美国FDA细菌学分析手册第八版(BAM)大肠埃希氏菌和大肠菌群

BAM:EnumerationofEscherichiacoliandtheColiformBacteriaSeptember2002BacteriologicalAnalyticalManualChapter4EnumerationofEscherichiacoliandtheColiformBacteriaAuthors:PeterFeng,StephenD.Weagant,MichaelA.GrantChapterContentsConventionalMethodforDeterminingColiformsandE.coliLST-MUGMethodforDetectingE.coliinChilledorFrozenFoodsExclusiveofBivalveMolluscanShellfishBottledWaterExaminationofShellfishandShellfishMeatsAnalysisforE.coliincitrusjuicesOtherMethodsforEnumeratingColiformsandE.coliReferencesEscherichiacoli,originallyknownasBacteriumcolicommune,wasidentifiedin1885bytheGermanpediatrician,TheodorEscherich(14,29).E.coliiswidelydistributedintheintestineofhumansandwarm-bloodedanimalsandisthepredominantfacultativeanaerobeinthebowelandpartoftheessentialintestinalflorathatmaintainsthephysiologyofthehealthyhost(9,29).E.coliisamemberofthefamilyEnterobacteriaceae(15),whichincludesmanygenera,includingknownpathogenssuchasSalmonella,Shigella,andYersinia.AlthoughmoststrainsofE.coliarenotregardedaspathogens,theycanbeopportunisticpathogensthatcauseinfectionsinimmunocompromisedhosts.TherearealsopathogenicstrainsofE.colithatwheningested,causesgastrointestinalillnessinhealthyhumans(seeChap.4A).In1892,ShardingerproposedtheuseofE.coliasanindicatoroffecalcontamination.ThiswasbasedonthepremisethatE.coliisabundantinhumanandanimalfecesandnotusuallyfoundinotherniches.Furthermore,sinceE.colicouldbeeasilydetectedbyitsabilitytofermentglucose(laterchangedtolactose),itwaseasiertoisolatethanknowngastrointestinalpathogens.Hence,thepresenceofE.coliinfoodorwaterbecameacceptedasindicativeofrecentfecalcontaminationandthepossiblepresenceoffrankpathogens.AlthoughtheconceptofusingE.coliasanindirectindicatorofhealthriskwassound,itwascomplicatedinpractice,duetothepresenceofotherentericbacterialikeCitrobacter,KlebsiellaandEnterobacterthatcanalsofermentlactoseandaresimilartoE.coliinphenotypiccharacteristics,sothattheyarenoteasilydistinguished.Asaresult,thetermcoliformwascoinedtodescribethisgroupofentericbacteria.ColiformisnotataxonomicclassificationbutratheraworkingdefinitionusedtodescribeagroupofGram-negative,facultativeanaerobicrod-shapedbacteriathatfermentslactosetoproduceacidandgaswithin48hat35°C.In1914,theU.S.PublicHealthServiceadoptedtheenumerationofcoliformsasamoreconvenientstandardofsanitarysignificance.Althoughcoliformswereeasytodetect,theirassociationwithfecalcontaminationwasquestionablebecausesomecoliformsarefoundnaturallyinenvironmentalsamples(6).Thisledtotheintroductionofthefecalcoliformsasanindicatorofcontamination.Fecalcoliform,firstdefinedbasedontheworksofEijkman(12)isasubsetoftotalcoliformsthatgrowsandfermentslactoseatelevatedincubationtemperature,hencealsoreferredtoasthermotolerantcoliforms.Fecalcoliformanalysesaredoneat45.5°Cforfoodtesting,exceptforwater,shellfishandshellfishharvestwateranalyses,whichuse44.5°C(1,3,30).ThefecalcoliformgroupconsistsmostlyofE.colibutsomeotherentericssuchasKlebsiellacanalsofermentlactoseatthesetemperaturesandtherefore,beconsideredasfecalcoliforms.TheinclusionofKlebsiellasppintheworkingdefinitionoffecalcoliformsdiminishedthecorrelationofthisgroupwithfecalcontamination.Asaresult,E.colihasreemergedasanindicator,partlyfacilitatedbytheintroductionofnewermethodsthatcanrapidlyidentifyE.coli.Currently,all3groupsareusedasindicatorsbutindifferentapplications.Detectionofcoliformsisusedasanindicatorofsanitaryqualityofwaterorasageneralindicatorofsanitaryconditioninthefood-processingenvironment.Fecalcoliformsremainthestandardindicatorofchoiceforshellfishandshellfishharvestwaters;andE.coliisusedtoindicaterecentfecalcontaminationorunsanitaryprocessing.AlmostallthemethodsusedtodetectE.coli,totalcoliformsorfecalcoliformsareenumerationmethodsthatarebasedonlactosefermentation(4).TheMostProbableNumber(MPN)methodisastatistical,multi-stepassayconsistingofpresumptive,confirmedandcompletedphases.Intheassay,serialdilutionsofasampleareinoculatedintobrothmedia.Analystsscorethenumberofgaspositive(fermentationoflactose)tubes,fromwhichtheother2phasesoftheassayareperformedandthenusesthecombinationsofpositiveresultstoconsultastatisticaltables(Appendix2),toestimatethenumberoforganismspresent.Typicallyonlythefirst2phasesareperformedincoliformandfecalcoliformanalysis,whileall3phasesaredoneforE.coli.The3-tubeMPNtestisusedfortestingmostfoods.The5-tubeMPNisusedforwater,shellfishandshellfishharvestwatertestingandthereisalsoa10-tubeMPNmethodthatisusedtotestbottledwaterorsamplesthatarenotexpectedtobehighlycontaminated(3).ThereisalsoasolidmediumplatingmethodforcoliformsthatusesVioletRedBileAgar,whichcontainsneutralredpHindicator,sothatlactosefermentationresultsinformationofpinkcolonies.Therearealsomembranefiltrationtestsforcoliformandfecalcoliformthatmeasurealdehydeformationduetofermentationoflactose.ThischapteralsoincludesvariationsofaboveteststhatusefluorogenicsubstratestodetectE.coli(18),specialtestsforshellfishanalysis,abriefconsiderationofbottledwatertestingandamethodfortestinglargevolumesofcitrusjuicesforpresenceofE.coliinconjunctionw