐性,如链霉素,诺氟沙星,氯霉素,红霉素,四环索,氨苄西林及一些其他的抗菌染剂利用 大肠埃希菌超敏菌株KAM32作为宿主,从鲍曼不动杆菌ATCC19606染色体DNA中克隆耐药基因,共获得9个使宿主细胞产生 耐药性的杂合质粒,其中1个为单一耐药,其余全部为多重耐药.根据药物特异性分析可知,具有不同耐药图谱的杂合质粒携带不 同类型的耐药基因.由此揭示鲍曼不动杆菌ATCC19606的多重耐药有多种机制参与. 关键词:多重耐药;基因克隆;鲍曼不动杆菌 收稿日期;2006—07—19 基金项目:教育部留学回国人员科研启动基金资助项目?中国矿业大学科技基金(2005B014). 作者简介;苏显中,男,生于1965年,博士,教授.研究方向;细菌耐药机制研究及耐药菌的有效药物开发.E—mail;xzhsu@126.com 鲍曼不动杆菌多药耐性分析及耐药基因的克隆苏显中等?689? Theappearanceandspreadofdrugresistanceinpathogenicorganismshavemademanyavail ableantibiotics ineffective[..Drugresistance,especiallymultipleresistance,inbacterialcellshascurrentlybecomean increasingseriousclinicalproblem.Anextensiveknowledgeofmolecularmechanismsunderlyingbacterialdrug resistanceisrequiredtocontrolmultipleresistantbacteriaandtotreatpatientsinfectedwithmultiple—resstant bacteriasuccessfully. CertainstrainsofA.baumanniiarenowresistanttoallcommonlyprescribedantibiotics(includingimipen— emandthenewfluoroquinolones),andreportsofahighprevalenceoffluoroquinoloneresistanceamongAcine— tobacterisolateshaveappeared[.?. ItisimportanttoinvestigatethemechanismsbywhichA.baumanniiacquiresdrugresistance.Inthis study,wereportpatternsofdrugresistanceinstrainA.baumanniiATCC19606andfunctionalgenecloningof thedrug—resistancesystems. 1Materialsandmethods 1.1Bacteriaandgrowth A.baumanniiATCC19606(generouslyprovidedbyProfessorShigeoYamamoto,FacultyofPharmaceuti— calSciences,OkayamaUniversity,Japan)andEscherichia.coliKAM32wereusedinthisstudy.E.coliTG1 andPsudomonasaeruginosaPAO1werealsoused.E.coliKAM32,aderivativeofE.coliTG1,lacksthemajor multipleeffluxpumpsAcrBtS3andYdhE[6],andshowshypersensitivitytomanyantimicrobialagentsandis usefulasahostforgenecloningofdrug— resistancesystems.BacterialcellsweregrowninI.Bmedium(Difco) withaerationat37? overnight.Thegrowthofcellswasmonitoredbymeasuringopticaldensityat650nm. 1.2Drugsusceptibilitysest Theminimalinhibitoryconcentrations(MICs)ofvariousdrugsweredeterminedbyserialtwo—folddilution ofantimicrobialsintheMueller— Hintonbroth(Difco)containingdifferentdrugsatvariousconcentrations.MIC valucsweredeterminedasaconcentrationofanantimicrobialthatcompletelypreventedcellgrowthduringan 24hincubationat37Cwithaeration. 1.3Genecloning Chromosoma1DNAwaspreparedfromcellsofA.baumanniiATCC19606bythemethodofChenand Kuot. TheDNAwaspartiallydigestedwithSau3AI,andfragmentsof4to10khpwereseparatedbysucrose densitygradientcentrifugation.TheDNAfragmentswereligatedintoplasmidpBR322(whichwasdigested withBamHIanddephosphorylatedwithshrimpalkalinephosphatase)usingaligationkit,Ver.2(TaKaRa Co.).TheligatedhybridDNAwastransformedintocompetentcellsofE.coliKAM32,thenthecellswere spreadontoagar(1.5%)platescontainingLBbroth,ampicillinl00/~g/ml,andoneofthefollowingdrugs: norfloxacin(O.05~g/m1),ethidiumbromide(10ttg/m1)or4,6-diamidino2phenylindole(DAPI)(0.5 t~g/m1).Theplateswereincubatedat37Cfor24h.Weobtained9candidatecoloniesontheplates+Eachcan— didatecolonywaspurifiedbysinglecolonyisolationontheplatecontainingeachdrug.Plasmidswereisolated fromeachofthecandidates.PlasmidDNAswereretransformedintheKAM32cells,spreadonthesametype ofplate,andtheirresistanceagainstvariousantimicrobialagentstested.Weconfirmedthatonetypeofplasmid washarboredineachcandidateclonebytheretransformationandbycheckingtherestrictionfragmentofeach plasmid.AllhybridplasmidscarriedDNAinsertsof3.0to11kbp. 2Resultsanddiscussion 2.1MultipleresistanceinA.baumannii Toinvestigatedrugresistancepatterns,wedeterminedtheMICsofvariousantimicrobialagentsinA-bau ,"d""iiATCC19606.WecomparedtheMICswiththoseinE.coliTG1andP.aeruginosaPAO1thatshowed intrinsicmuhip1eresistance.Theresultsshowedthisstrainwasresistanttoavarietyofantimicrobialagents includingaminoglycosides,quinolones,~-lactams,detergents,dyesandantiseptics(Tab?1)?Inaddition,the SDSlsodiumdodecy1suIfate;DAPIt4,6-diamidino一2一 phenylindoleTPPCIttetraphenylphosphoniumchloride? strainexhibiteda1owerleve1ofresistancetoimpenemandcarbenicillin,butnotampicillinin13-laetams,aswell ast0nalidixicacidandnotnorfloxacininquinolones.ThusitisclearthattheclinicallyisolatedstrainofA?bau一 ,d以打 areresistanttomanyantimicrobia1agents,andthelevelofresistanceishigherthanthatinE?coliTG1' butsimilarsubstratesspecificitywithP.aeruginosaPAO1,awell— knownmultipleresistantbacterium? 2.2Functionalcloningofgenesfordrugresistanceandclassification Wetriedtoc1oneA.baumanniiATCC19606genesresponsiblefordrugresistanceusingE.coliKAM32'a drughyper— sensitivestrain,asthehost.Weobtained9candidateclonesharboringrecombinantplasmidsUSlng s0meantimicrobialagentsfortheselectionofdrug— resistantclones.Threecloneswereobtainedfromtheno卜 floxacinplate,fourc1onesfromtheethidiumbromideplate,twoclonesfromtheDAPIplate(Tab?2)?They sh0weddifferentpatternsofdrugresistance.Onecloneobtainedfromthenorfloxacinplateshowedresistane onlvtonorfloxacinamongtheantimicrobialagentstested,andothersshowedresistancenotonlytono卜 floxacin,butalsotokanamycin,DAPI,chloramphenicol,andethidiumbromide— Drugresistancepatterns amongcl0nesobtainedfromtheethidiumbromideplatewereversatileandclassifiedintothreetypes.Thefirst type(oneclone)showedresistancetokanamycin,norfloxacin,DAPI,andethidiumbromide.Thesecondtype (onec1one)wasresistanttokanamycin,norfloxacin,erythromycin,DAPIandethidiumbromide.Thethird tvpe(tw0clones)wasresistanttoalltheantimicrobialagentstested.ThetwoclonesobtainedfromtheDAP1 plateshowedresistancen0tonlytoDAPIbutalsotokanamycin,norfloxacin,andethidiumbromide?We checkedtherestrictionfragmentsofeachplasmidandconfirmedthatplasmidsthatconferredthesamedrug sDecificitypossessedacommonDNAfragment.Thus,itappearsthatweclonedfivedifferenttypesofdrug resistancegene(s).Amongthem,onetypewasspecifictonorfloxacin,andotherswereresista nttomultiple 鲍曼不动杆菌多药耐性分析及耐药基因的克隆苏显中等.691. KMIkanamycinINFLX{norfloxacinEM:erythromycinCP:chloramphenicollDAPIt4.6-diamidin0—2一phenylind0Ie} EtBr;ethidiumbromide. +:cellsgrew;一tcellsdidnotgrow. structurallyandfunctionallyunrelatedantimicrobialagents.Thosefourtypesmayrepresentmuhipleefflux pumps,becauseonlythemultipleeffluxpumpisinvolvedinallmuhipleresistanceamongknowndrug-resis— tancemechanisms.InE.coliorP.aeruginosa,inwhichtheexistenceofmorethan30multipleeffluxpumpsis suggestedfromtheirgenomesequences,itisclearthatmultipleeffluxpumpsaremainlyinvolvedinintrinsic andacquiredmultipleresistance.Althoughwedonotknowatpresenthowmanysystemsofdrugresistanceare presentinA.baumannii,itappearsthatclonedgenesofA.baumanniicontributetotheobservedmultipleresis tanceofthismicroorganism.Geneticanalysisoftheclonedgenesandbiochemicalanalysisofthegeneproducts arenOWinprogress. 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