细菌的感染诊断防治消毒灭菌课件

BacterialInfectionBacterialInfectionNormalfloraOpportunisticpathogenPathogenicbacteria(virulentbacteria)DysbacteriosisExotoxinsEndotoxinsSeparation&CultureofbacteriaBacteriemiaSepticemiaPyemiaToxemiaEndotoxemiaCarrierColonyToxoidsAntitoxinKeyWordsNormalfloraBacteriemiaKeyWoNotallbacteriacausediseaseNormalfloraThenormalfloraarebacteriawhicharefoundinoronourbodiesonasemi-permanentbasiswithoutcausingdisease.Therearemorebacterialivinginoronourbodies,thanwehavecellsofourown.Ahumanbodycontainsaround1013cells.Thehumanbodyishometoaround1014bacteria.Onefourthoffecalweightismadeofbacteria!Thenormalfloraareparticularlyimportantinthelargeintestine.Normalfloraarealsofoundinthenose,mouth,throat,skin.Opportunisticbacteriacanbepartofthenormalflorabutnotpathogenicfornormalpersons,causediseaseonlyinimmunocompromisedpersons,orunderspecificconditions.Pathogenicbacteria(virulentbacteria)Normallynotmembersofthenormalflora,havemechanismstopromotetheirowngrowthintheexpenseofhoststissueororgansfunctionNotallbacteriacausediseaseThenormalfloraprotectusfromdiseaseby:1.Competingwithinvadersforspaceandnutrients.2.Producingcompounds(bacteriocins)whichkillotherbacteria.3.LoweringthepHsothatotherbacteriacantgrow.ThenormalfloraprotectusfrInadditiontotheabovewaysofprotectingusfromdiseaseournormalflorahelpusinotherways.Ofcourse,thereisadownsideaswell.TheGoodSideProducevitaminsweareunabletoproducesuchasvitaminB12.BoostourimmunesystemGermfreeanimals(bornandraisedinagermfreeplastictent)areverysusceptibletodiseasewhenremovedfromthegermfreeenvironmentbecausetheirimmunesystemisunderdeveloped.Helpdigestfood.InadditiontotheabovewaysTheBadSideIfthenormalfloraescapefromtheirnormallocation,theycancausedisease.Forexample,Escherichia coli,commonlyfoundintheintestine,cancauseurinarytractinfectionsifintroducedintothebladder.Immunosuppressioncanallowotherwiseharmlessbacteriatocausedisease.AIDS,somecancertreatmentsandtransplantrejectiondrugsallsuppresstheimmunesystemandallowthenormalfloratocauseoccasionallyseriousdisease.TheBadSide Threeconditionscorrelatewithopportunisticinfection:1.ImmuneSystemCompromise2.Dysbacteriosis3.ChangeofInhabitingPlaces:ThreeconditionscorrelatewiPathogenesisisamulti-factorialprocesswhichdependsonthenatureofthespeciesorstrain(virulencefactors),thePathwayofBacterialEntrance(gateway)andtheimmunestatusofthehost,aswellasthenumberoforganismsintheinitialexposureandtheEnvironmentalconditions.BacterialPathogenesisPathogenesisisamulti-factorBACTERIAL VIRULENCE FACTORS BACTERIALVIRULENCEFACTORS1.AdherenceFactorsSurfacehydrophobicity,pili,Capsule&glycocalyxBacterialBiofilms2.InvasionofHostCells&Tissues:Enzymes3.immuneevasion:Capsule&glycocalyx,IgA1Proteases,Antigenicvariation,InterferenceofcomplementactivityIntracellularpathogenicity.BacterialVirulenceFactorsAdherenceFactors.Bacterial4.ToxinsExotoxinsEndotoxinsExotoxinsl l proteinsproteinslusuallyenzymesusuallyenzymesl ldestroycellularstructuresdestroycellularstructuresl ldestroyextracellularmatrixdestroyextracellularmatrixl lAntibodies(anti-toxins)neutralizeAntibodies(anti-toxins)neutralize-vaccination-vaccinationActiveActiveBindingBindingA ACellsurfaceCellsurfaceB BA-B toxins4.ToxinsExotoxinsExotoxinsACholeratoxinandE.colilabiletoxinADP-ribosylationofregulatoradenylatecyclaseactivationcyclicAMPactiveionandwatersecretiondiarrheaCholeratoxinandE.colilabiTetanustoxinTetanustoxininhibitsglycinereleaseinhibitsglycinereleaseinactivatesinhibitoryneuronsinactivatesinhibitoryneuronsmusclesover-activemusclesover-activerigidparalysisrigidparalysis NormalconditionRigidparalysisRigidparalysisTetanustoxinNormalconditionR BotulinumtoxinBotulinumtoxininhibitsacetylcholinereleaseinhibitsacetylcholinereleaseinhibitsnerveimpulsesinhibitsnerveimpulsesmusclesinactivemusclesinactiveflacidparalysisflacidparalysis(Acetylcholine)Botulinumtoxin(Acetylcholine15ExotoxinsAntibodies(anti-toxins)neutralizevaccination15ExotoxinsAntibodies(anti-toEndotoxinsnLipopolysaccharide(LPS):atoxiclipidAacorepolysaccharideOantigenpolysaccharidesidechainsnCellwalllysisrequirednFormaldehydeandheatresistantnPoorantigenasfreemoleculenEndotoxineffectsEndotoxinsLipopolysaccharideEndotoxineffectslFever-pyrogen1microgram/kglLeukopeniaandleukocytosisnecrosislEndotoxemiaandshocklDisseminatedintravascularcoagulation(DIC).l lN Non-specificinflammationon-specificinflammation.l lC Cytokinereleaseytokinereleasel lC Complementactivationomplementactivationl lBcellmitogensBcellmitogensl lP PolyclonalBcellactivatorsolyclonalBcellactivatorsl lA AdjuvantsdjuvantsEndotoxineffectsNon-speci细菌的感染诊断防治消毒灭菌课件ExotoxinsToxinsproducedbybacteriaandsecretedtotheoutsideofthebacteriacell(differentfromtheendotoxin)actingoncellsurfaceBybindingtocertainreceptorsPossesssomedegreeofhostcellspecificitydenaturedbyformaldehydetreatmenttogeneratetoxoidswhichlacktoxicactivitybutstillinduceprotectiveimmunitywhenusedasvaccines.ManyexotoxinshaveanA-BsubunitstructureAsubunit-providesthetoxicactivityBsubunit-mediatesadherenceofthetoxincomplextoahostcellSummaryExotoxinsToxinsproducedbybEndotoxins(LPS)nderivedfromGcellwallsandareoftenliberatedwhenthebacterialyse.nheat-stable,nthreemainregionslatoxiclipidAlacorepolysaccharidelOantigenpolysaccharidesidechainsnThepathophysiologiceffectsofLPSaresimilarEndotoxins(LPS)derivedfro.PathwayofBacterialEntrancenContact:directorindirect(Sexualcontact)nInhalation:transmittedbytherespiratoryroute(Airbornedroplets)nIngestion:ingestedandtransmittedthroughintestinaltracttotheoutsideandgetnewinfectionsbycontaminatingoffoodandwater,whichiscalledfecal-oralspread(F Food,ood,WWaterater)nInoculation&bloodtransfusionnAnimalvectors:transmittedbyanimalvectors(insect bittinginsect bitting).PathwayofBacterialEntranResponsetoInfectioninfectionxdiseaseInnate immunityno diseaserecoveryadaptive immunityre-infectionno diseasexResponsetoInfectioninfectionImmunityofextracellularbacterialinfection:antibodies(IgG,IgM,SIgA);phagocytes(neutrophils);complement;humoralimmunitymainly.Immunity of intracellular bacterial infection:cell-mediatedimmunity(delayed-typehypersensitivity,DTHresponse(DTH)involvingTH1andmacrophages)mainly.ImmunityofextracellularbactNodiseaseHost defenseBacterial infectionBalance between Infection and Immunity(immunity)NodiseaseHostdefenseBacteriaDiseasedevelopsonlyintherighthostandundertherightconditionsDiseaseHostPathogenEnvironmentDiseasedevelopsonlyintherDiseaseHostdefenseBacterialinfectionEnvironmentalconditionscanhelptiltthebalance Environmentalsignalsoftencontroltheexpressionofthevirulencegenes.Commonsignals,include:Temperrature/Ironavailability:Cdiphtheriae/lowion/Osmolality/Growthphase/pH/SpecificionsDiseaseHostdefenseBacterialiSourcesofInfectionB.EndogenousInfection:opportunisticpathogeninfectionsA.ExogenousInfectionPatientsCarriersAnimalsSourcesofInfectionB.EndogenTypesofBacterialInfectionAccordingtoinfectiousstate:nInapparentinfection:withoutclearclinicsymptoms.nApparentInfection:haveevidentclinicsymptoms.lAccordingtoinfectioussites(LocalinfectionandGeneralizedorsystemicinfectionlBacteriemia:bacteriacirculatebutnotmultiplyintheblood.lSepticemia:Bacteriacirculateandmultiplyintheblood.lPyemia:producesepticemiawithmultipleabscessesininternalorgans.lToxemia:Bacteiamultiplyatinvadinglocationanddonotenterbloodstream,buttheexotoxinsenterbloodandcausecorrespondingtoxicsymptomslEndotoxemia:multiplyatlocationorinbloodstream,releasealotofamountendotoxinreleasedfrombacterialcellrupturenCarrier:Types of Bacterial InfectionAcPrinciplesofDiagnosisandPreventionofBacterialInfectionPrinciples of Diagnosis and PrnManifestationsofInfection:Signsandsymptomsvaryaccordingtothesiteandseverityofinfection.Diagnosisrequiresacompositeofinformation,includinghistory,physicalexamination,radiographicfindings,andlaboratorydata.nMicrobialCausesofInfection:Infectionsmaybecausedbybacteria,viruses,fungi,andparasites.Thepathogenmaybeexogenous(acquiredfromenvironmentaloranimalsourcesorfromotherpersons)orendogenous(fromthenormalflora).Manifestations of Infection:S1.Thequantitymaterialmustbeadequate.2.Collectfromappropriatesite.3.Preparesitetominimizecontamination.4.Wheneverpossible,collectspecimenspriortoantibiotics.5.Transportsystemmaximizedforpathogensurvivalshouldbeused.GeneralguidelinesforspecimencollectionThe quantity material must be BodyfluidforspecimencollectionnBloodsepticemianCerebrospinalfluidbacterialmeningitisnPeritoneal(abdominal)nPleural(chest)nSynovial(joint)nPericardial(heart)nUrineBody fluid for specimen collecSamplecollectiondevicesSample collection devicesMicroscopyandStains:cellularmorphologyandstainmaypermitpreliminaryidentification.Separation&Cultureofbacteria:Isolationofinfectiousagentsfrequentlyrequiresspecializedmedia.Nonselective(noninhibitory)mediapermitthegrowthofmanymicroorganisms.Selectivemediacontaininhibitorysubstancesthatpermittheisolationofspecifictypesofmicroorganisms.ColonymorphologycansometimesbeusefulinbacterialidentificationIdentificationofbacteria:qCulturalcharacteristics:Growthcharacteristicsundervariousconditions,lBiochemicalcharacterization:utilizationofcarbohydratesandothersubstrates,enzymaticactivitylSerologyIdentificationAhighorrisingtiterofspecificIgGantibodiesorthepresenceofspecificIgMantibodiesmaysuggestorconfirmadiagnosis.lGenomicMethodsAntimicrobialSusceptibility:Microorganisms,particularlybacteria,aretestedinvitrotodeterminewhethertheyaresusceptibletoantimicrobialagents.MicrobiologicExaminationMicroscopy and Stains:cellulaMicroscopyandStainsDirectexaminationofstainedorunstainedpreparationsbylight(brightfield)microscopyisarelativelysimple:lwitha100oilimmersionobjective,la5to10eyepiece,loptimallighting.Thetwomostimportantmethods,theGramandacid-fasttechniques,toclassifyaswellasstaintheorganism.lemploystaining,ldecolorization,lcounterstainingMicroscopy and Stains Direct eExamplesofGramStainsGramPositiveRodsandCocciGramNegativeRodsandCocciExamples of Gram StainsGram PoExamplesofAcid-FastStainsFullyAcid-FastRods(Mycobacterium species)PartiallyAcid-FastRods(Nocardia species)Examples of Acid-Fast StainsFutypesofmediaforroutineculture:Thestandardmediumforspecimens:bloodagar,-Usuallymadewith5%sheepblood.-MostaerobicandfacultativelyanaerobicorganismswillgrowonbloodagarAsecondnecessarymedium:Chocolateagar,-amediumcontainingheatedbloodwithorwithoutsupplements,-Someorganismsthatdonotgrowonbloodagar,includingpathogenicneisseriaandhaemophilus,willgrowonchocolateagar.Culturetypes of media for routine culSelectivemedianSelectivemediaisonethatgrowsonlycertainmicroorganismswhileinhibiting(orpreventing)othersfromgrowing,thatistosay,themediahascertainchemicalsthatallowoneorganismtogrowbutanotherorganismcannotgrowwiththoseingredientsinthemedia,thus,isselectivefortheorganismsthatcangrowinthatmedia.Therefore,mostcommonlygrowsonlyonetypeoforganism.Selective mediaSelective mediaDifferentialmedianDifferentialmediaisonethatdistinguishesonemicroorganismfromanother,itcangrowmorethanonemicroorganism,butdependingonhoweachorganismreactstothemedia(liketurnsred)itdifferentiatesfromanothermicroorganism.Inthistypeofmediaifyouaretryingtodistinguishbetween2typesofmicroorganisms,bothshouldgrowbuttheywillhavedifferentreactionstothemedia,andthrutheirvisiblereactionsyoucantellthemapart(differentiatethem).Differential mediaDifferentialqGrowthonselective-differentialmedia,suchasSalmonella-Shigella(SS)medium,eosin-methylene-blue(EMB)andMacConkeyagarTTheselecteffectofthemediainsuppressingunwantedgram-positiveorganismsisexertedbybilesaltsorbacteriostaticdyesintheagar.TThedifferentialabilityofthesemediaisbasedonlactosefermentation:normalflorapositive(coloredcolonies)andpathogensnegative(colorlesscolonies).Growth on selective-differentiSeparationofbacteria:platestreaking.Bacteriologicplatestreaking.Separation of bacteria:plate细菌的感染诊断防治消毒灭菌课件Colony:thevisiblegrowthofbacteriaonsolidgrowthmedia.Ideally,thecolonyistheprogenyofone,oratmost,afewbacteria.Acolonywillusuallycontainmillionsofbacterialcells.Colonymorphologycansometimesbeusefulinbacterialidentification.Coloniesaredescribedastosuchpropertiesassize,shape,texture,elevation,pigmentation,effectongrowthmedium.Colony Colony:the visible growth of细菌的感染诊断防治消毒灭菌课件细菌的感染诊断防治消毒灭菌课件Growthonbloodagartotestforhemolyticproperties-hemolytic:incompletelysisofredbloodcells,resultinginagreenishhaloaroundthecolony-hemolytic:completelysisofredbloodcells,resultinginaclearhaloaroundthecolony-hemolytic:non-hemolytic1.Culturalcharacteristics:luniquenutritionalrequirements,lpigmentproduction,lhemolyticpropertiesIdentificationofBacteriaGrowth on blood agar to test fn na a a a(alpha)(alpha)n npartialhemolysispartialhemolysisvvgreenishcolor(i.e.S.greenishcolor(i.e.S.pneumoniae)pneumoniae)n nb b b b(beta)(beta)n ncompleteclearingcompleteclearingn nGroupAandBGroupAandBn n (gamma)(gamma)n nnolysisnolysisn nEnterococcus(groupD)Enterococcus(groupD)hemolysisreaction-sheepbloodagara(alpha)hemolysis reaction-MacConkeysAgarContainslactoseandapHindicator,E.colifermentlactosetoproduceacid,whichturnsthepHdyered.So,E.colicoloniesappearred.2.BiochemicalcharacterizationIdentificationofBacterialtheabilitytoattackvarioussubstrateslortoproduceparticularmetabolicproductsMacConkeys Agar2.Biochemical2.BiochemicalcharacterizationTripleSugarIronAgarIdentificationofBacteria2.Biochemical characterizatio细菌的感染诊断防治消毒灭菌课件Asimpleapproachtorapiddiagnosis(asanexampleofantigendetection)isusedinmanydoctorsofficesforthegroupAstreptococcus.Thepatientsthroatisswabbedandstreptococcalantigenextracteddirectlyfromtheswab(withoutpriorbacteriologicalculture).Thebacterialantigenisdetectedbyaggregation(agglutination)ofantibodycoatedlatexbeads.Serologicidentificationofanantibodyresponse(inpatientsserum)totheinfectingagentcanonlybesuccessfulseveralweeksafteraninfectionhasoccurred.3.3.SerologyIdentificationuseofantibodiesofknownspecificitytodetectantigenspresentonwholebacteriaorfreeinbacterialextractsIdentificationofBacteriaA simple approach to rapid diaSerologicalmethodsusedtodetectbothantigenandantibodyinspecimensnThefastestandmostspecificwayqImmunofluorescencemicroscopy,FACSqEnzyme-immunoassayELISA,WesternblotqRadioimmunoassayquantitateantigen-antibodycomplexSerological methods used to FACS(fluorescenceactivatedcellsorter)FACS(fluorescence activated c4.GenomicMethodsn16SDNAsequencingqLabeledprobesspecificforthe16SrRNAofaspeciesareadded,andtheamountoflabelonthedouble-strandedhybridismeasured.qThistechniqueiswidelyusedfortherapididentificationofmanyorganisms.nFluorescenceinsituhybridization(FISH)nPolymeraseChainReaction(PCR)nNucleicAcidSequenceAnalysisnCheckerboardDNAhybridization(DNAmicroarray)IdentificationofBacteria4.Genomic Methods16S DNA sequELISA(Enzyme-LinkedImmunosorbentAssay)ELISA(Enzyme-Linked ImmunosorE.coli 16S RNAlThe16SrRNAofeachspeciesofbacteriahasstable(conserved)portionsofthesequence.lManycopiesarepresentineachorganism.E.coli 16S RNA The 16S rRNA oFISH FISH PCR(polymerasechainreaction)PCR(polymerase chain reactionBacteriamRNAcDNADNA microarraysBacteriamRNAcDNADNA microarray37oC cDNA labeledw/fluorescein tag25oC cDNA labeledw/rhodamine tagDNA array 6000 genes“Transcriptome”37oC cDNA labeled25oC cDNA细菌的感染诊断防治消毒灭菌课件SummarynDirectmicroscopyqFastqGivesomehintsonthetypeofbacteriaqLowsensitivitynCultureqHighsensitivityqCanmakedefinitiveIDqSlowqOnlyworksonculturablebacterianSerologicalassayqFastqHighspecificityqCandetectbothantigenandantibodyqEasy(canbeusedatchairsideorbedside)qLowsensitivitynGenomicbasedassayqFastqHighsensitivityandspecificityqWorksonbothculturableandnon-culturablebacteriaqEspeciallyusefulfordetectingslowgrowingbacteriasuchasT.b.qRequirestechnicalexpertiseqFalsepositiveornegativeSummaryDirect microscopyPreventionofBacterialInfectionPrevention of Bacterial InfectArtificialactiveimmunityVaccinesareantigenspreparedfrompathogensthatVaccinesareantigenspreparedfrompathogensthatcanraiseaprotectiveimmuneresponse,yetdonotcanraiseaprotectiveimmuneresponse,yetdonotcauseillness.Thesepreparedantigenswillstimulatecauseillness.ThesepreparedantigenswillstimulatebothBcellsandTcellsandhelptocreatememorybothBcellsandTcellsandhelptocreatememorycellsthatcanlatermountavigorousimmuneresponsecellsthatcanlatermountavigorousimmuneresponsetoanencounterwiththerealpathogen.toanencounterwiththerealpathogen.Artificial active immunityVaccnToxoids:amodifiedformofthetoxinthatpreservesitsantigenicitybuthaslostitstoxicity.Thishasbeenspectacularlysuccessfulwithtetanusanddiphtheria.nInactivatedvaccines:nAttenuatedlivevaccines:nSpecialvaccines:polysaccharidevaccine,subunitvaccine,(conjugatevaccine,bio-engineeredvaccine,chemicalvaccine,syntheticvaccine),nucleicacidvaccine,idiotypevaccine,autovaccine,etc.ArtificialactiveimmunityToxoids:a modified form of thArtificialpassiveimmunity nAntitoxin:e.g.Tetanusantitoxinanddiphtheriaantitoxin.Itisraisedinthehorse.Itismostimportanttogiveanintentedrecipientofequineserumapriortestdosetoexcludehypersensitivitysubjectswhomayhavebeensensitizedbyapreviousdoseofequineserum.nPooledimmunoglobulin:Itcontainsthenormalrepertoireofantibodiesforanadult,andcanprotectagainsthepatitisA,andmeasles.nSpecificimmunoglobulin:Preparationsofspecificimmunoglobulinareavailableforpassiveimmunizationagainsttetanus,hepatitisB,rabies,varicella-zoster.nCytokineArtificial passive immunity AnSterilizationandDisinfectionA.SterilizationAphysicalorchemicalprocessthatcompletelydestroysorremovesallmicrobiallife,includingspores.B.Disinfection treatmenttodestroyharmfulmicroorganisms.C.PreservationThepreventionofmultiplicationofmicroorganismsinformulatedproducts,includingpharmaceuticalsandfoods.Sterilization and DisinfectionSterilizationandDisinfectionBactericidalAspecifictermreferringtothepropertybywhichabiocideisabletokillbacteria.Bactericidalactiondiffersfrombacteriostasisonlyinbeingirreversible;ie,the“killed”organismcannolongerreproduce,evenafterbeingremovedfromcontactwiththeagent.BacteriostaticAspecifictermreferringtothepropertybywhichabiocideisabletoinhibitbacterialmultiplication;multiplicationresumesuponremovaloftheagent.Sterilization and DisinfectionDisinfectionnPhysicalMethodsqHeatqRadiationqFiltrationqUltrasonicandSonicqFreezingDisinfectionPhysical MethodsSterilizationandDisinfection.HeatnHeatenergycanbeappliedinthreeways,intheformsofqMoistheat(eitherboilingorautoclaving)qDryheatqPasteurizationSterilization and DisinfectionMoistHeat:farmorerapidandeffectiveinsterilizationthandryheat,nAtemperatureof100Cwillkillallbutsporeformsofbacteriawithin23minutesinlaboratory-scalecultures;121Cfor15minutesisutilizedtokillspores.nAutoclavesorpressurecookersareusedforthispurpose.nAutoclavesareusuallyoperatedat121C,whichisachievedwithapressureof15lb/sqnItsuse：anymaterialsthatarenotdamagedbyheatandmoisture,lheat-stableliquidslswabs,lmostinstruments,lculturemedia,lrubbergloves.HeatMoist Heat:far more rapid anDryHeat:nrequired:2hoursat180nItsuse:lsterilizationofglassware,lincinerationofdisposableobjectslpassageofbacteriologicneedles,coverslips,orsmallinstrumentsthroughthenameofaBunsenburner.nThemostwidelyusedtypeofdryheat:isthehot-airoven.HeatDry Heat:.Heat.HeatPasteurization,whichisusedprimarilyformilk,consistsofheatingthemilkto62for30minutesfollowedbyrapidcooling.(“Flash”pasteurizationat72for15secondsisoftenused).Thisissufficienttokillthevegetativecellsofthemilk-bornepathogens,eg,M bovis,Salmonella,Streptococcus,Listeria,andBrucella,butnottosterilizethemilk.Usedforfoods(milk,fruitDrinks,wine)andfragilemedicalequipment,tokillallvegetativebacteriaofsignificanceinhumandisease，butnotspores.Heat Pasteurization,which.RadiationnThetwotypesofradiationusedtokillmicroorganismsareqUltraviolet(UV)light:Themosteffectivebactericidalwavelengthisinthe240to280nmrange,withtheoptimumatabout260nm,qIonizingRadiations:havehigherenergyandpenetratingpowerthanUVradiation.RadiationThe two types of rUltravioletRadiation(UV)Mechanism:UVlightcausesdirectdamagetoDNA,formationofthepyrimidinedimers,Asaresult,DNAreplicationisinhibitedandtheorganismscannotgrow.UseofUVlightinmedicineislimitedlitspoorabilitytopenetratelsafety,becauseUVradiationcandamagethecorneaandskin,So,Itmustberememberedthatworkersexposedtoitmustbeappropriatelyprotected.itsmainapplication:lairofh