英文-学术报告

CharacteristicsofEscherichiacolibiolmproduction,genetictyping,drugresistancepatternandgeneexpressionunderaminoglycosidepressures,Escherichiacoli大肠杆菌infection感染Aminoglycoside氨基糖苷类antibiotic抗生素Pseudomonas假单胞菌aeruginosa聚合酶链反应技术Crystalvioletstainingmethod结晶紫染色法phenotype表现型drugresistancepattern耐药模式Antimicrobialagent抗菌药物molecularbiologyreagent分子生物学试剂Brothmicrodilutionmethod肉汤稀释法Susceptibilitytesting药敏试验Genotype基因型Real-timeqRT-PCR实时定量荧光PCRStreptomycin链霉素amikacin丁胺卡那霉素Gentamycin庆大霉素apramycin安普霉素,1.Newwords,Background,SeveraltypesofinfectioncausedbyEscherichiacoli(e.g.,urethralcatheter,biliarytractprosthesis,biliarytractinfection,lithangiuria,urinarytractinfectionandmanyotherdiseases)areassociatedwithbiolmformation,whichleadstoaninabilitytoeradicatetheinfectionduetoitsintrinsicnaturetoresisthighlevelsofantibiotics(FontaineandSmith,2006;Melchioretal.,2006).,Background,Inaddition,underextensiveandpersistentpressureofantibiotics,inordertosurvive,theE.coliformsbiolmtoevadetheimmuneclearanceandresultsinmultipleantibioticsresistance.Hoffmanetal.(2005)foundthatsubinhibitorylevelsoftheaminoglycosideantibioticscouldinduceE.coliandPseudomonasaeruginosabiolmformation.SomeotherstudiesalsorevealedthatbiolmformationbyE.coliinvitrocorrelatedwiththevirulencefactors(Navesetal.,2008;Rijavecetal.,2008).,Abstract,Inthisstudy,qualitative(scanningelectronmicroscope)andsemi-quantitative(modiedcrystalvioletstainingmethod)methodshadbeenusedtoevaluateEscherichiacolibiolm-formingability.Brothmicrodilutionmethodandenterobacterialrepetitiveintergenicconsensus-basedPCR(ERIC-PCR)wereperformedtostudyE.colidrugresistancepatternandgenetictyping.Basedontheresultsabove,westudiedthecorrelationbetweenbiolm-formingabilityphenotype,drugresistancepatternandgenetictypinginE.coli.Real-timeqPCR(qRT-PCR)wasusedtorevealmRNAexpressionlevelofE.colibiolmrelatedmultipleantibioticsresistancegenes(acrA,agn43,csgA,csgD,ompFandpgaA)underdifferentconcentrationsoffouraminoglycosidepressures.,Contents,1.Objective,Theprimaryaimsofthisstudyweretoinvestigate:(i)TheE.colibiolm-formingabilitybasedonthequalitative(scanningelectronmicroscope)andquantitative(modiedcrystalvioletstainingmethod)methods.(ii)ThecorrelationsbetweenE.coligenotype,drugresistancepatternandbiolm-formingabilityphenotype.(iii)ChangesofmRNAlevelofacrA,agn43,csgA,csgD,ompFandpgaAunderpersistentandextensiveantibioticpressuresusingqRT-PCRmethod.,2.Materialsandmethods,37C,64E.colistrains,trypticsoybrot胰蛋白酶大豆肉汤,2.1.Bacterialstrains,2.2.Antimicrobialagentsandmolecularbiologyreagents,10antimicrobialagents,TRIzolreagentKit,SYBRPrimeScriptRT-PCRKit,PrimeScriptRTreagentKit,othermolecularbiologyreagentsforqRT-PCRanduniversalPCR,2.3.Studydesignandexperimentalapproach,2.3.1.Semi-quantitativebiolmformationexperiment,Semi-quantitative半定量的,SpectrophotometerReader分光光度计,2.3.2.Scanningelectronmicroscope(SEM),SEM扫描电子显微镜,2.3.3.Correlationbetweendrugresistancepatternandbiolm-formingability,Brothmicrodilutionmethodwasusedforantimicrobialdrugsusceptibilitytestingof10antimicrobialagentsto64clinicalisolatesandtheexperimentaldatawasstatisticallyanalyzedbyWHONET5.3software.E.coliATCC25922wasusedasreferencestrainforqualitycontrolsBasedontheresultsofsemi-quantitativebiolmformationexperimentinSection2.3.1,wefocusedonwhetherbiolmformationwasrelatedtodrugresistancepatterns(Moskowitzetal.,2004;Franketal.,2007;Sumanetal.,2007).,2.3.4.Correlationbetweengenotypeandbiolm-formingability,2.3.5.RNAextractionandreal-timeqRT-PCR,3.1.Biolm-formingabilityof64E.coli,3.Results,3.2.SEMphotographofstrainE53,3.3.Drugresistancepatternandbiolm-formingability,3.4.ERIC-PCRandbiolm-formingability,3.5.mRNAlevelsofacrA,agn43,csgA,csgD,ompFandpgaAunderfouraminoglycosidepressures,3.5.mRNAlevelsofacrA,agn43,csgA,csgD,ompFandpgaAunderfouraminoglycosidepressures,3.5.mRNAlevelsofacrA,agn43,csgA,csgD,ompFandpgaAunderfouraminoglycosidepressures,4.Conclusions,Formaboveresults,wecandrawtheseconclusions:,(iii)qRT-PCRrevealedmRNAexpressionofacrA,agn43,csgA,csgD,ompFandpgaAgeneschangedaccordinglybystimulationofdifferentconcentrationsoffouraminoglycosides.DesignInc.,(ii)ERIC-PCRshowedthattherewassignicantcorrelationbetweenbiolm-formingabilityandgenotype;whiletherewasweakcorrelationbetweenbiolm-formingabilityanddrugresistancepatte