美国FDA细菌学分析手册第八版BAM致腹泻性大肠埃希氏菌.docx

美国FDA细菌学分析手册第八版BAM致腹泻性大肠埃希氏菌.docx

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美国FDA细菌学分析手册第八版BAM致腹泻性大肠埃希氏菌

BAM:

DiarrheagenicEscherichiacoli

February2011

-BacteriologicalAnalyticalManual

-Chapter4A

-DiarrheagenicEscherichiacoli

Authors:

PeterFeng,StephenD.Weagant

RevisionHistory:

∙February2011:

SectionM:

AdditionofprimersinTable2;SectionQ:

additionof5PMultiplexPCRforconfirmationofO157:

H7isolates

∙July2009:

SectionMmodifiedtoincludethereal-timePCRscreeningmethod

Escherichiacoliisoneofthepredominantspeciesoffacultativeanaerobesinthehumangutandusuallyharmlesstothehost;however,agroupofpathogenicE.colihasemergedthatcausesdiarrhealdiseaseinhumans.ReferredtoasDiarrheagenicE.coli（28）orcommonlyaspathogenicE.coli,thesegroupsareclassifiedbasedontheiruniquevirulencefactorsandcanonlybeidentifiedbythesetraits.Hence,analysisforpathogenicE.colioftenrequiresthattheisolatesbefirstidentifiedasE.colibeforetestingforvirulencemarkers.ThepathogenicgroupsincludesenterotoxigenicE.coli（ETEC）,enteropathogenicE.coli（EPEC）,enterohemorrhagicE.coli（EHEC）,enteroinvasiveE.coli（EIEC）,enteroaggregativeE.coli（EAEC）,diffuselyadherentE.coli（DAEC）andperhapsothersthatarenotyetwellcharacterized（21,28）.Ofthese,onlythefirst4groupshavebeenimplicatedinfoodorwaterborneillness.Somepropertiesandsymptomsofthese4subgroupsarediscussedbelowandsummarizedinTable1.

ETECisrecognizedasthecausativeagentoftravelers'diarrheaandillnessischaracterizedbywaterydiarrheawithlittleornofever.ETECinfectionsoccurscommonlyinunder-developedcountriesbut,intheU.S.,ithasbeenimplicatedinsporadicwaterborneoutbreaksaswellasduetotheconsumptionofsoftcheeses,Mexican-stylefoodsandrawvegetables.PathogenesisofETECisduetotheproductionofanyofseveralenterotoxins.ETECmayproduceaheat-labileenterotoxin（LT）thatisverysimilarinsize（86kDa）,sequence,antigenicity,andfunctiontothecholeratoxin（CT）.ETECmayalsoproduceaheatstabletoxin（ST）thatisoflowmolecularsize（4kDa）andresistanttoboilingfor30min.ThereareseveralvariantsofST,ofwhichST1aorSTpisfoundinE.coliisolatedfrombothhumansandanimals,whileST1borSThispredominantinhumanisolatesonly.TheinfectivedoseofETECforadultshasbeenestimatedtobeatleast108cells;buttheyoung,theelderlyandtheinfirmmaybesusceptibletolowerlevels.Becauseofitshighinfectiousdose,analysisforETECisusuallynotperformedunlesshighlevelsofE.colihavebeenfoundinafood.Also,ifETECisdetected,levelsshouldalsobeenumeratedtoassessthepotentialhazardofthecontaminatedfood.ProductionofLTcanbedetectedbyY-1adrenalcellassays（28）orserologicallybycommercialreversepassivelatexagglutinationassayandELISA（seeAppendix1）.TheproductionofSTcanalsobedetectedbyELISAorbyinfantmouseassay（35）.BothLTandSTgeneshavealsobeensequencedandPCR（37,41）andgeneprobeassays（seechapter24）areavailable.Analysisofcoloniesonplatingmediausinggeneprobe/colonyhybridizationalsoallowsenumerationofETECinfoods.

EIECcloselyresembleShigellaandcausesaninvasive,dysentericformofdiarrheainhumans（7）.LikeShigella,therearenoknownanimalreservoirs;hencetheprimarysourceforEIECappearstobeinfectedhumans.AlthoughtheinfectivedoseofShigellaislowandintherangeof10tofewhundredcells,volunteerfeedingstudiesshowedthatatleast106EIECorganismsarerequiredtocauseillnessinhealthyadults.UnliketypicalE.coli,EIECarenon-motile,donotdecarboxylatelysineanddonotfermentlactose,sotheyareanaerogenic.PathogenicityofEIECisprimarilydueitsabilitytoinvadeanddestroycolonictissue.Theinvasionphenotype,encodedbyahighmolecularweightplasmid,canbedetectedbyinvasionassaysusingHeLaorHep-2tissueculturecells（7,25）orbyPCRandprobesspecificforinvasiongenes（seechapter24）.

EPECcausesaprofusewaterydiarrhealdiseaseanditisaleadingcauseofinfantilediarrheaindevelopingcountries.EPECoutbreakshavebeenlinkedtotheconsumptionofcontaminateddrinkingwateraswellassomemeatproducts.ThroughvolunteerfeedingstudiestheinfectiousdoseofEPECinhealthyadultshasbeenestimatedtobe106organisms.PathogenesisofEPECinvolvesintiminprotein（encodedbyeaegene）thatcausesattachmentandeffacinglesions（14）;butitalsoinvolvesaplasmid-encodedproteinreferredtoasEPECadherencefactor（EAF）thatenableslocalizedadherenceofbacteriatointestinalcells（36）.ProductionofEAFcanbedemonstratedinHep-2cellsandthepresenceofeaegenecanbetestedbyPCRassays（28）.

EHECarerecognizedastheprimarycauseofhemorrhagiccolitis（HC）orbloodydiarrhea,whichcanprogresstothepotentiallyfatalhemolyticuremicsyndrome（HUS）.EHECaretypifiedbytheproductionofverotoxinorShigatoxins（Stx）.AlthoughStx1andStx2aremostoftenimplicatedinhumanillness,severalvariantsofStx2exist.TherearemanyserotypesofStx-producingE.coli（STEC）,butonlythosethathavebeenclinicallyassociatedwithHCaredesignatedasEHEC.Ofthese,O157:

H7istheprototypicEHECandmostoftenimplicatedinillnessworldwide（3,13,19,28）.TheinfectiousdoseforO157:

H7isestimatedtobe10-100cells;butnoinformationisavailableforotherEHECserotypes.EHECinfectionsaremostlyfoodorwaterborneandhaveimplicatedundercookedgroundbeef（3,13）,rawmilk（31）,coldsandwiches（19）,water（34）,unpasteurizedapplejuice

（2）andsproutsandvegetables（4,17）.EHECO157:

H7arephenotypicallydistinctfromE.coliinthattheyexhibitslowornofermentationofsorbitolanddonothaveglucuronidaseactivity（seechapter4.LST-MUGfordetails）;hence,thesetraitsareoftenusedtoisolatethispathogenfromfoods.TheproductionofStx1andStx2canbetestedbycytotoxicityassaysonveroorHeLatissueculturecellsorbycommerciallyavailableELISAorRPLAkits（seeAppendix1）.Geneprobes（seechapter24）andPCRassaysspecificforstx1andstx2andothertraitEHECmarkersarealsoavailable（12,15）（andseebelow）.

Table1.SomepropertiesandsymptomsassociatedwithpathogenicE.colisubgroups.

Properties/Symptoms

ETEC

EPEC

EHEC

EIEC

Toxin

LT/STa

-

ShigaorVerotoxin

（StxorVT）

-

Invasive

-

-

-

+

Intimin

-

+

+

-

Enterohemolysin

-

-

+

-

Stool

Watery

Watery,Bloody

Watery,verybloody

Mucoid,bloody

Fever

Low

+

-

+

Fecalleukocytes

-

-

-

+

Intestineinvolved

Small

Small

Colon

Colon,lowersmall

Serology

Various

O26,O111&others

O157:

H7,O26,O111&others

Various

IDb

High

High

Low

High

aLT,labiletoxin;ST,stabletoxin.

bID,infectivedose.

IsolationandIdentificationofPathogenicEscherichiacoli

-ExceptEHECofserotypeO157:

H7

SincepathogenicE.coliareidentifiedbasedonitsuniquevirulenceproperties,theanalyticalprocedureforthesepathogensinfoodsgenerallyrequirestheisolationandidentificationoftheorganismsasE.colibeforetestingforthespecificvirulencetraits.FollowingisageneralprocedureforenrichmentandisolationofpathogenicE.colifromfood（25）.

A.Equipmentandmaterials5

B.Media5

C.Reagents,inorganic,organic,andbiological5

D.Enumeration.6

E.EnrichmentforPathogenicE.coli6

F.Selection.6

G.ConventionalBiochemicalScreeningandidentification（8,30）7

H.TestsforEnterotoxigenicE.coli（ETEC）8

I.TestsforEnteroinvasiveE.coli（EIEC）8

J.TestsforEnteropathogenicE.coli（EPEC）8

K.ScreeningmethodforE.coliSerotypeO157:

H7fromFoods9

L.Equipmentandmaterials10

M.MediaandReagents11

N.SamplePreparationandEnrichmentProcedure14

O.Real-timePCRScreening15

P.CulturalIsolationandPresumptiveIsolateScreening.24

Q.IsolateConfirmationTests27

R.Screeningmethodfornon-O157STEC31

References33

A.Equipmentandmaterials

1.Balance,≥2kgwith0.1gsensitivity

2.Blender,Waringorequivalentmodelwithlowspeedoperationat8000rpm,with1literglassormetaljar

3.Incubators,35±0.5°Cand44±1°C

4.Petridishes20x150mm

5.Pipets,Pasteur

6.pHtestpaper,range6.0-8.0

B.Media

1.Tryptonephosphate（TP）broth（M1621）

2.Brainheartinfusion（BHI）broth（M242）

3.Levine'seosin-methyleneblue（L-EMB）agar（M803）

4.MacConkeyagar（M914）

5.Triplesugariron（TSI）agar（M1495）

6.Bloodagarbase（BAB）（M216）

7.Tryptone（tryptophane）broth（M1647）

8.Bromcresolpurplebroth（M268）supplementedindividuallywith0.5%（w/v）ofeach:

glucose,adonitol,cellobiose,sorbitol,arabinose,mannitol,andlactose

9.Ureabroth（M1719）

10.Lysinedecarboxylasebroth,Falkow（M8710）.

11.Potassiumcyanide（KCN）broth（M12611）

12.MR-VPbroth（M10412）

13.Indolenitritemedium（trypticnitrate）（M6613）

14.Acetateagar（M314）

15.Mucatebroth（M10515）

16.Mucatecontrolbroth（M10616）

17.Malonatebroth（M9217）

18.Koser'scitratebroth（M7218）

C.Reagents,inorganic,organic,andbiological

1.Sodiumbicarbonatesolution,10%,aqueous（sterile）（R7019）

2.ONPG（o-nitrophenyl-β-D-galactopyranoside）disks（R5320）

3.Phosphatebufferedsalinesolution,（sterile）（PBS）（R6021）,orButterfield'sPhosphate-buffereddilutionwater（BPBW）（R1122）.

4.Kovac'sreagent（R3823）

5.VPreagents（R8924）

6.Oxidasetestreagent（R5425）

7.Nitritedetectionreagents（R4826）

8.Mineraloil,heavysterile（R4627）

9.Gramstainreagents（R3228）

D.Enumeration.

Refrigeratesamplespromptlyafterreceipt.Donotfreezeexcepttoholdfrozenproductsuntiljustpriortoanalysis.Analyzesamplesassoonaspossible.Ifenumerationisrequired,prepareahomogenateof25