細(xì)菌分類-課件

Faculty:Dr.AlvinFoxSuggestedreading:Murray,Thirdedition Chapter2,16-18and21.TOPIC:CLASSIFICATION&IDENTIFICATIONOFINFECTIOUSAGENTS.Faculty:Dr.AlvinFoxSuggest1KeyTermsIsolation(culture)Agarplate/coloniesLiquidmediatesttube-bulkIdentification&taxonomyFamilyGenusSpeciesTypeStrainBiochemical(physiological)testsMoleculartestsDNA-DNAhomology16SrRNAsequencingChemicalprofiling NonculturebaseddetectionPolymerasechainreaction-(PCR)Agglutination(antigendetection)StainSerology(antibodydetection).KeyTermsIsolation(culture)Id2Bacterialidentificationinthediagnosticlaboratory1.Isolation&identificationaidstreatmentsinceinfectiousdiseases causedbydifferentbacteriahaveavarietyofclinicalcoursesandconsequences.Susceptibilitytestingofisolateshelpsinselectionofantibiotics fortherapy.Theidentificationisbasedontaxonomicprinciplesappliedto theclinicalmicrobiologicalsituation.4.Testsmustbeeasilylearned,lowincostandrapidlyperformed..Bacterialidentification1.3ClassificationFamily:agroupofrelatedgenera.Genus:agroupofrelatedspecies.Species:agroupofrelatedstrains.Type:setsofstrainwithinaspecies(e.g.biotypes,serotypes).Strain:onelineorasingleisolateofaparticularspecies..ClassificationFamily:agroup4StepsindiagnosticisolationandidentificationofbacteriaStep1.Samplesofbodyfluidsarestreakedoncultureplatesandisolatedcoloniesofbacteriaappearafterincubation.Observationofthesecoloniesforsize,texture,color,and(ifgrownonbloodagar)hemolysisreactions,ishighlyimportantasafirststepinbacterialidentification.Whethertheorganismrequiresoxygenforgrowthisanotherimportantdifferentiatingcharacteristic..Stepsindiagnosticisolation5BloodAgarPlate.BloodAgarPlate.6Step2.ColoniesareGramstainedandindividualbacterialcellsobservedunderthemicroscope..Step2.ColoniesareGramstai7Asinglecolonyisplacedonaslideanddried.Stage1.Stainingwithcrystalviolet.Stage2.Fixationwithiodinestabilizescrystalvioletstaining.Allbacteriaremainpurpleorblue.Stage3.Extractionwithalcohol.Decolorizessomebacteria (Gramnegative)andnotothers(Grampositive).Stage4.Counterstainingwithsafranin.Grampositive bacteriaarestainedwithcrystalvioletandremain purple.Gramnegativebacteriaarestainedpink.GramStain.Asinglecolonyisplacedona8..9GramstainmorphologyShapecocci(round)bacilli(rods)spiralorcurved(e.g.spirochetes)Singleormultiplecellsclusters(e.g.streptococci)chains(e.g.streptococci)Grampositiveornegative.GramstainmorphologyShape.10..11..12Step3.Thebacteriaarespeciatedusingtheseisolatedcolonies.

.Step3.Thebacteriaarespeci13TypicalCultureLaboratoryBench.TypicalCultureLaboratoryBen14Step4.Antibioticsusceptibilitytestingisperformed..Step4.Antibioticsusceptibil15TaxonomiccharacterizationofbacteriaComparisonsofspeciesinvolvecomparisonsof multiplestrainsforeachspecies.Comparisonsareprimarilybasedonchemicalor molecularanalysis..Taxonomiccharacterizationof16MolecularanalysisItwouldbeidealtocomparesequencesofentirebacterialchromosomalDNA.Alternatively,genomicsimilarityhasbeenassessedbytheguanine(G)+cytosine(C)content(%GC).Thishasbeenreplacedbytwoalternatives:HybridizationSequencingspecificgenes.MolecularanalysisItwouldbe17DNA-DNAhomology1.HowwelltwostrandsofDNAfromdifferent bacteriabind(hybridize)together. Thistechniqueisemployedtocomparethe geneticrelatednessofbacterialstrains/species.IftheDNAfromtwobacterialstrainsdisplaya highdegreeofhomology(i.e.theybindwell)the strainsareconsideredtobemembersofthesame species..DNA-DNAhomology1.Howwellt18“Goldstandard"inbacterialtaxonomySequencingof16SribosomalRNAmolecules(16SrRNA)The16SrRNAisapproximately1600nucleotidesinlength.The16SrRNAprovidesgenomicsimilarityabovethe specieslevelallowingcomparisonsofrelatednessacross theentirebacterialkingdom.CloselyrelatedbacterialspeciesoftenhaveidenticalrRNA sequences.5. Thetechniqueprovidescomplementaryinformationto DNA-DNAhybridization.

Thedevelopmentofprobesbasedon16Sdatahaveimproved identificationofsomebacterialpathogens..“Goldstandard"inbacterialt19ChemicalanalysisSophisticatedtoolsareavailableforstudyingthestructuralcompositionofbacteria(mostcommonlyfattyacid,carbohydrateorubiquinoneprofiling)ofbacteria.Characterizationofsecretedmetabolic

products(e.g.volatilealcoholsandshortchainfattyacids)isalsohelpful..ChemicalanalysisSophisticate20Approachestorapiddiagnosiswithoutpriorculture

Certainhumanpathogenseithercannotbeisolatedinthelaboratorygrowextremelypoorly.Successfulisolationcanbeslowandinsome instancesimpossible.3.Directdetectionofbacteriawithoutcultureis possibleforsomeorganisms..Approachestorapiddiagnosis21AsimpleapproachtorapiddiagnosisAntigendetection Usedinmanydoctor'sofficesforthegroupAstreptococcus.Thepatient'sthroatisswabbedandstreptococcalantigenextracteddirectlyfromtheswab(withoutpriorbacteriologicalculture).Thebacterialantigenisdetectedbyaggregation(agglutination)ofantibodycoatedlatexbeads..Asimpleapproachtorapiddia22BacterialDNAsequencesamplifieddirectlyfromhumanbodyfluidsThepolymerasechainreaction(PCR)canbeusedtoamplifylargeamountsofspecificgenesorportionsofgenes.Greatsuccesshasbeenachievedinrapiddiagnosisoftuberculosis..BacterialDNAsequencesamplif23DirectmicroscopicobservationCertainclinicalsamplessuchassputumcanbedirectlyassessedforthepresenceofbacteriacanbehelpful(e.g.detectionofM.tuberculosis

insputum)..Directmicroscopicobservation24SerologicidentificationSerologicidentificationofanantibodyresponse(inpatient'sserum)totheinfectingagentcanonlybesuccessfulseveralweeksafteraninfectionhasoccurred..SerologicidentificationSerolo25Faculty:Dr.AlvinFoxSuggestedreading:Murray,Thirdedition Chapter2,16-18and21.TOPIC:CLASSIFICATION&IDENTIFICATIONOFINFECTIOUSAGENTS.Faculty:Dr.AlvinFoxSuggest26KeyTermsIsolation(culture)Agarplate/coloniesLiquidmediatesttube-bulkIdentification&taxonomyFamilyGenusSpeciesTypeStrainBiochemical(physiological)testsMoleculartestsDNA-DNAhomology16SrRNAsequencingChemicalprofiling NonculturebaseddetectionPolymerasechainreaction-(PCR)Agglutination(antigendetection)StainSerology(antibodydetection).KeyTermsIsolation(culture)Id27Bacterialidentificationinthediagnosticlaboratory1.Isolation&identificationaidstreatmentsinceinfectiousdiseases causedbydifferentbacteriahaveavarietyofclinicalcoursesandconsequences.Susceptibilitytestingofisolateshelpsinselectionofantibiotics fortherapy.Theidentificationisbasedontaxonomicprinciplesappliedto theclinicalmicrobiologicalsituation.4.Testsmustbeeasilylearned,lowincostandrapidlyperformed..Bacterialidentification1.28ClassificationFamily:agroupofrelatedgenera.Genus:agroupofrelatedspecies.Species:agroupofrelatedstrains.Type:setsofstrainwithinaspecies(e.g.biotypes,serotypes).Strain:onelineorasingleisolateofaparticularspecies..ClassificationFamily:agroup29StepsindiagnosticisolationandidentificationofbacteriaStep1.Samplesofbodyfluidsarestreakedoncultureplatesandisolatedcoloniesofbacteriaappearafterincubation.Observationofthesecoloniesforsize,texture,color,and(ifgrownonbloodagar)hemolysisreactions,ishighlyimportantasafirststepinbacterialidentification.Whethertheorganismrequiresoxygenforgrowthisanotherimportantdifferentiatingcharacteristic..Stepsindiagnosticisolation30BloodAgarPlate.BloodAgarPlate.31Step2.ColoniesareGramstainedandindividualbacterialcellsobservedunderthemicroscope..Step2.ColoniesareGramstai32Asinglecolonyisplacedonaslideanddried.Stage1.Stainingwithcrystalviolet.Stage2.Fixationwithiodinestabilizescrystalvioletstaining.Allbacteriaremainpurpleorblue.Stage3.Extractionwithalcohol.Decolorizessomebacteria (Gramnegative)andnotothers(Grampositive).Stage4.Counterstainingwithsafranin.Grampositive bacteriaarestainedwithcrystalvioletandremain purple.Gramnegativebacteriaarestainedpink.GramStain.Asinglecolonyisplacedona33..34GramstainmorphologyShapecocci(round)bacilli(rods)spiralorcurved(e.g.spirochetes)Singleormultiplecellsclusters(e.g.streptococci)chains(e.g.streptococci)Grampositiveornegative.GramstainmorphologyShape.35..36..37Step3.Thebacteriaarespeciatedusingtheseisolatedcolonies.

.Step3.Thebacteriaarespeci38TypicalCultureLaboratoryBench.TypicalCultureLaboratoryBen39Step4.Antibioticsusceptibilitytestingisperformed..Step4.Antibioticsusceptibil40TaxonomiccharacterizationofbacteriaComparisonsofspeciesinvolvecomparisonsof multiplestrainsforeachspecies.Comparisonsareprimarilybasedonchemicalor molecularanalysis..Taxonomiccharacterizationof41MolecularanalysisItwouldbeidealtocomparesequencesofentirebacterialchromosomalDNA.Alternatively,genomicsimilarityhasbeenassessedbytheguanine(G)+cytosine(C)content(%GC).Thishasbeenreplacedbytwoalternatives:HybridizationSequencingspecificgenes.MolecularanalysisItwouldbe42DNA-DNAhomology1.HowwelltwostrandsofDNAfromdifferent bacteriabind(hybridize)together. Thistechniqueisemployedtocomparethe geneticrelatednessofbacterialstrains/species.IftheDNAfromtwobacterialstrainsdisplaya highdegreeofhomology(i.e.theybindwell)the strainsareconsideredtobemembersofthesame species..DNA-DNAhomology1.Howwellt43“Goldstandard"inbacterialtaxonomySequencingof16SribosomalRNAmolecules(16SrRNA)The16SrRNAisapproximately1600nucleotidesinlength.The16SrRNAprovidesgenomicsimilarityabovethe specieslevelallowingcomparisonsofrelatednessacross theentirebacterialkingdom.CloselyrelatedbacterialspeciesoftenhaveidenticalrRNA sequences.5. Thetechniqueprovidescomplementaryinformationto DNA-DNAhybridization.

Thedevelopmentofprobesbasedon16Sdatahaveimproved identificationofsomebacterialpathogens..“Goldstandard"inbacterialt44ChemicalanalysisSophisticatedtoolsareavailableforstudyingthestructuralcompositionofbacteria(mostcommonlyfattyacid,carbohydrateorubiquinoneprofiling)ofbacteria.Characterizationofsecretedmetabolic

products(e.g.volatilealcoholsandshortchainfattyacids)isalsohelpful..ChemicalanalysisSophisticate45Approachestorapiddiagnosiswithoutpriorculture

Certainhumanpathogenseithercannotbeisolatedinthelaboratorygrowextremelypoorly.Successfulisolati