Prevalence and relatedness of Escherichia coli O157_H7 strains in the feces

PrevalenceandRelatednessofEscherichiacoliO157:H7StrainsintheFecesandontheHidesandCarcassesofU.S.MeatGoatsatSlaughter

M.E.Jacob,aD.M.Foster,aA.T.Rogers,aC.C.Balcomb,aM.W.Sandersonb

DepartmentofPopulationHealthandPathobiology,NorthCarolinaStateUniversity,Raleigh,NorthCarolina,USAa;DepartmentofDiagnosticMedicine/Pathobiology,KansasStateUniversity,Manhattan,Kansas,USAb

WedeterminedtheprevalencesofEscherichiacoliO157:H7infeces,hide,andcarcassesofmeatgoatsataU.S.processingplant.Prevalenceswere11.1%,2.7%,and2.7%,respectively.Sixteenpulsed- eldgelelectrophoresis(PFGE)subtypeswereidenti edamong49E.coliO157:H7isolates,someofwhichwerepresentonmultiplesampletypesorcollectiondays.

S

higatoxin-producingEscherichiacoli(STEC)bacteria,includ-Hidesampleswerecollectedpostharvestbyswabbingverti-ingtheO157serogroup,arewell-describedhumanpathogenscally,atleastthreetimes,anareaofapproximately100cm2alongassociatedwithbloodydiarrhea,hemolytic-uremicsyndromeonesideandtherumpwithsterilegauzepads(2in. 2in.),(HUS),anddeath(1).Illnessesareoftenattributedtothecon-premoistenedwith5mlofsterilewater.Similarly,onesideandthesumptionofcontaminatedfoodorwater;however,transmissionrumpareaofcarcasseswereswabbedwithaSpeci-Spongespongebyanimalcontactmayalsobeimportant(2,3).Ruminants,par-(Nasco,FortAtkinson,WI)soakedwith15mlofbufferedpeptoneticularlycattle,areconsideredtheprimaryreservoirforE.coliwater.Carcasssampleswerecollectedimmediatelyafterhidere-O157:H7,wheretheorganismtypicallycolonizesthelowergas-movalandeviscerationbutpriortoUSDAinspection.Rectalcon-trointestinaltract(4)andisshedinthefeces.Recentoutbreaksoftentswerecollectedposteviscerationbycuttingopenintactrec-humanillnessassociatedwithsmall-ruminantcontactatlivestocktums.AllsampleswereplacedintoWhirl-Pakbags(Nasco)andexhibitions(5)havedemonstratedtheimportanceoftheseani-transportedinacoolerwithicepackstothelaboratoryforpro-malsasreservoirsforSTEC,includingE.coliO157:H7.However,cessingwithin24h.SamplingpersonnelworeanewnitrilegloveestimatesofE.coliO157:H7carriageinU.S.goatsislimited.betweeneachanimalandsampletype,andscissorsusedtocutPreviousworkincattlesuggeststhattheprevalenceofE.colirectumswererinsedwithwater,wipedtoremovegrossfecalcon-O157:H7inthefecesiscorrelatedtotheprevalenceonthehidetamination,andsoakedin0.1%chlorhexidinediacetatesolutionandcarcassesofanimalsatslaughter(6,7,8).Anincreasingnum-betweeneachanimal.

berofgoatcarcassesareinspectedinU.S.packingplants(9),sug-IsolationofE.coliO157:H7fromhides,carcasses,andfeces.gestingthatgoatmeatmaybeanincreasinglyimportanttransmis-Approximately1goffecalpellet(homogenizedwhenpossible)sionvehicleforfood-bornepathogens.EscherichiacoliO157:H7wasinoculatedinto9mlofGram-negativebroth(Difco;BD,contaminationofgoatcarcassesrepresentsapublichealththreatifSparks,MD)supplementedwithce xime(0.5mg/liter),cefsulo-thecontaminationisnotremovedpriortoconsumption.Theob-din(10mg/liter),andvancomycin(GNccv)(8mg/liter).SamplesjectiveofourworkwastodeterminetheprevalenceofE.coliwerevortexedandincubatedfor5.5hat37°C.Manualimmuno-O157:H7inthefecesandonthehidesandcarcassesofmeatgoatsmagneticbeadseparation(IMS)(Dynabeads;InvitrogenCorp.,presentedforslaughterataUSDA-inspectedplantinthesouth-Carlsbad,CA)wasperformedwith1mlofenrichmentbroth,andeasternUnitedStates.Inaddition,wewantedtodeterminethe50 lofproductwasplatedonsorbitolMacConkeyagarwithgeneticrelatednessoftheisolatestobetterunderstandtheecologyce xime(0.5mg/liter)andpotassiumtellurite(2.5mg/liter)(CT-oftheorganismwithinthisenvironment.

SMAC)(BD).Plateswereincubatedovernightat37°C,anduptoAnimalsandsamplecollection.Fecal,hide,andcarcasssam-sixsorbitol-negativecolonieswereselectedandstreakedontopleswerecollectedfrom300goatspresentedforharvestatabloodagarplates(BD).Afterovernightincubation,colonieswereUSDA-inspectedprocessingplantinthesoutheasternUnitedtestedforindoleproduction(Remel;Lenexa,KS)andlatexagglu-States.Matchedsampleswerecollectedfromeachanimal.FortinationwiththeO157antigen(Remel).Positivecolonieswerelabelingpurposes,fecal,hide,andcarcasssamplesfromeachani-furthercharacterizedbymultiplexPCRtodetecttherfbE(O157),malweregiventhesamenumber(differentiatedbyanF,H,orC),eae(intimin),stx1(Shigatoxin1),stx2(Shigatoxin2),hlyA(he-andanimalswerelabeledconsecutivelyastheyappearedinthemolysin),and iC(H7)genes(10).

plant.Sampleswerecollectedon9daysduringa12-weekperiodHideandcarcasssampleswereculturedforE.coliO157:H7asfromAugusttoOctober;between10and40animalsweresampledpreviouslydescribed(7).Brie y,20mlofbrilliantgreenbroth

oneachcollectionday(Table1).Theplanthadacapacityofap-proximately200goatsperweekandalsoprocessedsheepandcattle.Goatswerealwaysprocessed rstoneachcollectionday.Received

11March2013Accepted10April2013Therewasnoavailableinformationonthesource,age,diet,orPublishedaheadofprint12April2013

previoususe(dairy,meat,orpet)ofthegoats.Retrospectively,AddresscorrespondencetoM.E.Jacob,megan_jacob@http://wendang.chazidian.comrmationonmaximumdailytemperatureandprecipitationCopyright©2013,AmericanSocietyforMicrobiology.AllRightsReserved.foreachcollectiondaywereobtainedfromtheclosestreportingdoi:10.1128/AEM.00772-13

NationalOceanicandAtmosphericAdministration(NOAA)site.

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TABLE1Numbersofprocessedgoatssampled,maximumdailytemperatures,andprecipitationreadingson9collectiondaysataUSDA-inspectedfacilityinthesoutheasternUnitedStatesSamplingNo.ofTempAmtof

Collectiondatesamples(°C)precipitation(in.)115August2029.40.0220August4023.30.75327August4027.20.0410September4026.10.0512September3026.10.0617September4023.80.1171October4016.70.0489October4010.60.229

15October

10

22.8

0.57

(BGB)wasaddedtohideswabsamples,whichwereenrichedfor6hat37°C.Carcassspongeswereenrichedin90mlofBGBfor10hat37°C.Afterenrichment,IMSwasperformedwith1mlofen-richedBGB,followedbyplatingontoCT-SMACplates.Plateswereincubatedandevaluatedasdescribedabove.AllE.coliO157:H7isolatesobtainedfromfeces,hides,andcarcasseswerefrozenat 80°Cforfutureanalyses.

PFGE.IndividualE.coliO157:H7isolatesweregrownonbloodagarplatesfor24hat37°Cforpulsed- eldgelelectropho-resis(PFGE)analysis.AllisolatesweresubjectedtorestrictionenzymedigestionwithXbaIandprocessedaccordingtothePulseNetprotocol(11).GelimageswereobtainedusingaGelDocXR imagingsystem(Bio-Rad,Hercules,CA),andthePFGEbandingpatternswerenormalizedandevaluatedusingBioNumericssoftware(AppliedMaths,Inc.,Austin,TX).Isolatesdisplaying100%DicesimilaritywereconsideredtobethesamePFGEsubtype(12).

Statisticalanalysis.Thefecal,hide,andcarcassprevalencesofE.coliO157:H7weretheprimaryoutcomesofinterestinthisstudy.DescriptivestatisticswerecalculatedusingtheMicrosoftExcelprogram(Redmond,WA).Datawerefurtheranalyzeduti-lizingcommerciallyavailablestatisticalsoftware(Stata12;Stata-CorpLP,CollegeStation,TX)tocalculateaFisherexacttesttoevaluatedifferencesinprevalencebycollectionday.Theoddsofacarcassbeingpositiveifeitherthefecesorhidewaspositivewascalculatedfromcross-tabulatedtables.Datawereconsideredbi-nomial(positiveornegative).AFisherexacttestwasusedtoeval-uatetheindependenceofvariablesassociatedwithoneanother.Statisticalsigni cancewasconsideredtobeaPvalueof 0.05.PrevalenceofE.coliO157:H7.TheprevalenceofE.coliO157:H7inthefecesofgoatsatslaughterwas11.1%(33of296;95%con denceinterval[CI],7.5%to14.8%).PreviousreportsofgoatswithinandoutsidetheUnitedStateshavesuggestedthatfecalprevalenceingoatsislow,lessthan5%(13,14,15,16).Althoughfecalprevalenceinourstudywasremarkablyhigher,alargenumberofpositivesampleswereobtainedononecollectionday.Themeanprevalenceforeachcollectiondayrangedfrom0(5of9collectiondays)to52.5%(Fig.1)andwassigni cantlydiffer-entbetweendays(P 0.01).Althoughourgoalwasnottospecif-icallyevaluateenvironmentalconditionsondifferentcollectiondays,theredidnotappeartobealargedifferenceintemperaturesoramountsofprecipitationonthedayswhenweobtainedposi-tivesamples(Table1).DailyvariationintheprevalenceofE.coli

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E.coliO157:H7inU.S.MeatGoats

O157:H7incattleatslaughterhasbeenwelldescribed.Theimpactofcollectiondaysongoatsinotherstudiesisunclear.

TheprevalenceofE.coliO157:H7onhidesofgoatsatslaughterwas2.7%(8of300;95%CI,0.8%to4.5%)andrangedbetweencollectiondaysfrom0(6of9collectiondays)to12.5%,whichwassigni cantlydifferent(P 0.02).TheprevalenceofE.coliO157:H7onmeatgoatcarcasseswas2.7%(8of299;95%CI,0.8%to4.5%),similartoapreviousreportonEthiopiangoatcarcasses(16).Prevalencesincarcassesrangedfrom0(6of9collectiondays)to10.0%andtendedtobedifferentbetweencollectiondays(P 0.052).Inaddition,for43of300goats(14.3%)sampledinourstudy,atleast1site(feces,hide,orcarcass)waspositiveforE.coliO157:H7.Theriskofananimalhavingatleastonecollectionsitepositivewasdifferentbetweencollectiondays(P 0.01).For6goats,2siteswereculturepositiveforE.coliO157:H7;for3goats,positiveisolateswerefoundforthefecesandcarcass;2goatswerefecesandhidepositive;andfor1goat,E.coliO157:H7wasisolatedfromboththehideandcarcass.

Itiswellestablishedinbeefproductionthataspeci csamplingdayand/orloadofcattlewithhighfecalprevalenceiscorrelatedwithcarcasscontaminationwithE.coliO157:H7(6,7,17).Thedaysofhigherhideandcarcassprevalencealsocorrelatedwithdayswithhighfecalprevalenceinourstudy(Fig.1).ApreviousstudyofsheepandgoatsinEthiopiaidenti edasigni cantasso-ciationbetweenthecarcasscontaminationofanindividualanimalandthefecalpresenceofE.coliO157:H7inthesameanimal(16).Wefoundthissamecorrelation;theoddsofagoatcarcassbeingpositiveforE.coliO157:H7ifthefecesfromthatanimalwaspositiveornegativeweredifferent(P 0.01).Inourstudy,agoatwithapositivefecalculturewas5.2times(95%CI,1.15to23.1)morelikelytobecarcasspositive.

StudiesincattlehavealsosuggestedthathidesarethemajorsourceofE.coliO157:H7forcarcassesatslaughter,andcontrol-linghidecontaminationwillreduceprevalencesincarcass(18,19).Interestingly,wedidnotobserveastrongassociationbetweenhideandcarcassprevalencesinourstudy.Theoddsofahidebeingpositiveiftheanimalwasfecalpositivewas2.8(95%CI,0.5to14.4),whichwasnotstatisticallysigni cant(P 0.21),andananimalwithapositivehidetendedtohaveanincreasedodds(5.8;95%CI,0.6to54.5)ofbeingcarcasspositive(P 0.08).Alackof

FIG1ProportionsofEscherichiacoliO157-positivesamplescollectedfromthefeces,hides,andcarcassesofmeatgoatsatasoutheasternU.S.abattoiroverninecollectiondays.

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Jacobetal.

FIG2DendrogramdescribingtherelatednessofEscherichiacoliO157isolatesobtainedfromthefeces,hides,andcarcassesofmeatgoatsatasoutheasternU.S.abattoiroverninecollectiondays.Animalswereconsecutivelynumberedastheyenteredtheslaughterplant.Thesamenumberforfecal,hide,orcarcasssampleswouldindicatethatthesamplescamefromthesameanimal.

powercouldhavecontributedtoourabilitytofullyunderstandtoxin-producinggenesfromE.coliO157:H7isolatesinthisstudythisassociation.

resemblesreportsfromoutsidetheUnitedStates,whichsuggestGeneticrelatednessofisolates.All49E.coliO157:H7isolatesthatsheepandgoatsmorefrequentlycarrystxobtainedinthisstudywerepositiveforstx1,rfbE, iC,eae,andthanstx,21,22,23,24).Thedistribution1orstxof1andstxvirulence2hlyA.Oneisolateobtainedfromthehideofananimalwaspositivegenes,including2alone(20Shigatoxingenes,inSTECisimportant,andtheforstx2;allotherisolateswerenegative.ThedistributionofShiga

presenceofstx2hasbeenassociatedwithmore-severehumandis-4156

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ease(25,26,27).Thecompletevirulencepro leoftheE.coliO157:H7isolatesobtainedinourstudyarenotknown;however,allisolateswerealsopositivefortheeaegene(encodingintimin)andthehlyAgene(encodinghemolysin).Takentogether,itislikelythatatleastsomeoftheE.coliO157:H7bacteriaisolatedfromthesegoatscouldbeimportanthumanpathogens.

Weidenti ed16uniquePFGEsubtypes(Dicesimilarity 100%)amongthe49E.coliO157:H7isolates;adendrogramde-scribingtheirrelatednessisprovided(Fig.2).Therewere13uniquePFGEtypes,withaDicesimilarityof 95%,and8typeswithasimilarityof 90%.SomePFGEtypesdifferedacrossandbetweendaysandsampletypes.Oncollectionday4,therewere6E.coliO157:H7isolatesobtainedand3uniquePFGEsubtypes,or2withaDicesimilarityof 95%.Fourofthe6isolates,including1carcassisolate,hadthesamePFGEsubtype.Oncollectionday6,therewere14E.coliO157:H7isolatesand9uniquePFGEsub-types:7PFGEtypeswithasimilarityof 95%.Thesamesubtypesweredetectedonhidesandcarcasses,fecesandhides,andfecesandcarcasses.Similarresultswereseenoncollectionday7,with27E.coliO157:H7isolatesand10uniquePFGEsubtypes.Therewere7PFGEtypeswithasimilarityof 95%.Oncollectionday8,2uniquesubtypesweredetectedfrom2isolates,bothfromfeces.Thesewerenotrecoveredonthehideorcarcassesofanyanimal.Therewere5differentPFGEsubtypesthatappearedon2ormorecollectiondays.

Withineachsampletype,therewasalsoadistributionofuniquePFGEpatterns.Of33fecalsampleE.coliO157:H7isolates,13uniquePFGEsubtypes(9with 95%Dicesimilarity)wereidenti ed.Twenty-fourfecalisolates(72%)hadaDicesimilarityof 95%.Therewere5uniquePFGEtypesfromhideisolates(n 8),allwithDicesimilaritiesof 90%.EscherichiacoliO157:H7isolatesfromcarcasses(n 8)wereobtainedon3collectiondays,and7uniquePFGEsubtypes(6with 95%Dicesimilarity)werefound.Foranimalswithmorethan1sampletypepositive,only1animalhadanidenticalPFGEpatternidenti edforbothsampletypes,fecesandcarcass.Forallotheranimals,2siteswerepositive,withuniqueE.coliO157:H7PFGEsubtypes(Dicesimilarity 95%).

Unfortunately,theoriginsofthegoatsandtheirdurationofminglingbeforeprocessingarenotknownforthisstudy;there-fore,thenumberofgoatsarrivingattheabattoirwiththesameE.coliO157:H7PFGEsubtypeisnotknown.Still,thisstudydemon-stratesthepotentialforspreadofE.coliO157:H7throughoutaprocessingfacility,asevidencedbyisolatePFGEpro lesandtheirrelatednesstooneanother.Withinacollectionday,anindividualE.coliO157:H7PFGEsubtypecouldbeisolatedfromthefeces,hide,orcarcassofmultiplegoats.Inonlyoneinstancewasthesamesubtyperecoveredfrommultiplesitesonthesameanimal.Therewasnoobviousrelationshipbetweenwhereaspeci cPFGEtypewas rstidenti edintheslaughterorderandwhereitmightbefoundagaindownstream(Fig.2).ThepresenceofthesamePFGEsubtypeinthefecesofanimalsonmultiplecollectiondays(upto3weeksapart)isinterestingandsuggeststhattheremaybeacommonsourceofE.coliO157:H7immediatelypriortoslaugh-ter,thatthegoatpopulationreceivedbythisabattoirisintegrated,orperhapsthattheplantreceivedanimalsfromthesameopera-tiononmultipledays.IdenticalPFGEsubtypesofE.coliO157:H7oncattlecarcassesandinfecesatslaughterhavealsobeenfoundtocrossloadsandcollectiondays(12).ThereisevidenceincattlefecesthatmorethanonePFGEtypecanbepresentwithinthe

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E.coliO157:H7inU.S.MeatGoats

samesample(28,29).WedidnotevaluatethenumberofE.coliO157:H7PFGEsubtypespresentingoatfecesoronhidesandcarcassesinthisstudy.However,ifweexpectthesesamplestocontainmultiplesubtypes,wemaybefailingtocaptureacompleteunderstandingofhowE.coliO157:H7isspreadinthegoatpro-cessingenvironment.

BecauseofanincreasingdemandforgoatmeatintheUnitedStates,determiningtheriskofE.coliO157:H7contaminationofgoatmeatproductsiswarranted.WefoundpreliminaryevidencetosuggestthatE.coliO157:H7existsthroughouttheproductionchainofmeatgoats,includingoncarcassesintendedforcon-sumption.TheprevalenceanddistributionofPFGEsubtypeswithinandbetweenanimalssuggestthatcontaminationwithintheplantmaybewidespreadandnotdependentsolelyonthefecalstatusofanindividualanimal.Inaddition,thevirulencepro lesofU.S.goatisolatessuggesttheymaybeimportanthumanpatho-gens.Furtherworktocharacterizethevirulencepro leofE.coliO157:H7bacteriaisolatedfromgoatsandtodeterminetheirpo-tentialashumanpathogens,aswellasunderstandingmitigationefforts,iswarranted.

ACKNOWLEDGMENTS

ThisstudywasfundedbyaNorthCarolinaStateUniversityCVMintra-muralgrant.

WethankthepersonnelattheprocessingplantandMelinaLillichfortheircontributionstothiswork.

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