IDEXX Summary. Colilert* 250 vs ISO** :2000(E) membrane filtration method

Transcript

1 IDEXX Summary 16A Topic Title Source Colilert* 250 vsiso** :2000(E) membrane filtration method A Performance Comparison of Colilert 250 Presence/Absence Method and ISO :2000(E) in the Detection of Escherichia coli and Total Coliform Bacteria in Bottled Water Samples IDEXX Laboratories, Inc. Date September 2011 Highlights Colilert is approved for use in 24 countries worldwide and has been adopted by over 90% of US State laboratories. The Colilert 250 test provides bottled water facilities with a more straightforward and easy-to-use method over the ISO :2000(E) membrane filtration method. Colilert 250 test provides a definitive result after 24 hours of incubation and performed equivalently to ISO9308-1:2000(E) in the detection of E. coli and total coliform introduced into various bottled water types In this study ISO9308-1:2000(E) falsely identified the total coliform, Klebsiella oxytoca (ATCC 13182), as an E. coli (confirmation results: indole positive and oxidase negative) after 48 hours of total incubation and multiple confirmation steps. Colilert 250 provided a definitive total coliform result after 24 hours of incubation (yellow only reaction). Colilert 250 is extremely proficient in the suppression of high concentrations of nontarget organisms. ISO9308-1:2000(E) required the user to perform multiple confirmation steps, which included an additional 24 hour incubation period, and further delayed definitive results. *ColilertisatrademarkorregisteredtrademarkofIDEXXLaboratories,Inc.oritsaffiliatesintheUnited Statesand/orothercountries. **InternationalOrganizationforStandardization.

2 APerformanceComparisonofColilert250*Presence/Absence MethodandISO93081:2000(E)intheDetectionofEscherichiacoli andtotalcoliformbacteriainbottledwatersamples September2011 IDEXXLaboratories,Inc. OneIDEXXDrive Westbrook,Maine04092USA idexx.com *Colilert250,DefinedSubstrateTechnologyandDSTaretrademarksorregisteredtrademarks ofidexxlaboratories,inc.oritsaffiliatesintheunitedstatesand/orothercountries. TheVITEK 2systemisatrademarkofbioMérieux,Inc.

3 Introduction IDEXXenteredthewatertestingmarketin1993withColilert;thisinnovativemicrobiologicaltestkit quicklybecameaverypopularwatertestingsystemwhichisnowadoptedbyover90%ofusstate laboratories 1.TheIDEXXColilertreagentisapprovedforusein24countriesaroundtheworld(Table1) forthedetectionofescherichiacoliandtotalcoliformbacteriaindrinkingwater 2.Colilerthasbeen includedforusewithdrinkingandsourcewatersinstandardmethodsforexaminationofwaterand Wastewater 3,ajointpublicationoftheWaterEnvironmentFederation(WEF),AmericanWaterWorks Association(AWWA)andAmericanPublicHealthAssociation(APHA).TheUnitedStatesEnvironmental ProtectionAgency(EPA)hasapprovedColilertforusewithbothdrinking 4 andsource 5 waters.the InternationalapprovalsofColilertincludetheAssociationofOfficialAnalyticalChemistsInternational 6 (AOAC),InternationalBottleWaterAssociation 7 (IBWA),andEuropeanBottledWatercoolerAssociation 8 (EBWA). Table1 WorldwideProductApprovals/AcceptanceslistedbyCountry Approvals/AcceptancesvaryperregioneffectiveasofMay2011.ContactIDEXXforreferencesorupdatedinformation. Asindicatedabove,Colilerthasbeenthoroughlyevaluatedwithinthedrinkingwatercommunity an industrythatrequiresaccurate,rapidandcosteffectivee.coliandtotalcoliformtestingoptions.the bottledwatercommunityhasrecentlybecomeinterestedinadoptingthecolilerttestfortheirroutine testingregiment.therefore,idexxconductedthefollowingstudytoprovideadditionalinformationon theuseofcolilert250presence/absencemethodinbottledwatersamplesandcompareits performanceagainsttheiso93081:2000(e)standardtestmethod

4 TheColilert250presence/absencemethodusestheIDEXXpatentedDefinedSubstrateTechnology* (DST*)tosimultaneousdetectofE.coliandtotalcoliformbacteriain250mLvolumesofdrinkingand bottledwatersamplesafter24 28hoursofincubationat36 C±2 C.Thetwonutrientindicators,o nitrophenyldgalactopyranoside(onpg)and4methylumbelliferyldglucuronidase(mug)arethe primarynutrientsincolilertandcanbemetabolizedbythetotalcoliformenzymegalactosidaseand thee.colienzymeglucuronidase,respectively.astotalcoliformbacteriagrowincolilert,theyusethe enzymegalactosidasetometabolizeonpgandchangethereagentfromcolorlesstoyellow(see Figures1aand1b).AsE.coligrowinColilert,theyusetheenzymeglucuronidasetometabolizeMUG andcreatefluorescencewhenplacedunder365nmultraviolet(uv)light(seefigures2aand2b).the Colilert250presence/absencemethodisalsospecificallyformulatedtoselectivelysuppressthefew none.coliandnontotalcoliformbacteriathathavethegalactosidaseandglucuronidaseenzymes present. Figure1a Figure1b Figure2a Method Figure2b BottledWaterSampleTypes Thethirtybottledwatersamplesincludedinthisstudyoriginatedfromtwelvedifferentcountries aroundtheworldandweremadeupofthefollowingbottledwatertypes springwaterbrands, artesianwaterbrands,drinkingwaterbrands,stillmineralwaterbrands,andsparklingmineralwater brands(table2).colilert250requiresthatsparklingwatertypesbethoroughlydegassedpriorto testing.therefore,allsparklingbottledwatersamplesweremanuallydegassed,byshaking,priorto testing. Table2 BottledWatertypes,totalnumberofbrandsandgeographicallocationsusedinthisstudy Total#of waters Total#of brands Watersourcelocation BottledSpring 9 6 4xUSA 3xFrance 1xIceland 1xScotland BottledArtesian 8 5 3xCroatia 3xNewZealand 2xSouthPacific Islands BottledDrinking 7 5 7xUSA BottledSparklingMineral 5 5 1xGermany 3xItaly 1xUnitedKingdom BottledStillMineral 1 1 1xUnitedKingdom Totals 30 22

5 AnalyticalMethods Itisoptimal,wheneverpossible,tousenaturalsampleswiththetargetorganismswhencomparing methods.duetothelimitedavailabilityofbottledwatersamplescontaininge.coliandtotalcoliform bacteria,itwasnecessarytoperformthecomparisonbytheinoculationofrelevanttargetand nontargetreferenceorganismsintothewatermatrixinquestion.iso/cd11133:2009wasusedas guidancewhenselectingthetargetandnontargetorganismsforthespikechallengestudy(table3). QualitycontrolstrainslistedinAnnexF,TableF3oftheISO/CD11133:2009wereincluded,alongwith otherrelevantstrainslistedwithintheiso/cd11133:2009document.pureculturesofthereference strainsweresourcedfromamericantypeculturecollection(atcc)andwereanalyzedbybiomérieux s VITEK2systempriortouse(AppendixB,TableB1). Thetargetorganismpanelconsistedof9relevantorganisms;3strainsofE.coliand6strainsoftotal coliformbacteria.eachorganismwasinoculatedintoabottledwatersampleataconcentrationof 1 15cfu/250mL.EachbottledwatersamplewasthensplitandanalyzedinduplicateusingtheColilert 250presence/absencemethodandthereferencemethodISO93081:2000(E). Thenontargetorganismpanelconsistedof6relevantorganisms;6strainsofcommon,nonE.coli, noncoliform,grampositiveandgramnegativewaterbacteria.nontargetorganismswereinoculated intoabottledwatersampleasacocktail,eachataconcentrationof1050cfu/1mlforatotalof 250cfu/1mL.EachbottledwatersamplewasthensplitandanalyzedinduplicateusingtheColilert250 presence/absencemethodandthereferencemethodiso93081:2000(e). Table3 ReferencestrainsofE.coli,totalcoliformandnontargetsselectedforpureculturespike Bacterium Source Relevance Escherichiacoli ATCC IDEXXrecommendedQC 11 Strain Escherichiacoli ATCC AmericanTypeCultureCollection(ATCC) 10.ATCC11775,ATCC25922,andATCC27853arelistedasqualitycontrolorganismsintheColilert250*insert 11.QualityControl(QC) IDEXXrecommendedQCStrain; QCStrainforISO93081(ISO11133:2009) Escherichiacoli ATCC8739 QCStrainforISO93081(ISO11133:2009) Klebsiellapneumoniae ATCC13883 IDEXXrecommendedQCStrain; QCStrainforISO93081(ISO11133:2009) Enterobacteraerogenes ATCC13048 QCStrainforISO93081(ISO11133:2009) Enterobactercloacae ATCC13047 ListedinISO11133:2009 Citrobacterfreundii ATCC43864 ListedinISO11133:2009 Klebsiellaoxytoca ATCC13182 Relevanttotalcoliform Hafniaalvei ATCC13337 ListedinISO11133:2009 Enterococcusfaecalis ATCC29212 QCStrainforISO93081(ISO11133:2009) Staphylococcusaureus ATCC25923 QCStrainforISO93081(ISO11133:2009) Pseudomonasaeruginosa ATCC QCStrainforISO93081(ISO11133:2009) Aeromonashydrophila ATCC7966 ListedinISO11133:2009 Bacilluslicheniformis ATCC14580 ListedinISO11133:2009 Pseudomonasputida ATCC12633 ListedinISO11133:2009

6 Colilert250presence/absencemethodwasperformedasdescribedinthesuppliedproductinsert. Aspreviouslydescribed,eachinoculatedbottledwatersamplewassplitbetweenthetwomethods. ForColilert250,duplicate250mLsamplesweredispensedintoIDEXXsterileplastic290mLvesselsanda blisterpackofcolilert250mediumwasaddedtoeachvessel.colilert250sampleswereagitateduntil mediumwascompletelydissolvedandthenplacedintoanaircirculatingincubatorsetat36 C±1 C. Colilert250sampleswereobservedforcolorandfluorescenceundera365nmUVlampafter24and28 hoursofincubation.afterthe28hourobservationallcolilert250samples,positiveandnegative,were confirmedbytheconfirmationmethodslistedinappendixa,flowchartsa1&a2. ReferencemethodISO93081:2000(E)wasperformedaccordingtotheStandardTestmethoddescribed intherelatedisodocumentation.aspreviouslydescribed,eachinoculatedbottledwatersamplewas splitbetweenthetwomethods.foriso93081:2000(e),duplicate250mlsampleswereconcentratedby membranefiltration,membranewascarefullyplacedonthelactosettcagarandfinallyagarplates wereplacedintoanaircirculatingincubatorsetat36 C±1 C.Sampleswereobservedafter21±3hours andlactosepositivebacteriawerecountedandconfirmedthroughtheoxidaseandindoleconfirmation testsdescribediniso93081:2000(e).theconfirmediso93081:2000(e)membranefiltrationcountwas usedtodeterminetheactuale.coliortotalcoliforminoculumduringtesting. StatisticalAnalysis TheconfirmedISO93081:2000(E)membranefiltrationcountwasusedtodeterminetheactualE.colior totalcoliforminoculum,onlydatasetswithanaveragecountfallingbetween115cfu/250mlwere usedinthedataanalysis.onceusedforthedeterminationofactualinoculum,theiso93081:2000(e) quantitativedatasetswereconvertedtopresence/absencedatasetssothattheycouldbedirectly comparedtothecolilert250presence/absencedatasets.finally,allthecolilert250andiso9308 1:2000(E)resultswerethenpopulatedintoasimple2x2summarytableasdepictedinTable4. TheresultsineachtablewereanalyzedusingtheMcNemar stest 12,13,whichhasafinaloutputofa pvalue.summaryofthemcnemar stestwillbeshownintheformofatableasdepictedintable5. Thepvaluewasassignedusingthechisquareresultcalculated,withonedegreeoffreedom,fromthe numberofunmatchedpairsrepresentedbybandcintable4.itwasdeterminedthatasignificance levelof0.05wouldbeassigned,thereforerequiringa95%confidenceofthenullhypothesis. Therefore,itcanbesaid,witha95%confidencelevel,thatdatasetswithapvalueequaltoorgreater than0.05wouldbeconsideredstatisticallysimilarwhiledatasetswithapvaluelessthan0.05wouldbe consideredstatisticallydifferent,asseenintable5. Table4 Exampleof2x2tableusedinMcNemar stest ISO93081:2000(E) Confirmedresult Colilert250 Confirmedresult # Comments + + a Numberofmatchingresults + b ISOmethodmoresensitive + c Colilert250moresensitive d Numberofmatchingresults n=a+b+c+d 12.McNemar,Quinn(June18,1947)."Noteonthesamplingerrorofthedifferencebetweencorrelatedproportionsorpercentages".Psychometrika12(2): McNemar stestislistedasasuitablemethodforthestatisticalevaluationofpresence/absencemethodsiniso/dis17994:2002.

7 Table5 ExampleofMcNemar stestoutputbasedontable4 Study Colilert250ConfirmedResult vs. ISO93081:2000(E)ConfirmedResult #datapoints Chisquared (1degreeoffreedom) Pvalue (=0.05) n=a+b+c+d x y ResultsandDiscussion Conclusion y0.05=methodsequivalent y0.05=methodsdifferent E.coliRecovery Fiftyeightwatersampleswereinoculatedwiththe3differentE.colireferencestrains.Ofthe58 samples,52samplesfellwithintherequiredspikeconcentrationof1 15cfu/250mL(determinedby ISO93081:2000(E)colonycount).These52samplepairs(104actualdatapoints)wereusedtopopulate Table6representingthe24hourColilert250resultscomparedtotheISO93081:2000(E)results. Table6 E.coliRecoveryofISO93081:2000(E)vs.Colilert250after24hour ISO93081:2000(E) Confirmedresult Colilert250 Confirmedresult , n=104 Ofthe104datapoints,thenumberofconfirmedColilert250positiveresultsmatchingtheISO9308 1:2000(E)confirmedpositiveresultswas100after24hoursofincubationandincreasedupto103after 28hoursofincubation.The3sampledifferenceintheperformanceofColilert250inthedetectionof E.coliATCC8739wasdirectlyrelatedtotheuseofcarbonatedmineralbottledwatertypes.The carbonatedwatertypesweredegassedbymanualshaking,buttheseresultsmayindicatethatthe presenceofresidualcarbonationmayslightlydelaythedetectionofsomee.colistrains.resultslistedin Table6weresubsequentlyusedinthestatisticalanalysisbytheMcNemar stest.thestatisticalanalysis indicatedthatthecolilert250confirmedpositiveresultsafter24hoursofincubationwereequivalentto theiso93081:2000(e)confirmedpositiveresults,summarizedintable7. Table7 E.coliMcNemar stestoutputbasedontable6 Study #datapoints Chisquared (1degreeoffreedom) # Pvalue (=0.05) Conclusion Colilert250at24hoursvs.ISO93081:2000(E) Methodsequivalent 14.DelaytodetectE.coli(ATCC8739)inthreesparklingmineralwatertypesamples,resolvedby28hour 15.Klebsiellaoxytoca(ATCC13182)wasspikedinto14differentwaters(28samplespermethod);presenceofthistotalcoliformwascorrectlyidentifiedbyColilert250after24hoursof incubation(yellowonlyreaction)whileiso93081:2000(e)falselyidentifiedthistotalcoliformasane.coli(confirmationresults:indolepositiveandoxidasenegative).thefalsepositivee.coli resultsbyiso93081:2000(e)arefurthersummarizedinappendixc TableC4andnotincludedinthissummarytable.

8 TotalColiformRecovery EightyFourwatersampleswereinoculatedwiththe6differenttotalcoliformreferencestrains.Ofthe 84samples,72samplesfellwithintherequiredspikeconcentrationof1 15cfu/250mL(determined byiso93081:2000(e)colonycount).these72samplepairs(144actualdatapoints)wereusedto populatetable8representingthe24hourcolilert250resultscomparedtotheiso93081:2000(e) results. Table8 TotalcoliformRecoveryofISO93081:2000(E)vs.Colilert250after24hour ISO93081:2000(E) Confirmedresult Colilert250 Confirmedresult # ISO93081:2000(E)results. 16.Klebsiellaoxytoca(ATCC13182)wasspikedinto14differentwaters(28samplespermethod);presenceofthistotalcoliformwascorrectlyidentifiedbyColilert250after24hoursof incubation(yellowonlyreaction)whileiso93081:2000(e)falselyidentifiedthistotalcoliformasane.coli(confirmationresults:indolepositiveandoxidasenegative).thefalsepositivee.coli resultsbyiso93081:2000(e)arefurthersummarizedinappendixc TableC4andnotincludedinthissummarytable. 17.H.alveiwasspikedinto15differentwaters(30samples)andonlyculturedonLactoseTTCthreetimes(thesearelistedinAppendixC,TableC4).GrowthontheLactoseTTCwasatypicalin nature extremelysmallclearandredcolouredcoloniesbutconfirmedasatotalcoliform(indolenegative,oxidasenegative).itshouldbenotedthath.alveidoesnotgrowonembor MacConkeyagarandisarareenvironmentaltotalcoliform,consideredsobyitsabilitytoproducelowlevelsofacidbylactosefermentation n=144 Ofthe144datapoints,thenumberofconfirmedColilert250positiveresultsmatchingtheISO9308 1:2000(E)confirmedpositiveresultswere140after24hoursofincubation.The4sampledifferencein theperformanceofcolilert250andiso93081:2000(e)wasdirectlyrelatedtototalcoliformreference strain,hafniaalvei(atcc13337) 18.ResultslistedinTable8weresubsequentlyusedinthestatistical analysisbythemcnemar stest.thestatisticalanalysisindicatedthatthecolilert250confirmedpositive resultsafter24hoursofincubationwereequivalenttotheiso93081:2000(e)confirmedpositive results,summarizedintable9. Table9 TotalcoliformMcNemar stestoutputbasedontables9&10 Study #datapoints Chisquared (1degreeoffreedom) 8 Pvalue (=0.05) Conclusion Colilert250at24hoursvs.ISO93081:2000(E) Methodsequivalent NontargetSuppression Twentyonewatersampleswereinoculatedwithanontargetcocktailspikecontaining6differentnon totalcoliformandnone.colireferencestrains.ofthe21samples,21samplesfellwithintherequired totalnontargetspikeconcentrationof cfu/1mL(determinedbyspreadplatecolonycount ontrypticsoyagarwith5%sheepblood).appendixd,tabled1liststheaverageconcentrationof eachnontargetstrainalongwiththetotalcocktailconcentration.the21samplepairs(42actualdata points)wereusedtopopulatetable10representingthe24hourcolilert250resultscomparedtothe

9 Table10 NontargetSuppressionofISO93081:2000(E)vs.Colilert250after24hour ISO93081:2000(E) Confirmedresult Colilert250 Confirmedresult # n=42 Ofthe42datapoints,thenumberofconfirmedColilert250negativeresultsmatchingtheISO9308 1:2000(E)confirmednegativeresultswere42after24hoursofincubation.Allsamplesanalyzedusing ISO93081:2000(E)resultedinconfluentcolonygrowthonmembranefiltersandrequiredadditional confirmationstepswhichincludedanadditional24hourincubationperiod,delayingthecompletionof finalresult.colilert250productsignificantlyoutperformedtheiso93081:2000(e)byprovidinga definitiveandconfirmedresultafter24hoursofincubation,completelysuppressingthenontarget cocktailspike. Conclusions ThefollowingstudyshowedthatthedetectioncapabilitiesofColilert250,forE.coliandtotalcoliform bacteriuminvariousbottledwatermatricesisequivalenttothatofiso93081:2000(e),andisfurther summarizedintable11. Table11 SummaryofColilert250performanceindetectoftargetorganisms Study #datapoints Pvalue (=0.05) Conclusion E.coliColilert250vs.ISO93081:2000(E) Methodsequivalent TotalcoliformColilert250vs.ISO93081:2000(E) Methodsequivalent Inthisstudythepresenceoftotalcoliform,Klebsiellaoxytoca(ATCC13182) 18,wascorrectlyidentifiedby Colilert250after24hoursofincubation(yellowonlyreaction)whileISO93081:2000(E)falselyidentified thistotalcoliformasane.coli(confirmationresults:indolepositiveandoxidasenegative)after48hours oftotalincubationandmultipleconfirmationsteps.thefalsepositivee.coliresultbyiso93081:2000(e) isfurthersummarizedinappendixc TableC4. ThisstudyalsoshowedthattheColilert250productisextremelyproficientinthesuppressionofhigh concentrationsofnontargetorganisms.thisisanotherareathatiso93081:2000(e)fallsshort; requiringtheusertoperformmultipleconfirmationstepswhichincludesanadditional24hour incubationthatfurtherdelaysdefinitiveresults.additionally,theexcessivenontargetgrowthonthe membranefiltermayinterferewiththemethod sabilitytodetecttargetorganisms. 18.Klebsiellaoxytoca(ATCC13182),alongwithallotherreferencestrainsusedinthisstudy,wereanalyzedbybioMérieux svitek2systempriortouse(appendixb,tableb1). 9

10 References ISO/TR13843:2000(E)WaterQuality Guidanceonvalidationofmicrobiologicalmethods. Geneva:InternationalOrganizationforStandardization. ISO17994:2004WaterQuality Criteriaforestablishingequivalencebetweenmicrobiological methods.geneva:internationalorganizationforstandardization. ISO93081:2000(E)WaterQuality DetectionandenumerationofEscherichiacoliandcoliform bacteria Part1:Membranefiltrationmethod.Geneva:InternationalOrganizationfor Standardization. 10

11 11 AppendixA MethodsusedtoconfirmColilert250presence/absencesamples FlowchartA1 ConfirmationstepsperformedonpositiveandnegativeColilert250samplesinoculatedwithtotalcoliform At28hours 10uLstruckontoEMB (36 C±2 C;22±2hrs) SamplesInoculatedwithTotalColiformbacterium Colilert250*PositiveResultsColilert250*NegativeResults PerformOxidasetest Coliformsamples (readat24&28hrs) Nogrowth=FalsePositive Observecolonymorphologytypes,streak eachcolonialtypeontonutrientagar (36 C±2 C;22±2hrs) Oxidasepositive= NonColiform PresumptiveFalse Positive Oxidasenegative= Coliform TruePositive At28hours 10uLstruckontoEMB (36 C±2 C;22±2hrs) Observecolonymorphologytypes,streak eachcolonialtypeontonutrientagar (36 C±2 C;22±2hrs) Coliformsamples (readat24&28hrs) Nogrowth=TrueNegative PerformOxidasetest PFP isolate identifiedbyvitek Oxidasepositive= NonColiform TrueNegative Oxidasenegative= Coliform PresumptiveFalse Negative PFN isolate identifiedbyvitek

12 12 AppendixA(continued) MethodsusedtoconfirmColilert250presence/absencesamples FlowchartA2 ConfirmationstepsperformedonpositiveandnegativeColilert250samplesinoculatedwithE.coli SamplesInoculatedwithE.coli Colilert250*PositiveResultsColilert250*NegativeResults Coliformsamples (readat24&28hrs) Coliform samples (readat24&28hrs) Add5dropsofKovac s reagent Add5dropsofKovac s reagent Indolepositive= E.coli TruePositive Indolenegative= none.coli PresumptiveFalse Positive PFP isolateidentified byvitek Indolenegative= none.coli PresumptiveFalse Positive Indolepositive= E.coli PresumptiveFalse Negative 10uLinnoculatedinto Trytophanbroth (44 C±0.5 C;21±3hrs) PFN isolateidentified byvitek 10uL innoculatedinto Trytophanbroth (44 C±0.5 C;21±3hrs)

13 AppendixB biomérieux svitek2systemanalysisofpureculturereferencestrains TableB1 ResultsfromVITEK2SystemonReferenceStrainsusedinthisStudy Bacterium Source VITEK2Analysis SelectedOrganism Probability Bionumber Escherichiacoli ATCC11775 Escherichiacoli 99% Escherichiacoli ATCC25922 Escherichiacoli 98% Escherichiacoli ATCC8739 Escherichiacoli 99% Klebsiellapneumoniae ATCC13883 Klebsiellapneumoniae 99% Enterbacteraerogenes ATCC13048 Enterbacteraerogenes 99% Enterobactercloacae ATCC13047 Enterobactercloacae 95% Citrobacterfreundii ATCC43864 Citrobacterfreundii 99% VITEK2reportedLowDiscrimination(Pseudomonasaeruginosa(50%)/Pseudomonasputida(50%)).Teststoseparate,indicatedbyVITEK2report,werecompletedandresultedinafinal determinationofp.putida(99%).resultsoftheteststoseparateincludedalackofgrowthbyorganismat42 Candlackofpyocyaninproductionduringgrowth. Klebsiellaoxytoca ATCC13182 Klebsiellaoxytoca 99% Hafniaalvei ATCC13337 Hafniaalvei 89% Enterococcusfaecalis ATCC29212 Enterococcusfaecalis 99% Staphylococcusaureus ATCC25923 Staphylococcusaureus 99% Pseudomonasaeruginosa ATCC27853 Pseudomonasaeruginosa 98% Bacilluslicheniformis ATCC14580 Bacilluslicheniformis 96% Pseudomonasputida ATCC12633 Pseudomonasputida 99%

14 Spike Code 20 E3 E3 E3 AppendixC ISO93081:2000(E)withconfirmation,andColilert250after24&28hours TableC1:Legendforbacteriumcodes SpikeCode Bacterium Source E1 E.coli ATCC11775 E2 E.coli ATCC25922 E3 E.coli ATCC8739 EA Enterbacteraerogenes ATCC13048 EC Enterobactercloacae ATCC13047 KO Klebsiellaoxytoca ATCC13182 KP Klebsiellapneumoniae ATCC13883 HA Hafniaalvei ATCC13337 CF Citrobacterfreundii ATCC43864 NT Enterococcusfaecalis Staphylococcusaureus Pseudomonasaeruginosa Aeromonashydrophila Bacilluslicheniformis Pseudomonasputida ATCC29212 ATCC25923 ATCC ATCC7966 ATCC14580 ATCC12633 TableC2:Colilert250presence/absencemethodandISO93081:2000(E)resultsforE.colispike SampleID Bottled Water Code Duplicate LactoseTTCagar Yellow/ Orange colonies Red colonies INDOLE TEST Average ConfirmedISO E.colicount Colilert250@ 24hours Colilert250@ 28hours +/ Yellow FLR 22 Yellow FLR A B A B A B TableC3:Colilert250presence/absencemethodandconfirmationresultsforE.colispike SpikeCode SampleID Colilert250@24hours Colilert250@28hours INDOLETEST Bottled WaterCode Duplicate Yellow FLR Yellow FLR +/ Confirmed Result E3 5 A Yes B Yes 20.SeeAppendixC,TableC1forlegendofSpikeCodes 21.Eachofthebottledwaterswasassignedauniqueidentitynumber;pleaseseeTable2formoreinformationonthebottledwatertypesused 22.FLR=Fluorescentundera365nmUltravioletlamp 23.Averageinoculumwas1.5cfu/250mL;Colilert250replicate1negative,Colilert250replicate2positive(unevendistributionoflowbacterialconcentrationmayhavebeenthecause); Colilert250replicate1confirmedtobeabsentofE.colibyconfirmationtesting(SeeAppendixD,TablesD1&D3) 14

15 Spike Code KO KO KO KO KO KO KO KO KO KO KO KO KO KO AppendixC(continued) ISO93081:2000(E)withconfirmation,andColilert250after24&28hours TableC4 Colilert250presence/absencemethodandISO93081:2000(E)fortotalcoliformspike,Klebsiellaoxytoca(ATCC13182) SampleID Bottled Water Code Duplicate A B A B A B A B A B A B A B A B A B A B A B A B A B A B LactoseTTCagar Yellow/ Orange colonies Red colonies INDOLE TEST OXIDASE TEST 24.FalsePositiveReaction KlebsiellaoxytocaisincorrectlyconfirmedasanE.colibyISO93081:2000(E) Average Confirmed ISOE.coli count 24 Colilert250@ 24hours Colilert250@ 28hours +/ +/ Yellow FLR Yellow FLR

16 AppendixC(continued) ISO93081:2000(E)withconfirmation,andColilert250after24&28hours TableC4 Colilert250presence/absencemethodandISO93081:2000(E)fortotalcoliformspike,Hafniaalvei(ATCC13337) Spike Code HA HA SampleID Bottled Water Code Duplicate LactoseTTCagar Yellow/ Orange colonies Red colonies INDOLE TEST OXIDASE TEST Average Confirmed 24hours 28hours +/ +/ ISOTC 25 count Yellow FLR Yellow FLR A 0 3 B 6 A 4 5 B 6 25.TC=Totalcoliform 16

17 AppendixD Nontargetconcentrations TableD1 Nontargetspikeinoculumwasdeterminedbyaspreadplatecount Bacterium Set1(n=10) cfu/1ml Set2(n=10) cfu/1ml Set3(n=22) cfu/1ml S.aureus A.hydrophila E.faecalis B.licheniformis P.aeruginosa P.putida Total (allstrainscombined)