美国FDA细菌学分析手册第八版(BAM)弯曲菌

BAM:CampylobacterReturntoBAMtableofcontents1January2001BacteriologicalAnalyticalManualChapter7CampylobacterAuthors:JanM.Hunt,CarlosAbeytaandTonyTranRevisionHistory:Updatedandrevised:2000-DEC-29MediaInstructionsModifiedon2001-MAR-08IsolationofCampylobacterSpeciesfromFoodandWaterCampylobacterisconsideredbymanytobetheleadingcauseofentericillnessintheUnitedStates(20,26).Campylobacterspeciescancausemildtoseverediarrhea,withloose,waterystoolsoftenfollowedbybloodydiarrhea(7,20).C.jejuni,C.coli,andC.lariaccountformorethan99%ofthehumanisolates(C.jejuni90%).Otherspecieshavebeenassociatedwithhumanillnessinrecentyears(7,17,18,23,26,27).Campylobacterspeciesarehighlyinfective.TheinfectivedoseofC.jejunirangesfrom500to10,000cells,dependingonthestrain,damagetocellsfromenvironmentalstresses,andthesusceptibilityofthehost(4,6,7,20,27).OnlythemesophilicC.fetusisnormallyinvasive.Thermophilicspecies(optimum42°C)suchasC.jejuniareoccasionallyinvasive.Theinfectionsaremanifestedasmeningitis,pneumonia,miscarriage,andasevereformofGuillain-Barrésyndrome(6,20).ThermotolerantstrainsofC.fetusthatgrowat42°Chavebeenisolatedfrompatients(17).Campylobactersarecarriedintheintestinaltractofawidevarietyofwildanddomesticanimals,especiallybirds.Theycanestablishatemporaryasymptomaticcarrierstate,aswellasillness,inhumans.Thisisespeciallyprevalentindevelopingcountries(20).Consumptionoffoodandwatercontaminatedwithuntreatedanimalorhumanwasteaccountsfor70%ofCampylobacter-relatedillnesseseachyear.Thefoodsincludeunpasteurizedmilk,meats,poultry,shellfish,fruits,andvegetables,(1,8-11,17,19,20,22,25,26).C.jejunicansurvive2-4weeksundermoist,reduced-oxygenconditionsat4°C,oftenoutlastingtheshelflifeoftheproduct(exceptinrawmilkproducts).Theycanalsosurvive2-5monthsat-20°C,butonlyafewdaysatroomtemperature(5,8-11,20).Environmentalstresses,suchasexposuretoair,drying,lowpH,heating,freezing,andprolongedstorage,damagecellsandhinderrecoverytoagreaterdegreethanformostbacteria.Olderandstressedorganismsgraduallybecomecoccoidalandincreasinglydifficulttoculture(5,20).Oxygenquenchingagentsinmediasuchasheminandcharcoalaswellasamicroaerobicatmosphereandpreenrichmentcansignificantlyimproverecovery(2,14-16,21,25,28).Campylobactersaremicroaerophilic,verysmall,curved,thin,Gram-negativerods(1.5-5µm),withcorkscrewmotility.Theyoftenjointoformzigzagshapes(20,24).Campylobacterspp.arecurrentlyidentifiedbytestsdescribedbyHarvey(13)andBarretetal.(3).PCRgenusandspeciesidentificationmethodshavebeenpublished(12,18,30).ForadditionalinformationcontactCarlosAbeyta,FDA,PRLNW,Bothell,WA98041-3012.Phonenumber:[phone].SAMPLEPREPARATIONFLOWCHARTFORVARIOUSFOODSANDWATER(SECTIONC.2.a-h)*forsamplesproducedorprocessed10dayspreviously.†forfrozensamplesorsamplesproducedorprocessed≥10dayspreviously.A.Equipmentandmaterials1.Balances,6000gcapacity,accurateto0.1g;and200gcapacity,accurateto0.0001g2.Sterilestomacherbags,400and3500mlbagsand400mlfilterbags(otherbagtypesandsizesdescribedinrefs.16,21,and22)3.Whirl-pakbagracksandstainlesssteelbaskets4.Benchtopshaker5.Centrifuge,refrigerated,capableof20,000xg6.Polypropyleneorstainlesssteel250mlcentrifugebottlesand50mlcentrifugetubes,sterile7.Largefunnelswithcheeseclothlinings,sterile(forwholeseafoodandmeatsamplesoriffilterbagsareunavailable)8.Whiteororangegreasepencilstomarkblood-freeagarplates9.50mlsterileconicalcentrifugetubes10.Plastic5-10mltubeswithscrewcaplids,sterile11.Cryotubes,1ml,sterile12.Phase-contrastmicroscope,with100Xoilimmersionobjectiveordark-fieldmicroscopewith63Xobjectiveorlightmicroscopewith1000Xobjective13.Microscopeslides,1cmsqcoverslipsandimmersionoil14.Gastankassembly(5%O2,10%CO2,85%N2)andvacuumsource(Fig.1)15.Microaerobiccontainersystema.Anærobejarsandbags:1.Jarswithvacuum-pressuregaugeandSchradervalves.Theymaybeusedwitheitherthegastank/vacuumassemblyorwithgas-generatingenvelopes.3.4L,(Difco1950-30-2orOxoidHP11)maybeusedwitheitherthegastank/vacuumassemblyorwithgas-generatingenvelopes.2.Jarswithoutagaugeorvalves(2.5literBBLorEMDiagnostics[Remel,Lenexa,KS66215]and9.5literBBL)anaerobejars.Theseareusedwithgas-generatingenvelopes(2.5litertypesuchasOxoidN025AortheBBLandEMgaspaks).3.Rectangularjars2.5and5.5literanaeroberectangularjars(InternationalBioproducts,800-729-7611orMitsubishiGasChemicalAmerica,212-752-4620).4.Anærobepouchesorrectangularjars,0.4L,for2-plateincubation(InternationalBioproductsorMitsubishi).Pouchesfor1-plateincubationareavailablefromEMDiagnostics.5.Air-tightplasticbags(4milwt),12X16orlargerthatcanbeclosedbyheatsealingortapecanbeusedasanincubationchamber.b.Campygas-generatingenvelopesorpouches:for3.4literjars,OxoidBR56orCN035A;for2.5literand9.5literjars,OxoidCN025A,Difco1956-24-4,BBL71040or71034orEMDiagnostics53013678;forrectangularjars,Mitsubishi10-04;for2-plate,Mitsubishi20-04and1-plate,EMDiagnostics,53-13699.TheOxoidCNO25AandtheMitsubishiandEMenvelopesareusedwithoutwater.§CAUTION:ONLYGASPACSUSEDWITHOUTWATERARECOMPATIBLEWITHTHEITEMSINA.3)ANDA.4).THERECTANGULARJARSCANEXPLODEIFUSEDWITHTHEOXOIDBR56,BBLORDIFCOENVELOPES.c.AnAnær