细菌感染诊断防治消毒灭菌课件

BacterialInfectionBacterialInfection1Normal flora Opportunistic pathogenPathogenic bacteria(virulent bacteria)DysbacteriosisExotoxins EndotoxinsSeparation&Culture of bacteriaBacteriemiaSepticemiaPyemiaToxemiaEndotoxemiaCarrier Colony Toxoids AntitoxinKey WordsNormalfloraBacteriemiaKeyWo2NotallbacteriacausediseaseNormalfloraThenormalfloraarebacteriawhicharefoundinoronourbodiesonasemi-permanentbasiswithoutcausingdisease.Therearemorebacterialivinginoronourbodies,thanwehavecellsofourown.Ahumanbodycontainsaround1013cells.Thehumanbodyishometoaround1014bacteria.Onefourthoffecalweightismadeofbacteria!Thenormalfloraareparticularlyimportantinthelargeintestine.Normalfloraarealsofoundinthenose,mouth,throat,skin.Opportunisticbacteriacanbepartofthenormalflorabutnotpathogenicfornormalpersons,causediseaseonlyinimmunocompromisedpersons,orunderspecificconditions.Pathogenicbacteria(virulentbacteria)Normallynotmembersofthenormalflora,havemechanismstopromotetheirowngrowthintheexpenseofhoststissueororgansfunctionNotallbacteriacausedisease3The normal flora protect us from disease by:1.Competing with invaders for space and nutrients.2.Producing compounds(bacteriocins)which kill other bacteria.3.Lowering the pH so that other bacteria cant grow.Thenormalfloraprotectusfr4In addition to the above ways of protecting us from disease our normal flora help us in other ways.Of course,there is a down side as well.TheGoodSideProducevitaminsweareunabletoproducesuchasvitaminB12.BoostourimmunesystemGermfreeanimals(bornandraisedinagermfreeplastictent)areverysusceptibletodiseasewhenremovedfromthegermfreeenvironmentbecausetheirimmunesystemisunderdeveloped.Helpdigestfood.Inadditiontotheaboveways5TheBadSideIfthenormalfloraescapefromtheirnormallocation,theycancausedisease.Forexample,Escherichiacoli,commonlyfoundintheintestine,cancauseurinarytractinfectionsifintroducedintothebladder.Immunosuppressioncanallowotherwiseharmlessbacteriatocausedisease.AIDS,somecancertreatmentsandtransplantrejectiondrugsallsuppresstheimmunesystemandallowthenormalfloratocauseoccasionallyseriousdisease.TheBadSide6 Three conditions correlate with opportunistic infection:1.ImmuneSystemCompromise2.Dysbacteriosis3.ChangeofInhabitingPlaces:Threeconditionscorrelatewi7Pathogenesis is a multi-factorial process which depends on the nature of the species or strain(virulence factors),the Pathway of Bacterial Entrance(gateway)and the immune status of the host,as well as the number of organisms in the initial exposure and the Environmental conditions.BacterialPathogenesisPathogenesisisamulti-factor8BACTERIAL VIRULENCE FACTORS BACTERIALVIRULENCEFACTORS91.AdherenceFactors2.Surfacehydrophobicity,3.pili,4.Capsule&glycocalyx5.BacterialBiofilms6.InvasionofHostCells&Tissues:Enzymes7.immuneevasion:8.Capsule&glycocalyx,9.IgA1Proteases,10.Antigenicvariation,11.Interferenceofcomplementactivity12.Intracellularpathogenicity.Bacterial Virulence.Bacterial Virulence FactorsFactorsAdherenceFactors.Bacterial104.ToxinsExotoxinsEndotoxinsExotoxins proteins usually enzymes destroy cellular structures destroy extracellular matrix Antibodies(anti-toxins)neutralize -vaccinationActiveActiveBindingBindingA ACellsurfaceCellsurfaceB BA-B toxins4.ToxinsExotoxinsExotoxinsA11CholeratoxinandE.colilabiletoxinADP-ribosylationofregulatoradenylatecyclaseactivationcyclicAMPactiveionandwatersecretiondiarrheaCholeratoxinandE.colilabi12TetanustoxinTetanustoxininhibitsglycinereleaseinhibitsglycinereleaseinactivatesinhibitoryneuronsinactivatesinhibitoryneuronsmusclesover-activemusclesover-activerigidparalysisrigidparalysis Normal conditionRigid paralysisRigid paralysisTetanustoxinNormalconditionR13BotulinumtoxinBotulinumtoxininhibitsacetylcholinereleaseinhibitsacetylcholinereleaseinhibitsnerveimpulsesinhibitsnerveimpulsesmusclesinactivemusclesinactiveflacidparalysisflacidparalysis(Acetylcholine)Botulinumtoxin(Acetylcholine1415ExotoxinsAntibodies(anti-toxins)neutralizevaccination15ExotoxinsAntibodies(anti-to15EndotoxinsnLipopolysaccharide(LPS):natoxiclipidAnacorepolysaccharidenOantigenpolysaccharidesidechainsnCellwalllysisrequirednFormaldehydeandheatresistantnPoorantigenasfreemoleculenEndotoxineffectsEndotoxinsLipopolysaccharide16EndotoxineffectsFever-pyrogen1microgram/kgLeukopeniaandleukocytosisnecrosisEndotoxemiaandshockDisseminatedintravascularcoagulation(DIC).l lNon-specificinflammation.Non-specificinflammation.l lCytokinereleaseCytokinereleasel lComplementactivationComplementactivationl lBcellmitogensBcellmitogensl lPolyclonalBcellactivatorsPolyclonalBcellactivatorsl lAdjuvantsAdjuvantsEndotoxineffectsNon-speci17细菌感染诊断防治消毒灭菌课件18ExotoxinsToxins produced by bacteria and secreted to the outside of the bacteria cell(different from the endotoxin)acting on cell surface By binding to certain receptorsPossess some degree of host cell specificitydenatured by formaldehyde treatment to generate toxoids which lack toxic activity but still induce protective immunity when used as vaccines.Many exotoxins have an A-B subunit structureA subunit-provides the toxic activityB subunit-mediates adherence of the toxin complex to a host cell SummaryExotoxinsToxinsproducedbyb19Endotoxins(LPS)n derived from Gcell walls and are often liberated when the bacteria lyse.n heat-stable,n three main regions n a toxic lipid A n a core polysaccharide n O antigen polysaccharide side chains n The pathophysiologic effects of LPS are similarEndotoxins(LPS)derivedfro20.PathwayofBacterialEntrancenContact:directorindirect(Sexualcontact)nInhalation:transmittedbytherespiratoryroute(Airbornedroplets)nIngestion:ingestedandtransmittedthroughintestinaltracttotheoutsideandgetnewinfectionsbycontaminatingoffoodandwater,whichiscalledfecal-oralspread(Food,Water)nInoculation&bloodtransfusionnAnimalvectors:transmittedbyanimalvectors(insectbitting).PathwayofBacterialEntran21ResponsetoInfectioninfectionxdiseaseInnate immunityno diseaserecoveryadaptive immunityre-infectionno diseasexResponsetoInfectioninfection22Immunityofextracellularbacterialinfection:antibodies(IgG,IgM,SIgA);phagocytes(neutrophils);complement;humoralimmunitymainly.Immunity of intracellular bacterial infection:cell-mediated immunity(delayed-type hypersensitivity,DTHresponse(DTH)involvingTH1andmacrophages)mainly.Immunityofextracellularbact23NodiseaseHost defenseBacterial infectionBalance between Infection and Immunity(immunity)NodiseaseHostdefenseBacteria24DiseasedevelopsonlyintherighthostandundertherightconditionsDiseaseHostPathogenEnvironmentDiseasedevelopsonlyinther25DiseaseHostdefenseBacterialinfectionEnvironmentalconditionscanhelptiltthebalanceEnvironmental signals often control the expression of the virulence genes.Common signals,include:Temperrature/Iron availability:C diphtheriae/low ion/Osmolality/Growth phase/pH/Specific ionsDiseaseHostdefenseBacteriali26SourcesofInfectionB.EndogenousInfection:opportunisticpathogeninfectionsA.ExogenousInfectionPatientsCarriersAnimalsSourcesofInfectionB.Endogen27TypesofBacterialInfectionAccordingtoinfectiousstate:Inapparentinfection:withoutclearclinicsymptoms.ApparentInfection:haveevidentclinicsymptoms.Accordingtoinfectioussites(LocalinfectionandGeneralizedorsystemicinfectionBacteriemia:bacteriacirculatebutnotmultiplyintheblood.Septicemia:Bacteriacirculateandmultiplyintheblood.Pyemia:producesepticemiawithmultipleabscessesininternalorgans.Toxemia:Bacteiamultiplyatinvadinglocationanddonotenterbloodstream,buttheexotoxinsenterbloodandcausecorrespondingtoxicsymptomsEndotoxemia:multiplyatlocationorinbloodstream,releasealotofamountendotoxinreleasedfrombacterialcellruptureCarrier:Types of Bacterial InfectionAc28PrinciplesofDiagnosisandPreventionofBacterialInfectionPrinciples of Diagnosis and Pr29nManifestationsofInfection:Signsandsymptomsvaryaccordingtothesiteandseverityofinfection.Diagnosisrequiresacompositeofinformation,includinghistory,physicalexamination,radiographicfindings,andlaboratorydata.nMicrobialCausesofInfection:Infectionsmaybecausedbybacteria,viruses,fungi,andparasites.Thepathogenmaybeexogenous(acquiredfromenvironmentaloranimalsourcesorfromotherpersons)orendogenous(fromthenormalflora).Manifestations of Infection:S301.Thequantitymaterialmustbeadequate.2.Collectfromappropriatesite.3.Preparesitetominimizecontamination.4.Wheneverpossible,collectspecimenspriortoantibiotics.5.Transportsystemmaximizedforpathogensurvivalshouldbeused.GeneralguidelinesforspecimencollectionThe quantity material must be 31BodyfluidforspecimencollectionnBlood septicemia nCerebrospinal fluid bacterial meningitisnPeritoneal(abdominal)nPleural(chest)nSynovial(joint)nPericardial(heart)nUrine Body fluid for specimen collec32SamplecollectiondevicesSample collection devices33MicroscopyandStains:cellularmorphologyandstainmaypermitpreliminaryidentification.Separation&Cultureofbacteria:Isolationofinfectiousagentsfrequentlyrequiresspecializedmedia.Nonselective(noninhibitory)mediapermitthegrowthofmanymicroorganisms.Selectivemediacontaininhibitorysubstancesthatpermittheisolationofspecifictypesofmicroorganisms.ColonymorphologycansometimesbeusefulinbacterialidentificationIdentificationofbacteria:Culturalcharacteristics:Growthcharacteristicsundervariousconditions,Biochemicalcharacterization:utilizationofcarbohydratesandothersubstrates,enzymaticactivitySerologyIdentificationAhighorrisingtiterofspecificIgGantibodiesorthepresenceofspecificIgMantibodiesmaysuggestorconfirmadiagnosis.GenomicMethodsAntimicrobialSusceptibility:Microorganisms,particularlybacteria,aretestedinvitrotodeterminewhethertheyaresusceptibletoantimicrobialagents.MicrobiologicExaminationMicroscopy and Stains:cellula34MicroscopyandStainsDirectexaminationofstainedorunstainedpreparationsbylight(brightfield)microscopyisarelativelysimple:witha100oilimmersionobjective,a5to10eyepiece,optimallighting.Thetwomostimportantmethods,theGramandacid-fasttechniques,toclassifyaswellasstaintheorganism.employstaining,decolorization,counterstainingMicroscopy and Stains Direct e35ExamplesofGramStainsGramPositiveRodsandCocciGramNegativeRodsandCocciExamples of Gram StainsGram Po36ExamplesofAcid-FastStainsFullyAcid-FastRods(Mycobacteriumspecies)PartiallyAcid-FastRods(Nocardiaspecies)Examples of Acid-Fast StainsFu37typesofmediaforroutineculture:Thestandardmediumforspecimens:bloodagar,-Usuallymadewith5%sheepblood.-MostaerobicandfacultativelyanaerobicorganismswillgrowonbloodagarAsecondnecessarymedium:Chocolateagar,-amediumcontainingheatedbloodwithorwithoutsupplements,-Someorganismsthatdonotgrowonbloodagar,includingpathogenicneisseriaandhaemophilus,willgrowonchocolateagar.Culturetypes of media for routine cul38SelectivemedianSelective media is one that grows only certain microorganisms while inhibiting(or preventing)others from growing,that is to say,the media has certain chemicals that allow one organism to grow but another organism cannot grow with those ingredients in the media,thus,is selective for the organisms that can grow in that media.Therefore,most commonly grows only one type of organism.Selective mediaSelective media39Differential medianDifferential media is one that distinguishes one microorganism from another,it can grow more than one microorganism,but depending on how each organism reacts to the media(like turns red)it differentiates from another microorganism.In this type of media if you are trying to distinguish between 2 types of microorganisms,both should grow but they will have different reactions to the media,and thru their visible reactions you can tell them apart(differentiate them).Differential mediaDifferential40qGrowth on selective-differential media,such as Salmonella-Shigella(SS)medium,eosin-methylene-blue(EMB)and MacConkey agarqThe select effect of the media in suppressing unwanted gram-positive organisms is exerted by bile salts or bacteriostatic dyes in the agar.qThe differential ability of these media is based on lactose fermentation:normal flora positive(colored colonies)and pathogens negative(colorlesscolonies).Growth on selective-differenti41Separationofbacteria:platestreaking.Bacteriologicplatestreaking.Separation of bacteria:plate42细菌感染诊断防治消毒灭菌课件43Colony:thevisiblegrowthofbacteriaonsolidgrowthmedia.Ideally,thecolonyistheprogenyofone,oratmost,afewbacteria.Acolonywillusuallycontainmillionsofbacterialcells.Colonymorphologycansometimesbeusefulinbacterialidentification.Coloniesaredescribedastosuchpropertiesassize,shape,texture,elevation,pigmentation,effectongrowthmedium.Colony Colony:the visible growth of44细菌感染诊断防治消毒灭菌课件45细菌感染诊断防治消毒灭菌课件46Growthonbloodagartotestforhemolyticproperties-hemolytic:-hemolytic:incomplete lysis of red incomplete lysis of red blood cells,resulting blood cells,resulting in a greenish halo in a greenish halo around the colonyaround the colony-hemolytic:-hemolytic:complete lysis of red complete lysis of red blood cells,blood cells,resulting in a clear resulting in a clear halo around the halo around the colonycolony-hemolytic:non-hemolytic1.Culturalcharacteristics:luniquenutritionalrequirements,lpigmentproduction,lhemolyticpropertiesIdentificationofBacteriaGrowth on blood agar to test f47n na(alpha)a(alpha)a(alpha)a(alpha)n npartial hemolysis partial hemolysis partial hemolysis partial hemolysis n nreenish color(i.e.S.reenish color(i.e.S.reenish color(i.e.S.reenish color(i.e.S.pneumoniae)pneumoniae)pneumoniae)pneumoniae)n nb(beta)b(beta)b(beta)b(beta)n ncomplete clearincomplete clearincomplete clearincomplete clearinn n Group A and B Group A and B Group A and B Group A and Bn n(amma)(amma)(amma)(amma)n nno lysisno lysisno lysisno lysisn nEnterococcus(roupD)Enterococcus(roupD)Enterococcus(roupD)Enterococcus(roupD)hemolysisreaction-sheepbloodagara(alpha)hemolysis reaction-48MacConkeysAgarContainslactoseandapHindicator,E.colifermentlactosetoproduceacid,whichturnsthepHdyered.So,E.colicoloniesappearred.2.BiochemicalcharacterizationIdentificationofBacterialtheabilitytoattackvarioussubstrateslortoproduceparticularmetabolicproductsMacConkeys Agar2.Biochemical492.BiochemicalcharacterizationTripleSugarIronAgarIdentificationofBacteria2.Biochemical characterizatio50细菌感染诊断防治消毒灭菌课件51Asimpleapproachtorapiddiagnosis(asanexampleofantigendetection)isusedinmanydoctorsofficesforthegroupAstreptococcus.Thepatientsthroatisswabbedandstreptococcalantigenextracteddirectlyfromtheswab(withoutpriorbacteriologicalculture).Thebacterialantigenisdetectedbyaggregation(agglutination)ofantibodycoatedlatexbeads.Serologicidentificationofanantibodyresponse(inpatientsserum)totheinfectingagentcanonlybesuccessfulseveralweeksafteraninfectionhasoccurred.3.SerologyIdentificationuseofantibodiesofknownspecificitytodetectantigenspresentonwholebacteriaorfreeinbacterialextractsIdentificationofBacteriaA simple approach to rapid dia52SerologicalmethodsusedtodetectbothantigenandantibodyinspecimensnThefastestandmostspecificwaynImmunofluorescencemicroscopy,FACSnEnzyme-immunoassayELISA,WesternblotnRadioimmunoassayquantitateantigen-antibodycomplexSerological methods used to 53FACS(fluorescenceactivatedcellsorter)FACS(fluorescence activated c544.GenomicMethodsn16S DNA sequencingnLabeled probes specific for the 16S rRNA of a species are added,and the amount of label on the double-stranded hybrid is measured.n This technique is widely used for the rapid identification of many organisms.nFluorescence in situ hybridization(FISH)nPolymerase Chain Reaction(PCR)nNucleic Acid Sequence Analysis nChecker board DNA hybridization(DNA microarray)IdentificationofBacteria4.Genomic Methods16S DNA sequ55ELISA(Enzyme-LinkedImmunosorbentAssay)ELISA(Enzyme-Linked Immunosor56E.coli 16S RNAl The 16S rRNA of each species of bacteria has stable(conserved)portions of the sequence.l Many copies are present in each organism.E.coli 16S RNA The 16S rRNA o57FISH FISH 58PCR(polymerasechainreaction)PCR(polymerase chain reaction59BacteriamRNAcDNADNA microarraysBacteriamRNAcDNADNA microarray6037oC cDNA labeledw/fluorescein tag25oC cDNA labeledw/rhodamine tagDNA array 6000 genes“Transcriptome”37oC cDNA labeled25oC cDNA61细菌感染诊断防治消毒灭菌课件62SummarynDirectmicroscopynFastnGivesomehintsonthetypeofbacterianLowsensitivitynCulturenHighsensitivitynCanmakedefinitiveIDnSlownOnlyworksonculturablebacterianSerologicalassaynFastnHighspecificitynCandetectbothantigenandantibodynEasy(canbeusedatchairsideorbedside)nLowsensitivitynGenomicbasedassaynFastnHighsensitivityandspecificitynWorksonbothculturableandnon-culturablebacterianEspeciallyusefulfordetectingslowgrowingbacteriasuchasT.b.nRequirestechnicalexpertisenFalsepositiveornegativeSummaryDirect microscopy63PreventionofBacterialInfectionPrevention of Bacterial Infect64ArtificialactiveimmunityVaccines are antigens prepared from pathogens that Vaccines are antigens prepared from pathogens that can raise a protective immune response,yet do not can raise a protective immune response,yet do not cause illness.These prepared antigens will stimulate cause illness.These prepared antigens will stimulate both B cells and T cells and help to create memory both B cells and T cells and help to create memory cells that can later mount a vigorous immune response cells that can later mount a vigorous immune response to an encounter with the real pathogen.to an encounter with the real pathogen.Artificial active immunityVacc65nToxoids:amodifiedformofthetoxinthatpreservesitsantigenicitybuthaslostitstoxicity.Thishasbeenspectacularlysuccessfulwithtetanusanddiphtheria.nInactivatedvaccines:nAttenuatedlivevaccines:nSpecialvaccines:polysaccharidevaccine,subunitvaccine,(conjugatevaccine,bio-engineeredvaccine,chemicalvaccine,syntheticvaccine),nucleicacidvaccine,idiotypevaccine,autovaccine,etc.ArtificialactiveimmunityToxoids:a modified form of th66ArtificialpassiveimmunitynAntitoxin:e.g.Tetanusantitoxinanddiphtheriaantitoxin.Itisraisedinthehorse.Itismostimportanttogiveanintentedrecipientofequineserumapriortestdosetoexcludehypersensitivitysubjectswhomayhavebeensensitizedbyapreviousdoseofequineserum.nPooledimmunoglobulin:Itcontainsthenormalrepertoireofantibodiesforanadult,andcanprotectagainsthepatitisA,andmeasles.nSpecificimmunoglobulin:Preparationsofspecificimmunoglobulinareavailableforpassiveimmunizationagainsttetanus,hepatitisB,rabies,varicella-zoster.nCytokineArtificial passive immunity An67SterilizationandDisinfectionA.SterilizationAphysicalorchemicalprocessthatcompletelydestroysorremovesallmicrobiallife,includingspores.B.Disinfectiontreatmenttodestroyharmfulmicroorganisms.C.PreservationThepreventionofmultiplicationofmicroorganismsinformulatedproducts,includingpharmaceuticalsandfoods.Sterilization and Disinfection68SterilizationandDisinfectionBactericidalAspecifictermreferringtothepropertybywhichabiocideisabletokillbacteria.Bactericidalactiondiffersfrombacteriostasisonlyinbeingirreversible;ie,the“killed”organismcannolongerreproduce,evenafterbeingremovedfromcontactwiththeagent.BacteriostaticAspecifictermreferringtothepropertybywhichabiocideisabletoinhibitbacterialmultiplication;multiplicationresumesuponremovaloftheagent.Sterilization and Disinfection69DisinfectionnPhysicalMethodsnHeatnRadiationnFiltrationnUltrasonicandSonicnFreezingDisinfectionPhysical Methods70SterilizationandDisinfection.HeatnHeat energy can be applied in three ways,in the forms of nMoist heat(either boiling or autoclaving)nDry heatnPasteurization Sterilization and Disinfection71MoistHeat:farmorerapidandeffectiveinsterilizationthandryheat,Atemperatureof100Cwillkillallbutsporeformsofbacteriawithin23minutesinlaboratory-scalecultures;121Cfor15minutesisutilizedtokillspores.Autoclavesorpressurecookersareusedforthispurpose.Autoclavesareusuallyoperatedat121C,whichisachievedwithapressureof15lb/sqItsuse：anymaterialsthatarenotdamagedbyheatandmoisture,heat-stableliquidsswabs,mostinstruments,culturemedia,rubbergloves.HeatMoist Heat:far more rapid an72DryHeat:required:2hoursat180Itsuse:sterilizationofglassware,incinerationofdisposableobjectspassageofbacteriologicneedles,coverslips,orsmallinstrumentsthroughthenameofaBunsenburner.Themostwidelyusedtypeofdryheat:isthehot-airoven.HeatDry Heat:.Heat73.HeatPasteurization,whichisusedprimarilyformilk,consistsofheatingthem