Microbiological examination of nonsterile Cannabis ... · Medicinal Genomics, Woburn MA Cannabis...

MicrobiologicalexaminationofnonsterileCannabisproducts:MolecularMicrobialEnumerationTestsandthelimitationofColonyFormingUnits.KevinMcKernan,YvonneHelbert,HeatherEbling,AdamCox,LiamKane,LeiZhangMedicinalGenomics,WoburnMACannabismicrobialtestingpresentsuniquechallenges.Unlikefoodtesting,cannabistestinghastoconsidervariousroutesofadministrationbeyondjustoraladministration.Cannabisflowersproducehighconcentrationsofantimicrobialcannabinoidsandterpenoidsandthusrepresentadifferentmatrixthantraditionalfoods1,2.In2018,itisestimatedthat50%ofcannabisisconsumedviavaporizingorsmokingoilsandflowerswhiletheotherhalfisconsumedinMarijuanaInfusedProductsorMIPs.Therearealsotransdermalpatches,salvesandsuppositoriesthatallpresentdifferentmicrobialconsiderations.Severalrecentpublicationshavesurveyedcannabisflowermicrobiologicalcommunities3-5.ThesehavedetectedseveralconcerninggenusandspeciessuchasAspergillusniger,Aspergillusfumigatus,Aspergillusflavus,Aspergillusterreus,PenicilliumpaxilliandPenicilliumcitrinum,Clostridiumbotulinum,Eschericiacoli,SalmonellaandStaphyloccus.ThereareseveraldocumentedcannabiscomplicationsandevenfatalitiesduetoAspergillosisinimmuno-compromisedpatients6-18.ArecentpaperevendemonstratesacaseofcannabisderivedAspergillosisinanimmunecompetentpatient19.ItisunknowntowhatextentAspergillusproducesmycotoxinsincannabisandtowhatextentthosetoxinsenrichinthecannabisextractionprocess.Llewellynetal.publishedlaboratorysettingswhereto8.7ug/gofaflatoxincouldbeproducewithinoculated“marihuana”buttheworkwasperformedin1977andstillleavesmanyquestionsregardingifthiscanoccurinthewild20.ItisalsounknownifClostridiumbotulinumproducesbotulinumtoxinincannabisoils.E.coliorSalmonellafoodpoisoningeventsarelikelytotriggerfebrileseizuresinmanyepilepticpatientsrelyingonCannabisoils21.WhilemycotoxinproducingP.citrinumandP.paxilliDNAhavebeendetectedincannabisflowers,itisunknowntowhatextentthesenanomolar,lipidsolublecompoundsareevenexpressedoncannabisflowers.Cannabinoidsareoftenconsumedinmicromolarconcentrationsimplyingevenrarecontaminantsshouldbeconsidered.PaxillineinparticularisresponsibleforRyegrassstaggersinlivestockandintheorymaycomplicatemanyanti-epilepticpropertiesofcannabidiol22.Grainsilosofcannabis?Thereiscurrentlynopublishedcannabisfailurerateformycotoxintestingpresumablyduetothefastturnoverandshortshelfdurationofcannabisflowers.Mycotoxinproductionisusuallyasaprophyticstatefoundinagriculturalgrainsiloswherelongertermcropstorageismorecommonplace23.Whilelongsaprophyticstorageconditionsincannabisareraretoday,thereisnoreasontoassumetherewillnotbesuchthingsinthefuturegiventhecropsutilityandnutritionalvalue.Asproductionincreaseswithcontinuedcannabisnormalization,weanticipatecommercialagriculturalpracticeswillmoveintothemarkettoleveragecurrentinfrastructureandeconomiesofscale.TherecentpopularityofvaporizingoilspresentsmanyunansweredquestionsregardingAspergillussporeviabilityandmycotoxinconcentrationincurrentcannabinoidextractionsystems24,25.

Onemustalsorecognizethatstrictandnonspecificmicrobialregulationscaneliminatetheuseofbeneficialmicroorganismsinagricultureanddeliverunforeseenconsequencesinthemarketplace.Mandatedlaboratorytestingfornon-specifictotalyeastandmoldpetri-dishassays(TotalYeastandMold(TYM))willfailcommonlyusedchitinase(anti-fungal)producingmicroorganismslikeTrichodermahazarium.TricodermahazariumisknowntoreduceAspergillusgrowth26,27.ThesefalseTYMfailurescaninducegrowerstouselessbiologicalfungicidesandmorechemicalfungicides.Thesechemicalfungicidesareknowntoconcentratepreferentiallyovercannabinoidsinsomeextractionmethods28.Anti-fungalbeneficialmicrobesthatareharmlesstohumanhealtharefareasiertosterilizethansmallmoleculefungicideslikemyclobutanil.Myclobutanilconvertsintohydrogencyanideundermanysmokingandvaporizationconditionsandisnowacommoncontaminantincannabis29.Tofurthercomplicatemicrobialtesting,manygrowersareundertheimpressionthatcannabisextractiontechniquesredeemmicrobialcontaminatedcannabisproducts.Thereisnopeer-reviewedliteratureonthesurvivaloflipophilicClostridiumorAspergillussporesandmycotoxinsincannabisextractiontechniques.Manypesticides,insecticidesandfungicidesareknowntoconcentratewithcannabisextraction30.Littleisknownifmycotoxinsalsoconcentrateincannabisextraction.DuetotheseconcernstheDenverDepartmentofEnvironmentalHealthissuedaC.botulinumwarningin201731.CulturebasedmethodsversusmolecularmethodsWhileculturebasedmethodshavebeeninuseforover100years,acenturylaterpublicationscontinuetoremindusthatlessthan5%ofthemicrobialspeciesareculturable32,33.MolecularmethodsoftenleverageamplificationofrDNAinternaltranscribedspacersorITSregions3,4.Asaresult,thesePCRproductscandetectunculturableorganismsandorganismsthatclumpanddistortCFU/genumeration(Figure1andFigure2).ThisclumpingartifactisreferredtoasheterogeneousmacrocoloniesandisknowntooccurwithAspergillus34.ThiscreatessignificantquantificationissuesforstatesrequiringsingleCFU/gfailurethresholdsastheclumpingcreatessamplingbias.Thereislittleresolutionbetween0and1000colonieswithAspergillusplatedonSabDex,PotatoDextroseagaror3MTYMfilms.Inaddition,AspergillusarefairlyubiquitousintheenvironmentandnotallAspergillusspeciesareharmful.Asaresult,manystates(CA,AK,NV)mandateA.niger,A.flavus,A.fumigatusandA.terreustesting.Thesespeciessynthesizemycotoxinsandpasteurizationresistantpathogenicspores35.Thesecanbedifficulttodiscernwithplatingmethodswhilemolecularmethodscaneasilyitemizethem.ArecentcaseinAlaskahadalabusingagarmistakebenignAspergillusbrasiliensiswithAspergilllusniger36.Thelabusingcultureultimatelycloseditsdoorscitingbankingissues.C.botulinumalsoproducespasteurizationresistantsporesandisananaerobethatisincompatiblewithaerobicplatecounts37.

Figure1.ReprintedfromMcKernanetal.CFU/gcorrelationswithqPCRCqisfairlyconsistentwithATCCfungithatdonotformheterogeneousmacro-colonies.Aspergillusclumpsandcreatessamplingbiasduring

platingeffectivelylimitingthedynamicrangeoftheassayandcomplicatingsingleCFU/gaccuracy.NotethelargediscordancebetweenestimatedgenomiccopynumberswithAspergillusandcoloniesdetectedwith3M.

Figure2.Aspergillusnigerplatedon3MPetrifilm®andSabDexagar.Enlargedcoloniesaretheresultof100-1000conidiaclumpedintoaheterogeneousmacro-colony.ToproperlycalibratesingleCFU/gAspergillussensitivitywithqPCR,declumpingoftheconidiaisrequired.Thiscanbeachievedbyharvestingconidiain1%Tween-80.

Thoroughvortexingandfiltrationthrougha5umspincolumndeliversindividual,declumpedconidia(Figure2B).WhilethisfiltrationprocessishelpfulforsingleCFUquantitationandcalibrationitisimpracticaltoapplytorealcannabissamplesduetothesignificantlossinyieldseeninthefiltrationprocess.Thiswouldfailtoproperlymeasurethefullriskprofileoncannabis.Nevertheless,theprocessdoesenablethevalidationofsingleCFU/gsensitivity.Oncesporesaredeclumpedandeasytocount,itisimportanttodemonstratethatthelysisconditionscaninfactlysethespores.Onecanquantitatethiswithahemocytometersporecount

coupledwithqPCRofsolutionsthathavesporeswithandwithoutlysisbuffer.AcommonlycitedbutincorrectlimitationtomolecularmethodsisthattheycannotinformmicrobialviabilityordiscernliveversusdeadDNA.Asaresult,thereismisguidedconcernthatmolecularmethodswillpenalizegrowersthatproperlysterilizetheircannabis.ThereareseveralpublishedmethodsdescribingviabilityPCR38,39butthesimplest

Figure2B.AspergillusnigerHeterogenousmacrocoloniescanbedeclumpedbyvortexinginsurfactantslikeTween-80andfilteredfor5umsinglespores.ThisenablesproperenumerationonplatingtobringhigherqPCRconcordancewithAspergillusCFU/g.Thisisnotadvisedforroutinecannabissafetytestingduetothelossinyieldviafiltrationprocessbutitisrequiredtodemonstratesinglesporesensitivity.

methodsuggestedbytheEPAistosimplyretest(qPCR)allfailedsamples24hourslaterafterincubationinagrowthmedia40.Sincethisislimitedtothesamplesthatfailimmediatetesting,re-testing24hourslaterinTSBisrestrictedtofewersamplesandnotascumbersome.AnysamplethathasviablecellsorpartiallysterilizedmaterialwillgrowanddemonstrateanincreaseinDNAconcentration24hourslater.ItisimportanttounderscorethatthismethodisconstrainedtothoseorganismsthatcancultureinTSBinagiventimeframeandtemperature.Nevertheless,qPCRdelivershighergenusandspeciesspecificitythanculture-basedmethods.Inadditiontobeingabletodiscernlivefromdeadorganisms,qPCRassaysalsohavealysisandDNApurificationstep.Thisstepde-clumpsmacro-coloniesandprovidesbettersamplingwithAspergilluswhilealsoremovinganycannabinoids,terpenoids,sugarsandotherinhibitorsfromtheassaythatmightinterferewithplatecountchemistryorpolymerasechemistry.ThisisparticularlyimportantwiththediversematricesfoundinMIPswhichhavebeenshowntointerferewithcultureconditions41.

LiveversusDeadisnotabinarydebateBeuchatetal.demonstratetheimportanceofvaryingnutrients,pH,andwateractivityrequiredforresuscitationofstressedorpartiallyviablecells42.Driedbananachipsrequired120hourstofullyresuscitateorganismsin5differentculturemediums.Thedehydrationisbelievedtoleavethecellsandsporesinapartiallyviablestate.Cannabisisalsodried.120hoursismuchlongerthanmostculturebasedtechniquesbeingutilizedinthecannabisindustry(48-60hours)thuschallengingthevalidityofanyviabilitybenefitfromshortdurationplating.Thefivedaysrequiredtocapturethesepartiallyviablecellsleavesonetoquestionwhatwillthenbegrowingonthecannabisbeingtestedthatmanydayslater.MeasuringtheentireDNAloadontheplantcanbedoneinunder5hours.The

Figure3A-Comparisonofclassifiedreadpercentagesforbacterial16SDNAonsamples2and14,beforeandafterculturingon3MandBiomerieuxTEMPO®(BMX)media.Theresultsrepresentallspeciesobserveddownto1%ofclassifiedreads.Largeshiftsinspeciesprevalenceareseenaftergrowthonthetwoculture-basedplatforms.

pursuitofviabilityhasalsoleadtogentlehomogenizationtechniquesthatlikelyfailtoliberateendophytes.Fusariumisapathogenicendophyte.Molecularmethodstendtolysecellsandmoreaggressivelyliberateendophytesanddeclumpfilamentousfungi.MoleculartechniquesoftenutilizeliberatedcannabisDNAasaninternalDNApurificationcontroldemonstratingthatthelysisbufferiscapableofdissolvingplantwallsandanyrespectiveendophytes.Thusthepursuitofviabilitynotonlylimitsthesensitivityoftheassaybutalsoprovidesafalsesenseofsafetywhenitfailstoaddressdriedgoods.ClinicaluseofqPCRforAspergillusinbloodhasbeenmetwithsomeresistanceduetopoorstandardization.Whiteetal.describetheutilizationofqPCRforAspergillusdetectioninclinicalbloodisolatesandhowthesetechniqueshavefailedtopassclinicalmilestonesbytheEuropeanOrganizationforResearchandTreatmentofCancerandtheMycosisStudyGroupin2006(EORTC/MSG)43.Muchofthisresistancewasrelatedtothelackofcommerciallyavailablekitswithproperpositiveandinternalcontrols.ManylabswerehomebrewingtheirownLDTtestsandmostofthediscordancewasattributedtoDNAextractionprocedureselutingDNAinover100ul,lysinglessthan3mlofbloodandthelackofinternalcontrolstoidentifyfailedPCRreactions.ItisimportanttonotethatblooddetectionofInvasiveAspergillus(IA)hasaBeta-D-GlucanassaythatiscurrentlyFDAapprovedandthusthethresholdforqPCRadoptionismuchhigher44.Asof2015thesemethodshavereachedequivalenceandarebeingconsideredforinclusionintotheEORTC/MSG45.Itisnotcleartheblooddetectiondebateisveryinformativetothecannabisindustry.ThegoldstandardBeta-D-Glucan(Fungitell®)assaysusedinbloodareunlikelytoberelevantinfoodduetohighlevelsofnaturallyoccurringBeta-D-Glucansinvariousnonbloodbornandofftargetfungiandplants46.Asaresultthesemethodsarenotpublishedforuseinthefoodindustry.MicrobiomesfrombeforeandafterculturingarehighlydiscordantCannabisITSsequencingofmicrobesthatexistedbeforeandafterculturingon3MPetrifilm®andBiomerieuxTEMPO®culturingdeviceshavedemonstratedverylowspecificityanddrasticdifferencesinaspeciesprevalencefrombeforeculturetoafterculture3.Thisimpliesthetwodifferentmethodsofculturingaredifferentiallyalteringtheinitialriskprofilewhilealsosufferingfromspecificity.ThisisnotasurprisegiventheTEMPO®YMsystemuseschloramphenicolwhilethe3Msystemdoesnot.Asmuchas60%oftheDNAisolatedfromtheseTYM3MPetrifilm®assaysandtheBiomerieuxTEMPO®YMcartridgeswerebacteria.Manywereknowntobechloramphenicolresistant.Bacteriaalsotendtohavefasterdoublingtimes(30minutescomparedto2ormorehoursforyeastandmolds)suggestingbacteriamayformasaturatedcultureinthefirst24hourswhiletheyeastcontinuestogrowfor72hours.SequencingoftheseofftargetbacteriaoftenrevealsBacillusspeciesknowtoproducelacticacidorPseudomonasspeciesknowntoproducesalicylicacid.Thedegreetowhichthesecontaminatingspeciesaltertheyeastandmoldenumerationisunknown.TheBiomerieuxTEMPO®platformutilizesapHsensitivefluorescentdyetomonitorthegrowthcurve.AdecreaseinpHwilldecreasefluorescenceandinferhigheryeastandmoldcounts.ItisassumedthatthesecontaminatingacidproducingbacteriaaredecreasingthepHoralteringthefluorescenceandfalselyelevatingtheyeastandmoldcounts.BacillusiscommonlyusedinthecannabisindustryasafoliarsprayknownasSerenade®.Likewise,moldsmayproduceacidiccompoundsatvaryingrates.Penicilliumcitrinum

producesACCandTanzawaicacids47.ItisuncleartowhatextentthisexpressionaffectspHincultureandhowspecificpHisasaproxyforCFU/ginamixedmicrobiomegrowthenvironment.Totestthistheorywesentachloramphenicolresistant,salicyclicacidproducingPseudomonasaeruginosaculturetotwotestinglaboratoriesusingtheTEMPO®systemandreceivedfailingTYM(9x10^4)countsandcloudycartridgeimageswithpassingBTGNresults.ThisimplieschloramphenicolresistantbacteriathatcanalterthepHofthebrothcancreatefalsepositiveYeastandMoldtestswiththisplatform.PseudomonasshouldtriggeraBTGNaccordingtotheUSP.

Pseudomonasaeruginosaisresponsiblefor11-14%ofhospitalacquiredinfectionsandisparticularlyproblematicwithCysticFibrosispatients48.CFpatientsareexperimentingwithcannabinoiduseforthereductionoffibrosis.CorbusPharmaceuticalsispursuingmodifiedcannabinoidsforCFpatientsinFDAtrials.100,000CFU/gofTACareallowablebutonly1,000CFU/gareallowableforBTGN.AnylabusingonlyBiomerieuxwillbevulnerabletopassingsampleswith1001-99,999CFU/gofPseudomonas.

OnemustalsobemindfulthatmanyfoliarspraysusedtocombatyeastandmoldsutilizehydrogenperoxideorvinegarandmayaffectthepHofcannabistested.ItwouldbepreferabletohaveatoolthatmeasuresthemicrobesmorespecificallythanonethatisexposedtothepHimpactofthemultitudeofadditivesonemightfindonCannabis.SeveralstudieshavealsofoundspicesandotherfoodproductscanaltertheUVfluorescencechemistryoftheTEMPO®YMplatformandthuswillrequiredeeperdilutionstogetaccuratequantitation49,50.Kunikademonstrates5outof9spicedfoodscreatedsuchinterferenceontheTEMPO®YMplatform(oniongrits,cinnamon,yeastextract,groundcaraway,beetrootjuiceconcentrate)whileOwenetal.demonstratepeppercorninterference.Onions,caraway,cinnamonandpeppercornsallproduceBetaCaryophyllene,asesquiterpenoidalsofoundinhighconcentrationsinsomecannabiscultivars.Cannabisismorelikelytofallintothecategoryofaspicegiventhepotentexpressionofvariousacidiccannabinoidsandterpenoids.Othermolecularstudieshavealsofoundthatbackgroundoff-targetmicrobiotaproducediscordantresultsinculturebasedE.colienumerationcomparedtoqPCR51.PCRbasedmethodsdescribedbyMcKernanet.aldemonstratedover96%specificitywiththelimiteddiscordancebeingderivedfromfungalmitochondrial16SsequencebeingmisclassifiedasbacterialDNA3.PCRalsooffersthecapacitytosurveythemicrobialcommunityontheflowersamplewithoutexposingittoanewcarbonsourceorselectivebias.Thiscapacityto

Figure4:BLASTanalysisoftheorganismsfoundviasequencingtheITSPCRproductsobtainedfromBiomerieuxTEMPO®YMcartridges(BMX)and3MPetrifilm®YMplates.18Sprimersdeliverover99.7%fungalsequences.16Sprimersdeliverover96%bacterialsequences.Offtarget16Ssequenceswerepredominantly16Sfungalmitochondrialsequence.

accuratelyquantitateAspergillusandothernon-culturablefungiisturningouttobeimperativeforthecannabisindustry.Giventhesepublishedwarnings,andobservedconcernsovercontaminatingspeciesorcompoundsfromspices,arobustvalidatedstudyofcannabisincultureisrequiredbeforegeneralfoodguidelinesareassumedtohaveadequateinclusionandexclusioncriteria.ITSamplificationconsiderationsManymoldsproducemyceliumthathavemultiplenucleipercell.Whilethismayseemproblematicformolecularcounting,fragmentedmyceliaareculturable52-54.Givenmoldsaregenerallyamixtureofconidiaandmycelium(andhomogenizationtechniquespartiallyfragmentthesestructures),webelieveitisbesttomeasurethenumberofinfectivenucleiasopposedtonumberofcellsinagivenYeastandMoldtest.Whencountingnuclei,onemustbeawareoftheapproximatecopynumberofyourDNAtargetpernuclei.ITScopynumbercanvarybetweenorganisms.ITSregionscanhave20-100copiesinfungi55.Thiscanbebothabenefitandacurse.Whilehavingmorethanonetargetpergenomecanenablesub-CFUsensitivityandreducedigitalPCRartifacts,itcancomplicateCFUcalculations.TheseITScopynumbervariationsareusuallylessthanalogscaleinvariation56.ThevariationweseewithsinglesporeformingCFUsandsporeclumpingintoheterogeneousmacrocoloniescancreate3logscalesofvariance3,34,57,58.Filamentousfungiclumpinghascreatedrigorousstandardsbodiestodevelopmethodsthatensureinter-laboratoryconsistencyquantitatingtheseorganisms59.Thesemethodsareamenabletomonoculturesstudyinganti-fungalcompoundsbutcannotbeperformedonwildmicrobiomesamplesduetothecarefulpipettingandcentrifugationthatisrequiredtoisolatehomogenoussporesuspensions59.ITScopynumberisneverthelessimportanttokeepinconsiderationasyoucomparedtotalgenomiccopynumberstoITSCqandCFU.AnotherbenefitofITStargetsisthatthesearethefirstregionsofthegenometoreplicateinviablecellsandasaresulthavebeendemonstratedtoassistinviabilityPCR38,39.Whilesinglecopyregionscanbedesigned,thesearehardertofindconserveduniversallyinallFungi.SinglecopytargetsalsosufferfromdigitalPCRartifactsatlowdilutions.Implicationsofendofungaltoxigenicbacteria.Manyfilamentousfungiarehostsfortoxigenicbacteria.RalstoniaisaplantandhumanpathogenicbacteriafoundincannabisandisknowntoinfectmanyfungifoundonCannabis60,61.SeveralclinicalreportsofRalstoniainfectionsinexistinCancerpatients,CysticFibrosispatientsandotherimmune-compromisedpatients62-64.Itisunlikelythatbacteriainternaltoafungiwillproducecoloniesonbacterialpetridishes.Likewise,sampleswithlowfungalcountsonTYMpetridishescouldstillcontainhighlevelsoftoxigenicbacteria.Ralstoniafurthercomplicatesquantitatingmoldsasitinducestheformationofchlamydosporesinmanyfilamentousfungi60.Chlamydosporesaremulticellular,asexualsporeclumpsthatareinducedforenvironmentalsurvival.ThesearedifficulttohomogenizeandcreatesimilarquantitationproblemsdescribedearlierwithAspergillus.RhizopusisanexampleofamoldknowntobeahostforthetoxigenicendofungalsymbioteBurkholderia-Rhizopus65.Rhizopusmicrosporusisknowntobeinvolvedinfatalfungalinfectionsaffectingimmune-compromisedpatients66.RhizoxinisrequiredforRhizopuspathogenicityandcaninduceliverfailure67.

PowderyMildew,Botrytisandsterilization.BotrytiscinereaandpowderymildewarethetwomostcommonfungalplantpathogensfoundonCannabis.Neitherofthesecultureon48-houragarlawnsorPetrifilms®.Cultureconditionsoftenrequirespecializedmediaand2-6weeksofgrowth68-71.Somespeciesofpowderymildewcanbeculturedonotherplanttissue-basedmediasbutthishasnotbeenvalidatedoncannabisderivedpowderymildew(CDPM)andthedefinitionofacolonyformingunitisinquestion.CDPMhasrecentlybeensequencedandinoculationoncannabisplantandisbelievedtobeaGolovinomycesgenus72.Powderymildewsareknowntobeobligatebiotrophsthatoftenspeciatetogrowonasinglehostsspecies73.CulturingofCDPMmayhavetobeperformedonCannabisleafbasedmediasmakingforacomplicatedproductthatcannotshipacrossstatelines.Thereislittletonoevidenceintheclinicalliteratureregardinghumanhealthriskswithinhalationofpowderymildew,howeverlitigationregardinganemployeeallergicreactionduringprolongedcannabistrimmingisongoinginMassachusetts74.Botrytisisresponsibleforcannabisbud-rot.ItisoftenusedinwineproductionandknownasNoblerot.Thisimpliesitissafetoconsumeorallyhowever,Botrytiscinereacanalsoproduceallergicreactions75.TheCDChaspublishedITSsequencingworkdemonstratinghighBotrytissporeexposurewithcannabistrimmingenvironments76.ThesecommonplantfungiarenotbeingaccuratelyquantifiedbythecurrentCFU/gbasedregulationsandwebelievemolecularmethodswillbetheonlymechanismstodoso.Toaddressthisgap,MedicinalGenomicssequencedthegenomeofcannabis-derivedpowderymildew.Its’ITSsequenceis98%identicaltoP.macularisandGolovinomycesambrosiae.Pepinetal.havesincepublishedaGolovinomyceschichoracearumpowderymildewonCanadiancannabis72.Withthisinformation,wedevelopedaPCRbasedassayforthisspecies77.ValidationofthisassayviacorrelationtoCFU/gmetricsrequiresdevelopmentofCDPMculturingconditionsthatproduceenumerablecoloniesorcalibrationtoconidiacountsonahemocytometer.MedicinalGenomicsisalsodevelopingqPCRassaysforBotrytiscinerea.CryptococcusIn2018,Shapiroetal.publishedacasestudyof“adailycannabissmokerwithoutevidenceofimmunodeficiencypresentingwithconfirmedCryptococcusneoformansmeningitis”.FurtherITSsequencingofthepatientscannabissamplesrevealedseveralvarietiesofCryptococcusspecies78.Cryptococcusisaslowgrowingmoldthatalsorequiresspecializedcultureconditions79.TherearebenignformsofCryptococcussospeciationiscriticalanddifficulttoachievewithculturealone.PathogenicspeciesincludeC.neoformans,C.gatti.BothspeciesareaddressablewithITSPCR.PeripheralCrytococcosisinfectionsareusuallytreatablewithfluconazolewhileCNSinfectionsrequireintravenousamphotericinB.FusariumoxysporumandotherorganismsofconcernManyotherorganismshavebeenreportedonCannabisthatpresentculturingproblems.Fusariumoxysporumhasbeenreportedtocausefusariosis.HundredsofcasesofFusariosishavebeendescribedintheliterature80-84.Severalcannabisderivedcasesaredescribed85.Fusariumisalsoquiteubiquitousandmostlyariskforimmuno-compromisedpatientsbuthasaveryhighfatalityrate

whenacquired.ThisisofsignificantconcerngivenFusariumoxysporumisoneofthemorewidelyusedbiocontrolagentsforcannabiseradicationprograms86.StaphylococcusaureusandPseudomonasaeruginosahavealsobeenlistedasorganismsofconcerninsomejurisdictions.ThePseudomonasgeneraisfairlyubiquitousandinfectsbothhumansandplants.Manyspeciesarebenignandusedasbio-controlagentstolimitharmfulfungi.SpeciationofharmfulversusbeneficialPseudomonasiscritical87-90.PseudomonasaeruginosaisoftenaproblemforburnvictimsandCysticFibrosispatients.Cannabinoidsarepopularamongstcysticfibrosispatientsbutitisunknownifsuchpatientscouldorwouldwanttoresorttoinhalationbaseddelivery91-93.CorbusPharmaceuticalshasbeenadvancingmodifiedcannabinoidsthroughtheFDAtotreatCysticFibrosisandotherfibroticdiseases.GiventhelikelyuseofCannabisbyCysticFibrosispatients,Pseudomonasaeruginosisscreeningisavalidconcern.Despitebeingabacteria,Pseudomonasalsogrowson3MTYMPetrifilm®andinBiomerieuxTEMPO®YeastandMoldcartridges.PseudomonasaeruginosaischloramphenicolresistantandknowntoproduceSalicylicacidthatcaninterferewithpHbasedfungaldetection94.StaphylococcusaureusisalwaysahealthconcernregardingantibioticresistanceandMRSA(methicillinresistantStaphylococcusaureus).Nevertheless,cannabinoidsareprovingtobesomeofthemorepromisinganti-MRSAcompounds1,95,96.DistinguishingMRSAfrombenignhumanskin-commensalslikeCoagulase-negativestaphylococci(CoNS)ischallengingwithoutmoleculartechniques97.OthermycotoxinproducingfungihavebeenrecordedonCannabissuchasP.citrinumandP.paxilli.Themycotoxinproductionofthesespeciesoncannabishasnotbeenquantitatedtodatebuttheirpotencyisnanomolarcomparedtothemicromolarusageofcannabinoids.Lowlevelcontaminationoftheselipidsolublemycotoxinsisanunknownriskbutpublishedliteratureontheirinterferencewithcannabidiolexistsinratssuggestingcaution.Inthemostextremecasesfungalmycotoxinsareclassifiedasweapons98.MycobacteriumtuberculosisAnAustralianstudyfound29peopleinfectedbythesamestrainofM.tuberculosisbelievedtobespreadfromsharingthesamebong99.AdditionalM.tuberculosisreportsinSeattleWAwerediscoveredin2004100.Whilecannabisparaphernaliamaybeavectorforcertainhumanpathogens,thereisalsosomeevidencethatcannabiscanbeahostformycobacterium3,101.ThereareseveralformsofNon-Tuberculosismycobacteria(NTM)thatalsopresentclinicalrisk102.MoreworkisrequiredtounderstandwhichspeciesofMycobacteriumpresentriskstocannabispatients.MostofthesestudiesrelyongenotypingtospeciatetheMycobacteriaofclinicalconcern.MucorandmucormycosisMucorisafungalgenusconsistingofmanypathogenicandbenignspecies.Mucorinfectionscanbelifethreateninginimmunocompromisedanddiabeticpatients.90%ofcasesarecausedbyRhizopusoryzaeandPCRbasedtestsusuallytargetmultiplespecies.103.ItisimportanttounderscorethatRhizopusoryzaeisusedtofermentTempeandtheFDAcategorizesitasGRAS(generallyrecognizedassafe).Rhizopusorzyaehasalsobeenreportedtoreduceaflatoxinproductionofotherspecies104,105.Nevertheless,Rhizopusorzyaepresentsaverydifferentriskprofiletoimmunocompromisedpatients.Itusuallyrequiresalacerationorulcerationtobecomeinfectious.

Mucorinfectionsareusuallyfoundinsinuses,brainandlungs.AmphotericinBandIsavuconazoleareoftenusedtofighttheinfections106.Rhinocerebralmucormycosisisthemostcommonmanifestationoftheinfectionandhas30-100%mortalitydependingonthepatientpre-existingdiagnosis.MucorhasbeenfoundonCannabisflowersintwoindependentITSsequencingstudies3,5.Moreworkisrequiredtospeciatethegenusdetectedoncannabisandifthesespeciesarethesamespeciesreportedinhospitalinfections107.ListeriamonocytogenesListeriosisisthe3rdmostfrequentformoffoodborneillness108.Itisanintracellulargram-positivepathogenthatcangrowinrefrigerationtemperaturesandisknowntocreatebiofilms109.LargedosesofcannabinoidshavebeenshowntoweakenimmuneresponsetoListeriainmicemodelsofdisease110.Thedosagesstudiedinthisworkwereseveralordersofmagnitudehigher(38-150mg/kgTHC)thanrecreationalcannabisdosages(5mg/75kg).Listeriahasnotyetbeenrecordedoncannabisbutitisimportanttorealizetheexistingpublishedsequencingsurveysarestillverypreliminarywithsmallsamplesizes.Listeriaismorelikelyrelevanttocannabisediblemanufacturingandcleankitchenmethodologies.Listeriadetectionrequiresselectiveenrichmentandspeciationwithcolonymorphologyandsugarfermentation.MorerapidPCRbasedtoolshavebeenpublishedthatcanascertainspeciationandviabilityinunder48hours111.NojurisdictionscurrentlyrequireListeriatestingforCannabisbutitisbeingreviewedasaconcerninColorado112.Insummary,thereissubstantialevidenceforculturebasedsystemspresentingsignificantvulnerabilitiesforthemostseveremicrobialrisksfoundonCannabisandguidelinesanchoredinCFU/gwillcontinuetoinhibitproperquantificationoftheserisks113.Webelievecannabismicrobialenumerationguidelinesmovingforwardshouldconsidermolecularmethods.GenomictechniquesaregainingadoptionattheFDAGenomeTrakrnetwork114andwebelievethisisrelatedtotheunreliabilityofculturebasedplatformstoproperlyinformonmicrobialhealthhazardsinatimelymanner.TheremainderofthisdocumentwillfocusonprotocolandmethodsvalidationconsiderationsusedinvalidatingacommerciallyavailablecannabismicrobialdetectionplatformknownasPathoSEEK®.MethodsValidationTherearecurrentlyonlyfourpeer-reviewedpapersthatdescribeCannabismicrobialmethodsandtheyallutilizeITSPCR3-5,115.Whilethereisatemptationtoliftwhatiscommonlyusedinthefoodindustryandassumeitwillbehaveaccordinglyoncannabis,mostregulatorsandcertificationagencieswillnotallowthesuperimpositionofamethodvalidationbetweendifferentmatrices.Ifyouchangethetargetmatrix(milktocannabis),youneedtorevalidatetheassayonthenewmatrix.Asofthiswriting,therearenopeer-reviewedculture-basedtechniquesthathaveperformedacannabismatrixvalidationintheliterature.Likewise,noregulatorwillallowamanufacturersvalidationtotransfertoanotherlaboratorysetting.Manufacturersvalidationsneedtobere-examinedinthelaboratoriestheyareimplementedin.ThisisalsotruewithISOcertifications.Belowareafewguidelinesonhowtoperformsuchavalidation.

DependingontheCountryorState,someorallofthefollowingliveorganismsandgenomicDNA(Table1)shouldorderedfromATCCorothertissueculturebankstoserveasspikeincontrolsformethodsvalidation.ABioLevel2(BL2)Safetylabisrequiredbylawtohandlehumanpathogens.SomehumanandplantpathogenslikeFusariummayrequireUSDAapprovalbeforeshipment.Spike-insshouldbemeasuredonbothflowersandatleast3differentMIPmatrices(Chocolate,gummy,shatter)at3ormoredifferentdilutionlevels.MIPsthatareinhaledareoftenheldtotighterregulatorystandardsthanoralMIPs(California). Table 1, Live Organisms Evaluated:

Live Organisms Source Aspergillus flavus ATCC# 16870

Aspergillus fumigatus ATCC# 16903 Aspergillus niger ATCC# 15475

Aspergillus terreus ATCC# 16793 Aspergillus janponicus ATCC# 16873

Candida albicans ATCC# 10231 Candida catenulate ATCC# 10565 Candida glabralta ATCC# 15545

Candida krusei ATCC# 28870 Candida sphaerica ATCC# 8565

Debaryomyces hanseii ATCC# 10623 Rhodotorula mucilaginosa ATCC# 4557

Trichothecium Roseum ATCC# 90473 Yarrowia lipolytica ATCC# 18944

Salmonella ATCC# 700720 Saccharomyces cerevisiae The Gold Pitch, Giga Yeast, Inc.

E.coli DH10B (New England Biolabs, #C3020K)

Table 2, DNA Organisms Evaluated

DNA Organisms Source Aspergillus flavus ATCC# 9643D-2

Aspergillus fumigatus ATCC# 1022D-5 Aspergillus niger ATCC# 1015D-2

Aspergillus terreus ATCC# 20542D-2 E.coli ATCC# 8739D-5

Penicillium chrysogenum ATCC# 10106D-5 Klebsiella pneumoniae ATCC# 700721D-5

STEC O111 ATCC# BAA-2440D-5 Salmonella enterica ATCC# 700720D-5

Coliform/Entero Positive Control MGC# 420314 Total Yeast and Mold Positive Control MGC# 420303 Total Aerobic Count Positive Control MGC # 420306

E. coli/Salmonella Positive Control MGC# 420313 Californiaregulations(2018)requireinclusionandexclusionanalysisbeperformedforSTECE.coli,Salmonella,Aspergillusterreus,Aspegillusniger,Aspergillusflavus,Aspergillusfumigatus.ThisrequiresadditionalATCCstrainstodemonstratedifferentiationfrombenignandubiquitousAspergillus.Molecularmethodsarerequiredforthislevelofspeciesspecificity.ForinclusioncriteriaforSTECE.coli,ATCChaspublishedaveryhelpfulguidelinedescribingthe“BigSix”E.coli.Table3hasadditional“BigSix”E.colistrainstodemonstrateSTECassaysareinclusiveofthesehealthhazardsandthattheseprimersdon'tamplifyofftargetorganisms(https://www.atcc.org/~/media/PDFs/Big%20Six.ashx)

Table 3, List of species tested with PathoSEEK and corresponding results:

Species Type ATCC # Total Y&M TAC TC TE E.

coli Sal STEC A.fla A.fu

m A.nig A. terr

Aeromonas hydrophilia B 7965D

Pos (+)

Aeromonas

hydrophilia Strain CDC 359-60

B 7966D-5

Pos (+)

Aspergillus aculeatus M 24147 Pos

Neg (-)

Neg (-) Neg (-) Neg (-)

Aspergillus brasiliensis WLRI

034 (120) M 16404D-2 Pos

(+) Neg (-)

Neg (-)

Neg (-) Neg (-) Neg (-)

Aspergillus carbonarius M 1025 Pos

Neg (-)

Neg (-) Neg (-) Neg (-)

Aspergillus flavus M 16870 Pos (+) Neg

(-) Neg (-)

Pos (+)

Neg (-) Neg (-) Neg (-)

Aspergillus flavus SN3 M 9643D-2 Pos

(+) Neg (-)

Neg (-)

Pos (+)

Neg (-) Neg (-) Neg (-)

Aspergillus fumigatus M 16903 Pos

(+) Neg (-)

Neg (-)

Pos (+) Neg (-) Neg (-)

Aspergillus fumigatus 118 M 1022D-2 Pos

Neg (-)

Pos (+) Neg (-) Neg (-)

Aspergillus janponicus M 16873 Pos

Neg (-)

Neg (-) Neg (-) Neg (-)

Aspergillus niger M 15475 Pos (+)

Neg (-)

Pos (+) Neg (-) Aspergillus niger M 1015D-2 Pos

(+) Neg (-)

Neg (-)

Neg (-) Pos (+) Neg (-)

Aspergillus oryzae M 1010 Pos (+)

Aspergillus terreus M 16793 Pos

Neg (-)

Neg (-) Pos(+) Aspergillus terreus M 20542D-2 Pos

(+) Neg (-)

Neg (-)

Neg (-) Pos(+) Aspergillus ustus M 1041 Pos

Neg (-)

Neg (-) Neg (-) Neg (-)

Bacillus subtilis subsp. Spizizenii B 6633D-5 Neg

(-) Pos (+)

Neg (-)

Bacillus subtilis

subsp. Spizizenii B 6633

Neg (-)

Candida albicans Y 10231D-5 Pos

(+) Neg (-)

Neg (-)

Neg (-) Neg (-)

Candida albicans Y 10231 Pos (+)

Neg (-)

Candida catenulata Y 10565 Pos

Candida glabralta Y 15545 Pos (+)

Candida

guilliermondii Y 90197 Pos (+)

Candida krusei Y 28870 Pos (+)

Candida lusitiniae Y 344449 Pos

Candida sphaerica Y 8565 Pos (+)

Clostridium sporogenes B 11437FD

-5 Neg(-) Pos (+)

Neg (-)

Clostridium sporogenes B 11437

Neg (-)

Debaryomyces

hanseii Y 10623 Pos (+)

E.coli DH10BTM

Compentent Cells B NEB

Pos (+)

Enterobacter aerogenes B 15038D-5

Pos (+)

Escherichia coli B 8739D-5 Neg(-) Pos

(+) Pos (+)

Pos (+)

Neg (-)

Escherichia coli B 8739

Pos (+)

Neg (-)

Escherichia coli

Strain 2000-3039 B BAA-2193D-5

Neg (-)

Pos (+)

Neg (-)

Neg (-) Neg (-) Escherichia coli

Strain 2002-3211 B BAA-2219D-5

Neg (-)

Pos (+)

Neg (-)

Strain 2003-3014 B BAA-2196D-5

Neg (-)

Pos (+)

Neg (-)

Strain 2006-3008 B BAA-2215D-5

Neg (-)

Pos (+)

Neg (-)

Neg (-) Neg (-) Escherichia coli Strain 99-3311 B BAA-

2192D-5

Neg (-)

Pos (+)

Neg (-)

Strain O111 B BAA-2440D-5

Pos (+)

Neg (-)

Pos (+)

Neg (-)

Neg (-) Neg (-) Geotrichum Candidum M 74170 Pos

Geotrichum capitatum M 28575 Pos

Klebsiella pneumoniae B 700721D-

Pos (+)

Lactobacillus acidophilos B 4357

Neg (-)

Lactobacillus acidophilus B 4357D-5

Pos (+)

Neg (-)

Listeria

monocytogenes B 19115D-5

Pos (+)

Neg (-)

Mucor racemosus M 22365 Pos

Penicillium chrysogenum M 10106D-2 Pos

(+) Neg (-)

Neg (-)

Neg (-) Neg (-)

Penicillium citrinum M 36382 Pos (+)

Penicillium paxilli M 96516 Pos

Penicillum Citrinum M 36382 Pos (+)

Pseudomonas

aeruginosa B 9027D-5 Neg (-)

Pos (+)

Neg (-)

Pseudomonas

aeruginosa B 9027

Pos (+)

Pseudomonas

syringae pathovar tomato

B BAA-871D-5

Pos (+)

Neg (-)

Rhodotorula mucilaginosa Y 4557 Pos

Saccharomyces cerevisiae Y - Pos

Salmonella enterica serova

Typhimurium LT2 B 700720D-

5 Neg (-)

Pos (+)

Neg (-)

Pos (+)

Neg (-)

Neg (-) Neg (-) Salmonella

enterica subsp. B 700720

Neg (-)

Pos (+)

aureus Salmonella

enterica subsp. houtenae (IV)

B BAA-1580D-5

Neg (-)

Pos (+)

Neg (-)

enterica subsp. salamae (II)

B BAA-1582D-5

Neg (-)

Pos (+)

Neg (-)

enterica subsp. indica (VI)

B BAA-1578D-5

Neg (-)

Pos (+)

Neg (-)

Neg (-) Neg (-) Neg (-)

Staphylococcus aureus subsp.

Aureus B 6538D-5 Neg

(-) Pos (+)

Neg (-)

Staphylococcus aureus subsp.

aureus B 6538

Pos (+)

Neg (-)

Trichoderma virens M 13213 Pos (+)

Trichothecium

Roseum M 90473 Pos (+)

Vibrio cholerae B 39315D-5

Pos (+)

Neg (-)

Pos (+)

Yarrowia lipolytica M 18944 Pos

Yersinia pestis K25 B BAA-1511D-5

Pos (+)

Neg (-)

Pos (+)

Species Type ATCC# Total

Y&M TAC TC TE E.coli Sal STEC A.fla A.fum A.nig A.terr

Aeromonashydrophilia B 7965D Pos(+)

Pos(+)

AeromonashydrophiliaStrainCDC359-60 B 7966D-5 Pos

(+)Pos(+)

Pos(+)

Aspergillusaculeatus M 24147 Pos(+) Neg(-) Neg(-) Neg(-) Neg(-)Aspergillusbrasiliensis

WLRI034(120) M 16404D-2 Pos(+) Neg(-) Neg(-) Neg

(-) Neg(-) Neg(-) Neg(-) Neg(-)

Aspergilluscarbonarius M 1025 Pos(+) Neg(-) Neg(-) Neg(-) Neg(-)

Aspergillusflavus M 16870 Pos(+) Neg(-)

Neg(-) Pos(+) Neg(-) Neg(-) Neg(-)

AspergillusflavusSN3 M 9643D-2 Pos(+) Neg(-) Neg

(-)Neg(-) Pos(+) Neg(-) Neg(-) Neg(-)

Aspergillusfumigatus M 16903 Pos(+) Neg(-)

Neg(-) Neg(-) Pos(+) Neg(-) Neg(-)

Aspergillusfumigatus118 M 1022D-2 Pos(+) Neg

(-)Neg(-) Neg(-) Pos(+) Neg(-) Neg(-)

Aspergillusjanponicus M 16873 Pos(+) Neg(-) Neg(-) Neg(-) Neg(-)

Aspergillusniger M 15475 Pos(+) Neg(-)

Neg(-) Neg(-) Neg(-) Pos(+) Neg(-)

Aspergillusniger M 1015D-2 Pos(+) Neg(-)

Neg(-)

Neg(-) Neg

(-)Neg(-) Neg(-) Neg(-) Pos(+) Neg(-)

Aspergillusoryzae M 1010 Pos(+)

Aspergillusterreus M 16793 Pos(+) Neg(-)

Neg(-) Neg(-) Neg(-) Neg(-) Pos(+)

Aspergillusterreus M 20542D-2 Pos(+) Neg(-)

Neg(-)

Neg(-) Neg

(-)Neg(-) Neg(-) Neg(-) Neg(-) Pos(+)

Aspergillusustus M 1041 Pos(+) Neg(-) Neg(-) Neg(-) Neg(-)Bacillussubtilissubsp.

Spizizenii B 6633D-5 Neg(-)

Pos(+)

Neg(-)

Neg(-) Neg(-) Neg

Bacillussubtilissubsp.Spizizenii B 6633 Neg

(-)Neg(-)

Candidaalbicans Y 10231D-5 Pos(+) Neg(-) Neg(-) Neg

(-) Neg(-) Neg(-) Neg(-)

Candidaalbicans Y 10231 Pos(+) Neg(-)

Neg(-)

Candidacatenulata Y 10565 Pos(+) Candidaglabralta Y 15545 Pos(+)

Candidaguilliermondii Y 90197 Pos(+) Candidakrusei Y 28870 Pos(+)

Candidalusitiniae Y 344449 Pos(+) Candidasphaerica Y 8565 Pos(+)

Clostridiumsporogenes B 11437FD-5 Neg(-) Pos(+)

Neg(-)

Neg(-) Neg(-) Neg

Clostridiumsporogenes B 11437 Neg(-)

Neg(-)

Debaryomyceshanseii Y 10623 Pos(+) E.coliDH10BTMCompententCells B NEB Pos

(+)Pos(+)

Pos(+)

Enterobacteraerogenes B 15038D-5 Pos(+)

Pos(+)

Escherichiacoli B 8739D-5 Neg(-) Pos(+)

Pos(+)

Pos(+) Pos(+) Neg

(-)Neg(-)

Escherichiacoli B 8739 Pos(+) Neg(-)

EscherichiacoliStrain2000-3039 B BAA-2193D-

5 Neg(-)

Pos(+) Neg(-) Neg(-) Neg(-) Neg(-)

5 Neg(-)

EscherichiacoliStrainO111 B BAA-2440D-

5 Pos(+)

Pos(+)

Pos(+) Pos(+) Neg

(-)Pos(+) Neg(-) Neg(-) Neg(-) Neg(-)

GeotrichumCandidum M 74170 Pos(+) Geotrichumcapitatum M 28575 Pos(+)

Klebsiellapneumoniae B 700721D-5 Pos(+)

Pos(+)

Lactobacillusacidophilos B 4357 Neg

Lactobacillusacidophilus B 4357D-5 Pos

(+)Neg(-)

Neg(-)

Listeriamonocytogenes B 19115D-5 Pos(+)

Neg(-)

Mucorracemosus M 22365 Pos(+) Penicillium

chrysogenum M 10106D-2 Pos(+) Neg(-) Neg

(-) Neg(-) Neg(-) Neg(-)

Penicilliumcitrinum M 36382 Pos(+) Penicilliumpaxilli M 96516 Pos(+) PenicillumCitrinum M 36382 Pos(+)

Pseudomonasaeruginosa B 9027D-5 Neg(-) Pos

(+)Neg(-)

Neg(-) Neg(-) Neg

Pseudomonasaeruginosa B 9027 Pos

Pseudomonassyringaepathovartomato B BAA-871D-5 Pos

(+)Neg(-)

Neg(-) Neg(-) Neg

Rhodotorulamucilaginosa Y 4557 Pos(+)

Saccharomycescerevisiae Y - Pos(+)

SalmonellaentericaserovaTyphimurium

LT2B 700720D-5 Neg(-) Pos

(+)Pos(+)

Pos(+) Neg(-) Pos

(+)Neg(-) Neg(-) Neg(-) Neg(-)

Neg(-)

Salmonellaentericasubsp.aureus B 700720 Neg(-) Pos

Salmonellaentericasubsp.houtenae(IV) B BAA-1580D-

5 Neg(-) Pos(+)

Neg(-) Neg(-) Neg(-) Neg(-) Neg(-)

Salmonellaentericasubsp.salamae(II) B BAA-1582D-

5 Neg(-) Pos(+)

Salmonellaentericasubsp.indica(VI) B BAA-1578D-

5 Neg(-) Pos(+)

Staphylococcusaureussubsp.Aureus B 6538D-5 Neg(-) Pos

(+)Neg(-)

Neg(-) Neg(-) Neg

Staphylococcusaureussubsp.aureus B 6538 Pos

(+)Neg(-)

Neg(-)

Trichodermavirens M 13213 Pos(+) TrichotheciumRoseum M 90473 Pos(+)

Vibriocholerae B 39315D-5 Pos(+)

Neg(-)

Pos(+)

Yarrowialipolytica M 18944 Pos(+)

YersiniapestisK25 B BAA-1511D-5 Pos

(+)Neg(-)

Pos(+)

Table3Legend:B=bacteria,M=Mold,Y=yeast

Note:Ablankcellmeansthatthespecieswasnottestedwiththatassay

Afilledwellmeansthatthespecieswastestedwiththeassayandshowsresultsofthetest.

In-SilicoInclusionAnalysisSinceAspergillusspeciescanbedifficulttoobtainandcultureandthosethatcanbepurchasedfromATCCdonotalwayshavegenomesequencingavailable,wechosetoalsoperformanin-silicoanalysisofWholeGenomeShotgunassembliesinNCBI.FourdifferentWholeGenomeShotgunassembliesforAspergillusflavusweredownloadedfromNCBI.PathoSEEK®positivecontrolsequencewascomparedwithBLASTtofind100%identicalsequenceto3ofthestrains.ThesingleSNPfoundinoneofthegenomeassemblieswasNOTunderanyPathoSEEK®primerorprobesequencessuggestingall4ofthesesubspecieswillamplifywithourprimers.ThevariantstrainwasisolatedfrompeanutsinGeorgiabutwasadraftassemblyat26Xcoverageinover6,423contigs.Itisverylikelythatthisisasequencingerrorinthisreferenceduetolowcoverage.Othergenomesweresequencedover50Xandinlessthan900contigs.ThisanalysiswasrepeatedforAspergillusterreus,fumigatusandniger.OnlyoneAspergillusterreusgenomeassemblyshowedpolymorphismsinthepositivecontrolsequence(isolatedfromtheinternationalspacestationtoassessmicrogravitymutationrates)andNONEofthesepolymorphismswerefoundinPathoSEEK®primersandprobesequencesimplyingidenticalamplificationshouldoccur.

Table4. In-silico BLAST analysis of Whole Genome Shotgun datasets in NCBI. AsimilaranalysiswasperformedwithourSalmonellaPrimersandProbesequences.146SalmonellaSubspecieswereidentifiedwith100%identicalsequence.34areshowninthebelowtableforreference.Table5.in-silicoSalmonellaBLASTanalysisoftargetsequence

SampleHomogenization250mgto1gofhomogenizationisrequiredinmoststates.Homogenizationof250mg/1gofflowerin3.55ml/14.2mlTrypticSoyBroth(TSB)canbeperformedinaWhirlPakbag.Thesebagshavea300ummeshfilterinthemtoeliminatelargedebrisfromthehomogenizationprocess.Thisfilteristoolargetopreventtrichomes(50-150um)fromfilteringthroughandtheseoftenrequirecentrifugationtofullyeliminate.Lysisbuffershouldbeaddedbeforeanycentrifugationstepsorbacterialandmoldcellswillpelletwiththetrichomesandresidualplantdebris.Homogenizationtechniquesneedtobecarefullyscrutinized.Somehomogenizationprotocolsusedforcannabinoidandterpenoidtestingmaygrindtheplantmatterandmicrobesintoanunviablestateinmethanolorotherorganicsolvents.Liquidnitrogenorotherformsofaggressivehomogenizationcanaltertheviabilityofthemicrobesandpresentfalsenegativetestsforculturebasedtechniques.ExtremehomogenizationcanalsocreatefinepowdersthatobstructorsaturatevarioussolidphaseparticlesorsurfacesoftenusedforDNApurification.Filterporesizesneedtobescrutinizedtoensurehomogenizationdebrisisproperlyfilteredwhilebacterialandmoldcellsarenotomittedfromthesample.CommonlyusedphosphaterichbuffersandbrothsshouldbeavoidedforhomogenizationandgrowthifyourmicrobialquantificationmethodrequiresDNApurification.MostDNAisolationtechniquesusesolidphasesthatareattractedtothephosphodiesterbondinDNA.ManyoftheseDNAisolationtechniquesactuallyusephosphatestoelutetheirDNAfromthesolidphase.Asaresult,theuseofphosphaterichhomogenizationbufferscansaturatethedownstreamDNApreps’solidphasewithphosphatessuchthatlittletonoDNAiscaptured.InternalDNApurificationcontrolsareadvisedsoonecandifferentiateaDNApurificationfailurefromanamplificationfailure.Somekits(PathoSEEK®)targetampliconsuniquetothecannabisplantasaninternalpurificationcontrol.Ifthissignaldoesnotappear,thehomogenizationandDNApurificationshouldbescrutinizedandrepeated.

SamplingBiasWhetherusingcultureormolecularmethodssuchasPCR,neitherapproachaffordsassayingtheentire3.55mlor14.2mlhomogenate.UsuallyasmallsubsamplingofthistotalhomogenateisplatedorplacedintoqPCR.ThissubsamplingneedstobeaccountedfororsingleCFU/gmeasurementscannotbemaderegardingtheentirecannabissample.Forexample,ifonly1/10thoftheoriginalsampleisplatedorplacedintoPCRandtheoriginalsamplewasat1CFU/g,90%ofthetimeyoupipettethatsubsamplingyouwillgetnegativeresultsandonlyonetimewillyougetapositivehit.ForthisreasonmostsingleCFUteststhatsubsample,useanenrichmentstepwheretheyallowtheE.colitoincubateforasetnumberofdoublingtimestocompensateforthesubsampling.AssumingE.colidoubleseveryhalfhourat37CinstaticgrowthwithTSB,8doublings(2^8=256)willcompensatefor100foldsubsampling.Thisenrichmentstepwillalsoensurethatmolecularmethodsareonlymeasuringviablecells.Where

possible,growthratesshouldbemeasuredinthepresenceofacannabismatrixandthebrothselectedduetotheinhibitorynatureofcertaincannabismatrixcomponents(Figure6).PCREfficiencyandLimitofDetection(LOD)InordertoachieveconcordancewithCFU/goneneedstomeasurePCRefficiencyandthelimitsofdetection(LOD)acrossabroaddynamicrange.Toachievethis,2foldserialdilutionsacross12datadilutionpointsareperformedintriplicate.%RSD,PCRefficiencyandRsquaredcanbecalculatedfromthereplicateddilutions.AOACguidelinesshouldbefollowedforproper

concordancewithlimiteddynamicrangetechniqueslikeplating(http://multimedia.3m.com/mws/media/241188O/3m-petrifilm-plate-certificates-recognitions-validations.pdf).Table6.

Assay Copies of STEC O111 DNA Cq Value (FAM) %RSD E.coli Specific 5,000 26.23

0.50 E.coli Specific 5,000 26.00 E.coli Specific 5,000 26.01 E.coli Specific 2,500 26.68 0.16

Figure6-E.colidoublingrateinTSBWhirlpakbagswithcannabismatrixpresent.

Figure5-DiagramofsubsamplingforSenSATIVAxDNApurificationfromFlower.

E.coli Specific 2,500 26.63 E.coli Specific 2,500 26.72 E.coli Specific 1,250 27.69

0.28 E.coli Specific 1,250 27.66 E.coli Specific 1,250 27.81 E.coli Specific 625 28.55

0.32 E.coli Specific 625 28.73 E.coli Specific 625 28.63 E.coli Specific 313 29.68

2.26 E.coli Specific 2 36.50 E.coli Specific 2 37.74 E.coli Specific 0 Not Detected

Not Applicable

E.coli Specific 0 Not Detected E.coli Specific 0 Not Detected

Figure 7: E.coli qPCR Dilution Curves and qPCR Efficiency (E)

CorrelationoftheseresultswithplatinglivespecieswillresultinanequationthatenablesconversionofCq/gtoCFU/g.Eachassayshouldhaveitsownequationandtheequationmaydifferbasedontheuseofextractversusflowerduetotheimpactofthematrixasacarbonsourceforgrowthonplates.ThebroaderspectrumassayslikeTACandTYMwillhavelowerconcordancethanthemorespecificassayssince95-99%ofmicrobesdon'tcultureandmanymoldsdon'tformcolonies116.Thusthebroaderthescopeofthetest,thehigherlikelihoodunculturableorganismswilltriggerqPCRsignalsandthehigherlikelihoodsomeorganismswillgrowthatdonotPCRwithITSor16Sprimersequences.Whiletheunculturableorganismswillneverbeknownwithplating,DNAsequencingcanprovidealistofthousandsoforganismsyourprimersareknowncapture.ThiscannotbeunderscoredenoughaseachorganismorderedfromATCCforspikeinassessmentoncannabisis$300makingtheacquisitionofalistofspikeinorganismsthatcancultureinthepresentofcannabismatrixveryexpensive.Asaresultthereiscurrentlynopeerreviewedpublishedvalidationdataregardingwhatorganismsderivedfromcannabiscangrowonplatesor3MPetrifilm®.TherearemultiplestudiesusingPCRandDNAsequencingtoacquirethisinformationwithandwithoutculture.SincemanyregulatorsarelookingforstrongcorrelationswithCq->CFU,theonlywaytoachievethisistodesignprimersthatdonotamplifyunculturablemolds.Commonlyfoundnon-culturablebutharmlessmoldslikeBotrytiscinereaandCannabisderivedpowderymildew(CDPM)canbedesignedtonotamplifywithcertainprimersets.Itisrecommendedthatspeciesspecificassaysexistforthesecommonmoldsintheeventregulatorybodiesdecidetheirallergenicpotentialneedstobemonitoredinthefuture.Table 7, Cq to CFU/g Equations for Flower, MIP/Extract and Gummy

Matrix Microbial Test Cq to CFU/g Conversion Equation Plant Total Yeast and Mold CFU/g = 10[(36.671 – Cq Value)/3.1194] Plant Total Aerobic Count CFU/g = 10[(35.111 – Cq Value)/2.8883] Plant Total Coliform CFU/g = 10[(40.073 – Cq Value)/3.3417] Plant Total Enterobacteriaceae CFU/g = 10[(41.218 – Cq Value)/4.3708]

MIP/Extract Total Yeast and Mold CFU/g = 10[(54.972 – Cq Value)/5.8485] MIP/Extract Total Aerobic Count CFU/g = 10[(38.076 – Cq Value)/3.2249] MIP/Extract Total Coliform CFU/g = 10[(41.935 – Cq Value)/3.6274]

MIP/Extract Total Enterobacteriaceae CFU/g = 10[(38.407 – Cq Value)/3.3041]

Gummy Total Yeast and Mold CFU/g = 10[(52.989 – Cq Value)/4.9718] Gummy Total Aerobic Count CFU/g = 10[(37.235 – Cq Value)/2.356] Gummy Total Coliform CFU/g = 10[(52.888 – Cq Value)/5.9643] Gummy Total Enterobacteriaceae CFU/g = 10[(44.81 – Cq Value)/4.9665]

OnewillnoticesignificantlyalteredCq->CFUconversionswithvariousExtractsorMIPs.TobetterunderstandthisweperformedspikeinplatingswithvariousMIPmatricestobetterunderstandtheimpactofthesecarbonsourcesonculturingconditions. MarijuanaInfusedProducts(MIP)interferenceandimpactonvalidationMIPsareaverydiverseclassofmatricesthatbehaveverydifferentlythancannabisflowers.Gummybears,chocolates,oilsandtincturesallpresentdifferentchallengestoculturebasedtechniquesasthesugarsandcarbohydratescanradicallyalterthecarbonsourcesavailableforgrowth.SomeoillovingmicrobeslikeClostridiumbotulinumhavebeenfoundincannabisandtheserequireanaerobicconditionstoculture.ThesevaryingmatricesalsopresentchallengestohomogenizationandDNApurificationtechniques.Toaddressthis,itisimportanttounderstandthevariousinhibitorstoyourplatingandPCRtechnologies.3MColiformplateslistcitrateasaninhibitortotheirreporterassay.CitrateiscommonlyusedincannabiscultivationintheformofCal-MagorCalciumandMagnesiumcitrate.Citricacidiscommonlyusedasafoliarspraytothwartoffplantpathogens.Likewise,certainterpenoidshavebeenlistedaspolymeraseinhibitorssuggestingdirectPCRwithoutDNApurificationmaypresentvariableresultswithdifferentcannabischemotypes117.Thelistofpotentialadditives,chemotypesandtheirimpactoncultureisanearinfinitelist.AnethanolbasedDNApurificationstepsimplifiesthisinhibitorycomplexitygreatly.ToassesstheimpactofMIPsonCFU/genumeration,wespikedinliveE.colicellsintovariousMIPstomeasuretheqPCRsignalandcomparedthesetothecolonycountswithandwithoutMIPs(Figure8).TooursurpriseE.colicellsspikedintomanyoftheMIPsfailedtogrowdespitegrowthintheTSBcontrols.ThisimpliestheMIPsareinterferingwiththereporterassayonthefilmsorthattheMIPsareantisepticinnature.ManyMIPsusecitricacidasasourflavoringingredientandcancauseinterferencewith3Mreporterchemistry.qPCRsignalwasconstantimplyingthereisDNAonthefilmsbutthecolonyformationorreportingisinhibited.Thisunderscorestheimportanceof

Figure8-MIPinhibitionofPetrifilm®

revalidationwithnovelmatrices.Morecanbefoundonthistopicathttps://www.medicinalgenomics.com/mips-and-extracts-negative-impact-on-plating/. PrecisionPrecisioncontainstwosub-tasks:repeatabilityandrobustness.Repeatabilityisdefinedastheprecisionunderthesameoperatingconditions(intra-assayprecision).Robustnessisdefinedasthedegreeofprecisionoftestresultsobtainedbytheanalysisofthesamesamplesunderavarietyoftypicaltestconditions,suchasdifferentdaysandanalysts.Withthisinmind,itwasnecessaryforustodisplaytherepeatabilityandrobustnessoftheSenSATIVAx™Flower/LeafDNAExtractionKitandtheSenSATIVAx™MIP/ExtractDNAExtractionKitandthePathoSEEK®qPCRassays.PrecisionofAspergillusspecies-specificandmultiplexassaysTheAspergillusmultiplexassaycombinestheAspergillusA.flavus,A.fumigatus,A.nigerandA.terreusdetectionassaystogetherinonereaction.ThedetectionprobeforeachspeciesisontheFAMchannel;therefore,ifapositiveresultweretooccur,theonlywaytodeterminewhichspecieswaspresentwouldbetorunallfourindividualAspergillusassays(presumablyonmanyfewersamplesasAspergillusfailureratesareusuallyunder3%(personalcommunicationwithCannabistestinglaboratories).LiveAspergillusflavus,fumigatus,nigerandterreusorganismsweregrowntosaturationinTSB.Thespecies-specificassayvs.multiplexassaywastestedbyadding100μLofthespecies-specificcultureinto3.45mLofTSBwith0.25gofflowerpresent.Themultiplexassaywasalsotestedbyadding100μLofeachspeciescultureto3.15mLofTSBwith0.25gofflowerpresent.Foreachmatrixtested,anon-spikedcontrolwasincludedtoshowthattheproductbeingtestedwascleanandtheqPCRsignalwasfromthespikedliveorganism.ThiswasfollowedbyDNAextractionusingtheSenSATIVAx™Flower/LeafDNAExtractionKit.TheextractedDNAwastestedusingthePathoSEEK™Aspergillusflavus,A.fumigatus,A.niger,A.terreusandorAspergillusmultiplexassayinreplicatesofsixforthesinglespeciesspike-insandintriplicateforthemultiplespeciesspike.ThedatapresentedinTable26showsthatAspergillusflavus,A.fumigatus,A.nigerandA.terreuscanbedetectedatsimilarCtvalueswheneithertheAspergillusspecies-specificassayortheAspergillusmultiplexassayisused.ThetablealsopresentstherepeatabilityoftheextractedDNAwhentestedusingtheAspergillusflavus,A.fumigatus,A.niger,A.terreusandorAspergillusmultiplexassays. Table 8, Precision of Aspergillus Assays

Sample Organism Spiked qPCR Assay Cq %RSD Flower Aspergillus flavus Aspergillus flavus 25.20

Flower Aspergillus flavus Aspergillus flavus 25.06 Flower Aspergillus flavus Aspergillus flavus 24.94 Flower Aspergillus flavus Aspergillus flavus 24.88 Flower Aspergillus flavus Aspergillus flavus 24.76 Flower Aspergillus flavus Aspergillus flavus 25.19 Flower Aspergillus flavus Aspergillus Multiplex 24.57

Flower Aspergillus flavus Aspergillus Multiplex 24.40 Flower Aspergillus flavus Aspergillus Multiplex 24.47 Flower Aspergillus flavus Aspergillus Multiplex 24.68 Flower Aspergillus flavus Aspergillus Multiplex 25.03 Flower Aspergillus flavus Aspergillus Multiplex 24.58 Flower A. flavus, fumigatus, niger, terreus Aspergillus flavus 25.43 1.91

Flower A. flavus, fumigatus, niger, terreus Aspergillus flavus 24.48 Flower A. flavus, fumigatus, niger, terreus Aspergillus flavus 25.01 Flower None Aspergillus flavus Not Detected

Not Applicable Flower None Aspergillus flavus Not Detected Flower None Aspergillus flavus Not Detected Flower Aspergillus fumigatus Aspergillus fumigatus 18.60

Flower Aspergillus fumigatus Aspergillus fumigatus 18.60 Flower Aspergillus fumigatus Aspergillus fumigatus 18.72 Flower Aspergillus fumigatus Aspergillus fumigatus 18.47 Flower Aspergillus fumigatus Aspergillus fumigatus 18.53 Flower Aspergillus fumigatus Aspergillus fumigatus 18.90 Flower Aspergillus fumigatus Aspergillus Multiplex 20.36

Flower Aspergillus fumigatus Aspergillus Multiplex 21.04 Flower Aspergillus fumigatus Aspergillus Multiplex 20.32 Flower Aspergillus fumigatus Aspergillus Multiplex 19.59 Flower Aspergillus fumigatus Aspergillus Multiplex 18.82 Flower Aspergillus fumigatus Aspergillus Multiplex 20.39 Flower A. flavus, fumigatus, niger, terreus Aspergillus fumigatus 21.55

0.15 Flower A. flavus, fumigatus, niger, terreus Aspergillus fumigatus 21.54 Flower A. flavus, fumigatus, niger, terreus Aspergillus fumigatus 21.49 Flower None Aspergillus fumigatus Not Detected

Not Applicable Flower None Aspergillus fumigatus Not Detected Flower None Aspergillus fumigatus Not Detected Flower Aspergillus niger Aspergillus niger 26.66

Flower Aspergillus niger Aspergillus niger 26.56 Flower Aspergillus niger Aspergillus niger 26.47 Flower Aspergillus niger Aspergillus niger 26.65 Flower Aspergillus niger Aspergillus niger 26.52 Flower Aspergillus niger Aspergillus niger 26.52 Flower Aspergillus niger Aspergillus Multiplex 26.97

Flower Aspergillus niger Aspergillus Multiplex 26.92 Flower Aspergillus niger Aspergillus Multiplex 26.95 Flower Aspergillus niger Aspergillus Multiplex 27.15 Flower Aspergillus niger Aspergillus Multiplex 26.98 Flower Aspergillus niger Aspergillus Multiplex 27.09 Flower A. flavus, fumigatus, niger, terreus Aspergillus niger 32.06

0.53 Flower A. flavus, fumigatus, niger, terreus Aspergillus niger 32.14 Flower A. flavus, fumigatus, niger, terreus Aspergillus niger 32.39 Flower None Aspergillus niger Not Detected

Not Applicable Flower None Aspergillus niger Not Detected Flower None Aspergillus niger Not Detected Flower Aspergillus terreus Aspergillus terreus 25.43

Flower Aspergillus terreus Aspergillus terreus 25.35 Flower Aspergillus terreus Aspergillus terreus 24.88 Flower Aspergillus terreus Aspergillus terreus 24.91 Flower Aspergillus terreus Aspergillus terreus 24.88 Flower Aspergillus terreus Aspergillus terreus 25.07 Flower Aspergillus terreus Aspergillus Multiplex 27.34

Flower Aspergillus terreus Aspergillus Multiplex 25.03 Flower Aspergillus terreus Aspergillus Multiplex 25.21 Flower Aspergillus terreus Aspergillus Multiplex 25.01 Flower Aspergillus terreus Aspergillus Multiplex 25.26 Flower Aspergillus terreus Aspergillus Multiplex 26.30 Flower A. flavus, fumigatus, niger, terreus Aspergillus terreus 26.62

0.94 Flower A. flavus, fumigatus, niger, terreus Aspergillus terreus 26.81 Flower A. flavus, fumigatus, niger, terreus Aspergillus terreus 27.12

Flower None Aspergillus terreus Not Detected Not Applicable Flower None Aspergillus terreus Not Detected

Flower None Aspergillus terreus Not Detected Flower A. flavus, fumigatus, niger, terreus Aspergillus Multiplex 24.04

5.73 Flower A. flavus, fumigatus, niger, terreus Aspergillus Multiplex 23.68 Flower A. flavus, fumigatus, niger, terreus Aspergillus Multiplex 21.59

RobustnessRobustnessshouldmeasuredaytodayvarianceandlaboperatortolaboperatorvarianceforbothFlowerandMIPsandpreferablyitemizehomogenizationandDNApurificationrelatedvariancesfromqPCRvariancewithspikeincontrols.AfewexamplesareprovidedinTable9,10,11. Table 9, Sampling Repeatability:

Sample qPCR Assay Cq Extract Sample 2 Total Coliform 21.07 Extract Sample 2 Total Coliform 20.93 Extract Sample 2 Total Coliform 20.80 Extract Sample 2 Total Coliform 20.99 Extract Sample 2 Total Coliform 21.07

Cq Percent RSD 0.534 Table 10, Sample Robustness:

Sample & Well qPCR Assay Cq Extract Sample 1 Total Coliform 20.58 Extract Sample 2 Total Coliform 20.47 Extract Sample 3 Total Coliform 20.56 Extract Sample 4 Total Coliform 20.62 Extract Sample 5 Total Coliform 21.05 Extract Sample 6 Total Coliform 20.81

Cq Percent RSD 1.034 Table 11, User-to-User Robustness:

Sample User qPCR Assay Cq Extract 1 User 1 Total Yeast & Mold 23.95 Extract 1 User 2 Total Yeast & Mold 22.14 Extract 1 User 3 Total Yeast & Mold 21.90 Extract 2 User 1 Total Yeast & Mold 24.45 Extract 2 User 2 Total Yeast & Mold 24.59 Extract 2 User 3 Total Yeast & Mold 25.21 Extract 3 User 1 Total Yeast & Mold 32.13 Extract 3 User 2 Total Yeast & Mold 32.18 Extract 3 User 3 Total Yeast & Mold 33.70

Extract 1 (3 users) Cq Percent RSD 4.94

Extract 2 (3 users)

Cq Percent RSD 1.63 Extract 3 (3 users)

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