病原微生物學(xué)課件：6- September Laboratory Diagnosis and Prophylaxis of Bacterial Infection-2

LaboratoryDiagnosisofBacterialInfectionLecture62ClinicalMicrobiologyLaboratoryBenchSpecimenscollection

ThequantitymaterialmustbeadequateSpecimensareselectedonthebasisofsignsandsymptoms,shouldberepresentativeofthediseaseprocessContaminationofthespecimenmustbeavoidedbyusingonlysterileequipmentandasepticprecautionsThespecimenmustbetakentothelaboratoryandexaminedpromptly.Specialtransportmediamaybehelpful.Meaningfulspecimenstodiagnosebacterialinfectionsmustbesecuredbeforeantimicrobialdrugsareadministered.

Generalprocedureforcollectingandprocessingspecimensforaerobicand/oranaerobicbacterialculture

5Isolation(culture)Agarplateplate/coloniesLiquidmediaIdentification&taxonomyFamilyGenusSpeciesTypeStrainAftercultureBiochemical(physiological)testsGenetictestsSequencing,Polymerasechainreaction(PCR)DNA-DNAhomologyRestrictionenzymesdigestChemical-fattyacid/proteinprofiling Serology(antigendetection)Directdetection(i.e.withoutculture)PCRStaining(e.g.Gramstain)Serology(antigenorantibodydetection)LaboratoryDiagnosisofBacterialInfection

LaboratoryDiagnosisofBacterialInfection

Conventionalmethods(stainingandmicroscopy)Biochemical(physiological)testsSero-diagnosisAntibioticsusceptibilitytestingMoleculardiagnosisAnimalTest(invivo)7I.Morphologyidentification

StainingGramstainingAcidfaststainingMicroscopy:ColoniesGramstainedcellsobservedmicroscopicallySheepbloodagarplatecultureBacillusanthracisBacilluscereus.BacterialMorphology-I3mostcommonshapes:Spherical/Rods/SpiralGramStaining:Gram-Positive/NegativeGram+Gram-12MixedSamplesDistinguishescellsthathavemycolicacidincellwall,fromthosethatdonot:

-Bluestaining,Nonacid-fastcells(NAF) -brightpinkstaining,Acid-fastcells(AF)Createasmearoforganismyouaretesting.Coversmearwithablottingpaper.SaturatepaperwithZiehl’scarbolfuchsin(sayfyook-sin).Heat3–5minutes.Removeblottingpaper.Rinseslidewithtapwater,thendecolorizethesmearfor10-15secondswithacidalcohol.Rinse.Applycrystalvioletfor1minute,wash,blotdry.ACID-FASTSTAININGPROCEDUREBlottingpaperZiehlscarbolfuchsin(3–5minheat)>

rinse

AcidAlcohol(10–15sec)>

rinse

crystalviolet(1min)>

rinse

&blotdryFromtheVirtualMicrobiologyClassroomonScienceProfOAcid-faststain14II.Biochemicalidentification

-BasedonthecatabolicproductsBacteriametabolism-Potentialapplicationfordifferiation

Theproductofthecatabolicprocessesmakeitpossibleforustodistinguishdifferentkindsofbacteria.SugarfermentationtestVoges-Proskauer(VP)testMethylred(MR)testCitratetestIndoletestH2StestUrealytictestThetestbasedontheproductsofthesugarThetestbasedontheproductsoftheproteinThetestbasedontheotherproductsBiochemicalidentificationCarbohydrateutilityH2S

testCitrateassay

UREASETESTDoneinureasemedium.Thistestisusedtodetectorganismsthatproduceurease.UreaseproducedbytheorganismssplitureaintoammoniaandCO2.Ureasepositive–pinkcolourUreasenegative–yellowcolourPositive–Proteus,KlebsiellaNegative–E.coli,SalmonellaAPI–20ETheAPI-20EtestisusedtoIDGram-negative

bacillifromthefamilyEnterobacteriacea.Image:API-20Eresultsfordifferentspeciesofbacteria,Dr.CharlesGerba,UniversityofArizonaFromtheVirtualMicrobiologyClassroomonScienceProfOBacteriametabolismIII:IMViCtestTheIMViCtestcanbeusedtodistinguishdifferentcategoryofintestinalbacilli.

I

M

Vi

CIndoletestMethylred(MR)testVPtestCitratetestE.coliEntericbacillus++++----E.ColiandEntericbacillus:Rod/Gram/Isolatedfromintestinaltract.Howtodistinguishthem?FromtheVirtualMicrobiologyClassroomonScienceProfOIntensepinkLight,unevenpinkSpeciesidentificationhierarchyinclinicalsettingsIII.Serological

DiagnosisDetectAntigen(pathogen)orantibodyagainstpathogenAgglutinationtestPrecipitationtestComplementfixationtestNeutralizationtestELISA(Enzyme-linkedImmunesorbentAssay)Fluorescent-AntibodyTechnique23StreptococcalAgglutinationTestLatexbeadsStreptococcalantigenicextractAntibodyTheenzyme-linkedimmunosorbentassay(ELISA)InaqualitativeorquantitativeformatSpecificSensitiveScreeningtest26IV.Antibioticsusceptibilitytesting28NogrowthAntibioticsusceptibilitytestingSusceptibleNotsusceptibleBacteriallawnGrowthAntibioticdiskBacteriadisplaysdifferentantibioticsensitivityprofilesV.MolecularDiagnostics

PolymeraseChainReaction(PCR)andreal-timePCRDifferiatingbacteriaspeciesusing16SrRNAMoleculartypingNucleicAcidSequenceAnalysisRestrictionenzymedigestDNAHybridizationDNAChips(Microarray)NextgenerationsequencingDevelopmentofclinicaltestsbasedonsequence(e.g.PCR)DifferentiatesbacterialspeciesMoleculartypingsuchasMLSTandmolecularserotypingPolymeraseChainReaction(PCR)PCRAmplificationofDNAfragmentDNA

MicroarrayNextGenerationSequencingAnimalsTestProphylaxisofBacterialInfection

ArtificialactiveimmunityVaccinesareantigenspreparedfrompathogensthatcanraiseaprotectiveimmuneresponse,yetdonotcauseillness.ThesepreparedantigenswillstimulatebothBcellsandTcellsandhelptocreatememorycellsthatcanlatermountavigorousimmuneresponsetoanencounterwiththerealpathogen.42Milestonesinimmunization1780ADEdwardJennerdiscoverssmallpoxvaccine43EdwardJennerDiscoveryofsmallpoxvaccine44EdwardJenner

AmongpatientsawaitingsmallpoxvaccinationToxoids:amodifiedformofthetoxinthatpreservesitsantigenicitybuthaslostitstoxicity.Thishasbeenspectacularlysuccessfulwithtetanusanddiphtheria.Inactivatedvaccines:Theproductionofprotectiveantibodiesisstimulatedbyusingthekilled(inactivated)organismsThisisdoneasaroutinewithvaccinesagainstpertussis(whoopingcough),typhoidandinfluenza.Thereisalsoaninactivatedpoliovaccine.Attenuatedlivevaccines:Theapproachistousesuspensionsoflivingorganismsthatarereducedintheirvirulence(attenuated)butstillimmunogenic.Thisstrategyhasyielded:BCG,mumps,measles,andrubellavaccines(nowcombined);theliveviruspoliovaccine.Specialvaccines:polysaccharidevaccine,subunitvaccine,(conjugatevaccine,bio-engineeredvaccine,chemicalvaccine,syntheticvaccine),nucleicacidvaccine,idiotypevaccine,autovaccine,etc.DNAvaccineArtificialpassiveimmunity

Antitoxin:e.g.Tetanusantitoxinanddiphtheriaantitoxin.Itisraisedinthehorse.Itismostimportanttogiveanintentedrecipientofequineserumapriortestdosetoexcludehypersensitivitysubjectswhomayhavebeensensitizedbyapreviousdoseofequineserum.Pooledimmunoglobulin:Itcontainsthenormalrepertoireofantibodiesforanadult,andcanprotectagainsthepatitisA,andmeasles.Specificimmunoglobulin:Preparationsofspecificimmunoglobulinareavailableforpassiveimmunizationagainsttetanus,hepatitisB,rabies,varicella-zoster.CytokineActive-passiveimmunityInvolvesgivingbothavaccinetoprovidelong-termprotection(preventiveinfection)andimmuneglobulintoprovideimmediateprotection(therapeuticandpreventiveinfectiousdisease).ANTIMICROBIALCHEMOTHERAPYChe