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GenØk-CentreforBiosafety,Tromsø,NorwayDecember2017

PrevalenceofAntibioticResistanceMarkerGenes(ARMG)inSelectedEnvironmentsinNorway–Reindeer

Projectmanager:LiseNordgård,ResearchleaderandScientist,GenØk

Contributors:ElisabethOlsen,LabEngineer,GenØk

IdaSofieFuruholmen,StaffScientist,GenØkProf.MortenTryland,SeniorAdvisor,GenØkProf.KaareM.Nielsen,SeniorAdvisor,GenØk

Suggestedcitation:Nordgård,L.etal(2017)PrevalenceofAntibioticResistanceMarkerGenes(ARMG)inSelectedEnvironmentsinNorway–Reindeer.Projectreport,M-909|2017,GenØk,Tromsø,Norway.

GenØk-Senterforbiosikkerhet,SivaInnovasjonssenter,P.B.6418,9294Tromsø,Norway,Tel.:+4777646620–post@genok.no–www.genok.no

Frontpagephoto:adobestock.com/DmitryChulov

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ThisprojectwascommissionedbytheNorwegianEnvironmentAgencyDevelopment of bacterial resistance to antibiotics is a growing problem in theworld. ThenationalstrategyoftheNorwegiangovernmentagainstantibioticresistancefor2015-2020highlights that this issuemust be considered in a holistic perspective, where human andanimalhealthandtheenvironmentinteractandmustbeseenincontexttoeachother.Thepresenceofresistantbacteriaindifferentnaturalenvironments,suchassoil,freshwater,seasedimentsandwildanimals,hasonlybeensporadicallystudied,althoughtheymaycontributetothedevelopmentofresistanceofclinicalimportance.Thereisthereforeaneedformoreknowledgeaboutantibiotic-resistantbacteriaindifferentnaturalenvironmentsingeneralandinNorway inparticular.Thenational strategy isbasedon the report “Antibiotikaresistens-kunnskapshullogaktuelletiltak(2014)”preparedbyanexpertgroup.Inthisreport,theneedtoassesstheriskofapossibledeliberatereleaseofgeneticallymodifiedorganisms(GMOs)withantibioticresistancemarkergenes(ARMG)isidentifiedasoneoftheareaswheremoreinformationisnecessary.GenØk–CentreforBiosafety(www.genok.no)isanindependentresearchinstitutefoundedin1998andlocatedinTromsø,Norway.GenØkisengagedinthefieldofbiosafetyandgeneecology research on modern biotechnology, synthetic biology and other technologiesemergingfromthese.Thisinstitutionalsoworksoncapacitybuildingandadvisoryactivitiesrelatedtobiosafety.GenØktakesaprecautionary,holisticandinterdisciplinaryapproachtobiosafety. In 2007, GenØk was appointed national competence center on biosafety byNorwegianauthoritiesAcknowledgementWethankBeritTømmeråsforassistanceinthelabandKatrineJaklinforeditorialwork.

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ContentSummary.................................................................................................................................................5

Norsksammendrag.................................................................................................................................5

Abbreviations/descriptions.................................................................................................................6

Background.............................................................................................................................................7

Methods..................................................................................................................................................9

Results...................................................................................................................................................11

Discussion..............................................................................................................................................12

Limitations.............................................................................................................................................14

Conclusions...........................................................................................................................................15

Appendix...............................................................................................................................................15

TotalDNAisolationfromreindeerstoolsamples.........................................................................16

DNAisolationfrombacterialcontrolstrains.................................................................................16

PCRamplificationoftheDNAextractioncontrol16SrRNA..........................................................16

PCRamplificationofnptIIandnptIIIgenesintheKmRisolatesandenvironmentalsamples......17

Controlsusedinthisstudy............................................................................................................18

References.............................................................................................................................................19

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SummaryThis projectwas commissioned by theNorwegian Environment Agency, and grantedwith0,175mill.NOK,, in order to map the naturally occurring prevalence of two antimicrobialresistance marker genes (ARMG), coding for kanamycin resistance, used in plant genetechnologyinNorwegianwildanimals.Thetwogenesare;neomycinphosphotransferaseII(NptII/aph(3`)-IIa)andneomycinphosphotransferaseIII(NptIII/aph(3`)IIIa(1,2).Faecalsamplesfromreindeersfromrespectivelythreedifferentgrazingdistrictshavebeenanalysed.Thesampleswerecollectedoveraperiodofthreeyears(2013-2015).Thereasonforthechoiceofthereindeer-grazingdistrictisbasedonassumeddifferencesinexposureofdriversforantimicrobialresistance.Thetotalnumberofsamplesfromeachdistrictperyear,rangedfrom7-17samples.Intotal76sampleswereanalysedforthepresenceofARMG.Fromthe76 samplesanalyzed,noneof the samples testedpositive fornptII and three samplestestedpositivefornptIII.Theresultsindicatethatthenaturaloccurrence/backgroundofnptIIandnptIIIgenesintheselectedsamplesarenotprevalentduringthetestingperiod.

NorsksammendragDetteprosjektetbleinitiertpåoppdragavMiljødirektoratet,ogbevilget0,175millNOK,foråkartlegge den naturlige forekomsten av to antibiotikaresistensmarkørgener (ARMG),neomycin phosphortansferase II (nptII/aph(3`)-IIa) og neomycin phosphotransferase III(nptIII/aph(3`)IIIa),,hosviltlevendearterinorsknatur.Idetteprosjektetharvistudertfeces-prøverfrareinsdyritreulikereinbeitedistrikter.Prøveneblehentetinnienperiodeovertreår(2013-2015).Bakgrunnenforvalgavreinbeitedistrikterbasertpåantattforskjellerieksponeringavhumanaktivitetogdrivereforresistensutvikling.Dettotaleantallprøverfrahvertdistriktperårvariertefra7-17prøver.Totaltbledetanalysert76prøverforforekomstavARMG.Avdetotalt76prøvenesombleanalyservaringenpositivefor nptII og kun tre prøver testet positiv for nptIII. Resultatene viser at den naturligeforekomsten/bakgrunnen avnptII ognptIII gener i de utvalgtemiljøene er lav eller underdeteksjonsgrenseniperiodenviharhentetprøver.

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Abbreviations/descriptions AMR AntimicrobialresistanceARB AntimicrobialresistantbacteriaARG AntimicrobialresistancegeneARMG Antimicrobialresistancemarkergenebp BasepairDNA Deoxyribonucleicacidg GramGM GeneticallymodifiedGMO GeneticallymodifiedorganismHGT HorizontalgenetransferKmR Kanamycinresistanceml Millilitreng NanogramNORM/VET Nasjonaltovervåkningssystemforantibiotikaresistenshosmikroberfra

fôr,dyrognæringsmidlerNptII(Aph(3’)-IIa NeomycinphosphotransferaseIIgeneNptIII(Aph(3’)-IIIa NeomycinphosphotransferaseIIIgenePCR PolymerasechainreactionrRNA RibosomalribonucleicacidWWTP Wastewatertreatmentplantµl Microlitre

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BackgroundOverthe lastdecade, increasedresistanceagainstantibiotics (AB)amongbacteria inmanydifferent ecological niches like soil, wastewater treatment plants (WWTPs), river water,drinkingwater,seawater,sedimentsetc.hasemerged(3-11).Inthesedifferentenvironments,antibiotics, antimicrobial resistant bacteria (ARB) and antimicrobial resistant genes (ARGs)haveroutinelybeenobserved.Theenvironmentcanbeseenasameltingpointforantimicrobialresistance,andthereasonsfor this are complex. First of all, it is important to remember that the occurrence ofantimicrobial resistance in the environment may also result from the production ofantimicrobialsbycertaingroupsofmicroorganismsindifferentecologicalniches(12-14).Inaddition, the increaseduseofantibiotics ingeneral combinedwithexposure toadditionalresistancepromotingsubstances, suchasdisinfectants,biocidesandsomeheavymetals issignificant(6,15,16).Antimicrobial resistance (AMR) is usually studied in bacteria from humans and domesticanimals.However,forfurtherinsightintothedisseminationandoccurrenceofAMRwildlifehas been suggested as a good source of information. It is well known that antimicrobialresistantbacteriaareabletospreadfromcontaminatedsourceslikeWWTPs,farmhandlingandotherhumansourcestootherecologicalnichesandprobablyalsowildanimals(17,18).Wildanimalsarenotnaturallyexposedtoantimicrobials,butcaninteractwithhumanwasteandinfrastructureinurbanareasandfunctionasareservoirofARBandARGaswellasprovidedisseminationoverlongdistances(19).ThepresenceofARMGsingeneticallymodified(GM)plantsandpossiblelarge-scalereleaseintheenvironment,ortheuseasGMplantsinfoodorfeed,hasraisedconcernsoverthepastyearsregardingpossiblerisksforhumanhealthandtheenvironment(20-22).NptII,oneofthemost abundantly used ARMG in plant gene technology, encodes the neomycinphosphotransferaseenzyme,thatconfersresistancetokanamycin,neomycinandgeneticin.Kanamycin ispresentlynot licensed for the treatmentof infectiousdiseases inhumansoranimalsinNorway,mainlybecauseofadversesideeffects.Thedevelopmentofresistancetootherantibioticsmayhowevermakeitmoreimportantinthefuture(23).NptIIIisamongthemostprevalentaminoglycosidephosphotransferasesinGram-positivebacteriaandthegenealsogivesresistancetoamikacin,licidomycinandisepamicinwhicharemoreclinicallyrelevantthankanamycin(24).BothnptII andnptIII arepresent ina rangeof synthetic vectorsand innaturallyoccurringplasmidsandtransposons,andmaybehorizontallytransferredbetweendifferentbacterialcellsandstrains(1,25-29).OneofthemainconcernsregardingtheuseofARMGsinGMplants,istheriskofhorizontalgenetransfer(HGT)ofplant-derivedARMgenestosoilorgutbacteria

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resultinginareducedantimicrobialtreatmentofanimalandhumaninfectiousdiseases.Thisriskhasbeenclaimedtobeverylow,butcannotbeexcluded(30,31).Althoughaneventmaybe rare, itmay have an ecological impact if the transferred gene alters the fitness of therecipientbacteriaorcell(32).Inthiscontext,theneedtomonitorthepresenceandlevelofantibioticsandARMGsindifferentecologicalnicheshasbeenidentifiedasoneoftheareaswheremoreinformationisnecessary.NorwayisoneofthemostrestrictivecountrieswithregardtoimportanduseofGMOs,andnoneoftheseplantsaregrowninNorwaytoday.Thismightbechangedinthefuture,andtobeabletoevaluatethesignificanceofanyreleaseofGMOscontainingARMGsitisimportanttomapandmonitorthealreadyexistingbackgroundlevelofthespecificARMGsinmicroorganismsindifferentNorwegianenvironments.Theaimof this studywas todescribe thenaturalbackground loadofantibiotic resistancemarkergenes(ARMGs)infecalsamplesfromreindeersgrazinginthreedifferentNorwegianareaswithdifferentexposureofhumanactivities.Reindeersliveinthenorthernpartsoftheworld,arehighlymigratoryandtravelinlargeherds.Theyareeatingmostlygrass-likeplantsand shrubs in summer,and lichen,whichcarpets the snow-covered forests, in thewinter.Depending on their grazing area, some reindeers may interact with human waste andinfrastructurewhileothersarenotexposedtohumanactivityatall.Inthisstudysamplesfromthree different reindeer-grazingdistricts,with assumed differences in exposure of humanactivity and drivers for antimicrobial resistance, have been analysed. Themethodologicalapproach fordetectionofARMGs in this study isbasedonpreviousprojectspublishedbyNordgårdetal.(22,33).

Photo: adobestock.com/ belov3097. Reindeer used in the tourism industry in Tromsø.

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MethodsThreedifferentgrazingareasinNorwaywerechosenwiththeintentiontoestablishabaselinefrequencyofnptIIandnptIIIinfecessamplesfromreindeers.Thedifferentareas,indicatedinfigure1,wereselectedbasedonestimateddifferentdegreesofexposurefromhumanactivityandresistantdriversubstances.Area1:Tønsvik (Tromsdalen/Mauken,Tromsø) isconsideredashighlyexposedtohumanactivity.Partsoftheherdthatgrazehereareinvolvedinatouristenterprisewithfrequentinteractionswithpeoplefromallovertheworldinthewinterseason.Moreover,thereisanindustry and offshore harbor located in old Grøtsund fort in Tønsvik. We therefore alsoassumethatresistancedriversmaybepresentintheenvironment.Area2:Røros(Riast-Hylling)isconsideredasanareasubjecttosomehumancontact,butlessthaninarea1.Area3:Varangerhalvøya(Rakkonjarga)isalargesub-arctictolow-arcticmountainterritorywithverylittleornohumanactivity.Allthreeareasselectedinthisstudyarealreadyestablishedsamplingareasforvariousstudiesofreindeerswithintheproject“"Infeksjonssjukdommerhosrein ikystnærebeiteområder"(UiT/Reindriftensutviklingsfond;2013-2016) ledbyProfessorMortenTryland (UiT). In thisproject, there was a joint sampling and data call for the various subprojects at alreadyestablished monitoring stations for climate research. Agreements for collection of thesamplingmaterialweremadewithreindeerherdersinthevariousdistricts.Thenumberofsamplesanalyzedarelistedintable1.Polymerasechainreaction(PCR)wasusedfordetectionofthespecificresistancegenes.Themethodsaredescribedintheappendix.Table1:Numberofsamplesincludedinthisstudy 2015 2014 2013Tønsvika 17 7 -Røros 17 - -Tana 9 12 14

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Figure1:Illustratesthesamplessitesinred.Thedifferentcoloursofthemapindicatespopulationdensity.Tana,thenorthernmostsamplingsiteislocatedinanareawithlowpopulationdensity,andRørosandTønsvikahaveintermediateandhighpopulationdensities,respectively.Source:StatisticsNorway."Folkemengdeogbefolkningsendringar",CreativeCommonsAttribution-ShareAlike4.0Internationallicense

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ResultsEachoftheindividualsampleswereanalysedinduplicatesbyPCR,forthepresenceofnptIIandnptIII.TwodifferentprimersetswereusedforbothnptIIandnptIII(Table2).Table2:PCRbaseddetectionofnptIIandnptIIIgenesindifferentenvironmentalsamplesDistrict Numberof

samplestested(n=76)

16Spositive(571bp)(n=75)

NptIIshortpositive(129bp)(n=0)

NptIIfullpositive(795bp)(n=0)

NptIIIshortpositive(82bp)(n=1)

NptIIIfullpositive(523bp)(n=2)

Tana2015 9 8/9 0/9 0/9 0/9 1/9Tana2014 12 12/12 0/12 0/12 0/12 0/12Tana2013 15 14/14 0/14 0/14 0/14 0/14 Røros2015 17 17/17 0/17 0/17 0/17 0/17 Tønsvik2015 17 17/17 0/17 0/17 1/17 1/17Tønsvik2014 7 7/7 0/7 0/7 0/7 0/7

Photo: adobestock.com/Juhku

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DiscussionTo provide information on kanamycin resistance in the total bacterial assemblage in fecalsamplesfromreindeersgrazinginthreedifferentpartsofNorway,totalDNAwasextractedandexaminedbyPCRfortheprevalenceofnptIIandnptIII.Theresultsindicatesthatthesespecificantibioticresistancemarkergenes(ARMG)arenotprevalentinthesamplestestedinthisstudy.The16sRNAgenewasusedasanamplificationinhibitioncontrolinthisscreeningstudy.Allsamples,exceptone,testedpositivefor16sRNAbyPCR.TheNptIIgenewasnotdetectedinanyof thesamples tested.Thedetection limitofagivenmolecularmethod is rarely zero,whichmeansthatthegenewasnotdetectedorthatitwasunderdetectionlimitofthespecificmethod.Fromthe76samplesanalyzed,threesamples(3,9%)testedpositiveforthepresenceofnptIIIgene,twofromTønsvikaandonefromTana.ThesamplesthatarenptIIIpositivewillbesubmittedforfull-lengthsequencingtogather informationofresistancegenesequencevariation,inafollowupstudy.A review of the scientific literature retrieves only few studies that have investigated theprevalence of nptII and nptIII genes in total DNA isolated from different environmentalsamples(21,22,24,34-36).Thesestudiesshowonlyavery lowprevalenceofthesegenesfrom total DNA isolated from non-clinical environments like soil, riverwater, sewage andmanure,whichsupportsourfindings.Inaddition,theresultsaresupportedbythefactthatnoneofoursampleareasareaffectedbyGMplantcultivationorrelease.TheprevalenceofthesespecificmarkergenesintotalDNAisolatedfromfecalsamplesfromwildanimalshasnotbeendescribedintheliterature,whichmakesthecomparisonofourresultswithothersimilarstudiesdifficult.Phenotypic antimicrobial resistance to different classes of antibiotics in faecal bacterialcommunitiesofdifferentwildanimals(birds,mammalsandothers)andindifferentcountriesisdescribedintheliterature(17,19,37-41).Amongthedifferenttypesofantibioticstestedfor,kanamycinresistancehasalsobeenreported.Inastudyfrom2005(41),alowpercentageof antimicrobial resistance in faecal enterococci from 77 wild animals in Portugal wasdemonstrated.Inanotherstudy,faecalbacterialisolatesfromwildwesternlowlandgorillasinGabon,antimicrobialresistanceagainst12differentantibioticswasobserved,andamongthemalsokanamycinresistance(38).Highlevelofkanamycinresistancewerealsoobservedinapproximately14%ofE.coli isolatestestedfromwildbirdsspecies inPoland(37).Thesekanamycin resistant isolates harboured the nptIII gene. A study from Spain by Guerrero-Ramos, a low percentage of high level resistance (HLR) to kanamycin was observed inenterococcispeciestestedfromwildgamemeetinSpain.Allenterococcal isolatesshowinghighlevelkanamycinresistancecontainedthenptIIIgene(19).Highlevelresistance(HLR)tokanamycinwiththepresenceofthenptIIIgene,wasalsoobserved inenterococcal species

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from wild animals in Spain (39). Some recent publication have also reported multidrug-resistant bacteria in wild birds and mammals from remote areas with no exposure ofantibiotics (42,43),whichmay indicate thatAMR isawiderenvironmental issueofpublichealthconcern.Inmanyofthestudiesofwildanimals,thesamplesizesaresmall.However,the prevalence of AMR in environmental samples indicates the need to monitor theantimicrobial resistance in wildlife and environments that are not directly exposed toantibioticorantimicrobialresistancegenes.Onlyafewstudieshavebeenconductedrelatedtoantimicrobialresistanceinfecalsamplesfromreindeers.InNorwayajointNORM/NORM-VETreportisissuedannuallywiththefocustopresentdataontheoccurrenceofantimicrobialresistanceandtheusageofantimicrobialagentsinhumansandanimalsforeveryyear.Sincetheprevalenceofantimicrobialresistanceamong certain bacteria of the normal entericmicroflora can serve as an indicator for theselective antimicrobial pressure in various populations, studies onE.coli strains from fecalsamplesalsofromreindeershasbeenconducted.TheNORM/NORM-VETreportfrom2003(44),moderateoccurrenceofantimicrobialresistanceamongE.colifromfecalsamplesfromwild reindeerswas reported. In another study conducted in 2012, 107E. coli isolateswasisolated from faecal samples from reindeer. The results indicated that the prevalence ofresistantE.coliinreindeersislow(45).E.colihasalsobeenisolatedfrom42wildreindeerbyLillehaugetalin2005(46).Theresultsinthisstudy,demonstratedthatten(24%)ofthereindeerisolateswereresistanttooneormoreoftheantibioticstestedfor.Ingeneral,theantibioticresistancepatternfoundinthisstudyindicatedlowlevelsofresistantstrainsamongtheE.coliisolatestested.InastudybyRamstadetal(47),E.colistrainsfromreindeers,livinginremoteenvironmentsinNorwayandSvalbardwascharacterizedforantimicrobialresistance.Themainconclusionfromthisstudywasthatknownresistancegenesweredetected.Somestrainsalsohadseveralresistancegenescloselyarrangedintheirgenomeandsomehadresistancegenesarrangedinconjunctionwithrepliconsthatmayindicateplasmidlocation.Inaddition,resistanceregionswithhighhomologytoplasmidregionspreviouslydescribedinbacteriafromhumansand/oranimalswereidentified,inseveralstrains.TheprevalenceofkanamycinresistanceandnptIIgenesintheE.coliisolateshas,however,notbeeninvestigatedinthesestudies.Ingeneral,comparisontopreviousresultshasbeendifficultinthisreport,sincewecouldnotfindanyotherstudiesintheliteraturethathasinvestigatedtheprevalenceofARMGinwildanimals. This studywas designed tomeet the requirements from the assignment text todescribe theprevalenceARMGs in reindeers.Cultivablebacteriawerenot included in thisstudyduethenaturalmovementofthereindeerinthewinterseasonandthelimitedprojectperiod.Byonlyusingonemethodologicalapproach,therearecertainlylimitations.Cultivationof bacteria from reindeer faeces on selective media and further screening of kanamycinresistantcoloniesforthespecificARMGs,willbeincludedinfollowupstudies.However,thePCRresultsoftotalDNAextractedfromfecalsamplesalsoincludesthebacteriathatweare

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notable tocultivate.This illustrates thateachmethodandapproachhas itsstrengthsandweaknesses,andtheimportanceofcombiningdifferentmethods.

LimitationsThere are many limitations/uncertainties related to how to assess ARMGs in naturalenvironments. Many of these uncertainties are due to technical limitations, datainterpretationandthefactthatthisstudyonlyrepresenta“snapshot”oftheenvironmenttested.Inthisstudy,someofthelimitationsarealsoduetothelimitedprojectperiodandcaneasilybefollowedup.Samplingandsamplesize: samples frommorethanthreereindeergrazingareascouldbeincludedtoincreasethenumberofsamples.Isolation of total DNA for PCR: may need optimization. Low yields and issues withamplificationsuggestthepresenceofinhibitorswhichmayhavereducedtheprobabilityofdetectingthespecificgenesinoursamples.Positive resistance-gene-specific amplicons has not yet been confirmed by furthersequencing due to limited project period, but will be included in follow up studies. Thesequencinganalysiswillgiveinformationofresistancegenesequencevariabilityinthesewildlifesamples.QuantitativePCR:couldbeincludedtodeterminetheabundancesofthespecificARMGsCultivationbasedmethods:Cultivablebacteriawasnotincludedinthisstudyduethenaturalmovementofthereindeerinthewinterseasonandthelimitedprojectperiod.Thiswillbeincludedinfollowupstudies.

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ConclusionsThisstudysuggeststhatthenaturallyoccurringbackgroundlevelofnptIIandnptIIIgenesinreindeersfromthreedifferentareasinNorwayisloworbelowthedetectionlimit.Fromthe76samplesanalysed,noneofthesamplestestedpositiveforthenptIIgene,andthreeofthesamples,twofromTønsvikaandonefromTana,testedpositiveforthepresenceofthenptIIIgene.Giventhelowlevelsofthesespecificgenesitcannotbeexcludedthattheintroductionofnew/externalsourcesofsuchgeneswillnotincreasetheprevalencelevelovertime.WiththelimitednumberofexperimentalstudiesavailabletoresolvetheuncertaintiesarisingfromintroductionofARMGs, there are still significant knowledge gaps, aswell as uncertaintiesregardingARMGsinnaturalenvironments.FurtherinformationonthenaturaloccurrenceofnptII/nptIIIindifferentrelevantNorwegianenvironmentsisnecessarytobeabletoprovidequantitativeassessmentofthepossibleriskof introducing ARMGs into Norwegian environments. In addition,more experimental andepidemiologicaldataareneededonthedistributionofARMGsandARGsingeneral,inwildlife.

Photo: adobestock.com/Sam

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Appendix

TotalDNAisolationfromreindeerstoolsamplesDuplicatesof0.2gfrozensampleswasusedtoisolatethetotalDNAfromreindeerfaeces.DNAfromthealiquotswasextractedusingtheQIAmpDNAstoolkitforpathogendetection(Qiagen), according to the manufacturer’s instructions. In addition, DNeasy Power CleanClenupkitfromQiagen,wasusedaccordingtothemanufacturers’protocol.Quantity and quality of the purified DNA were determined using a NanoDrop™ 2000spectrophotometer(ThermoFisherScientific,Wilmington,DE,USA).TheelutedDNAextractswerestoredat-20°Cuntilfurtheranalysis.50/100ngoftheresultingDNAsolutionservedastemplateforthenptII/nptIIIscreeningaswellasforthe16SrRNAPCRassay.

DNAisolationfrombacterialcontrolstrainsDifferentAcinetobacterbaylyi(A.baylyi)isolatesservedascontrolsinthisstudy(seetable4).The bacterial isolates were extracted using Quickextracttm DNA Extraction Solution 1.0(Epicentre Biotechnologies) according to the manufacturer`s instructions, and theconcentrationandqualitywereassessedusingNanoDrop.TheDNAextractswerestoredat-20°Cuntilfurtheranalysis.Oneµlofa50ng/µldilutionoftheresultingDNAsolutionservedastemplateforthenptII/nptIIIscreeningaswellasforthe16SrRNAPCRassay.

PCRamplificationoftheDNAextractioncontrol16SrRNATheelutedDNAfromenvironmentalsamplesandbacterialcoloniesservedastemplateforthe bacterial 16S rRNA gene to confirm the general absence of PCR inhibitors and thesuccessfulisolationofbacterialDNA.Thereactionswereperformedinatotalvolumeof20µlcontainingthefollowing:1µlofeachprimer(SigmaAldrich)at10µMconcentration,10µlmastermix (DreamTaqPCRMastermix,ThermoFisher),5µlwaterand50/100ng templateDNA.Maximum3µltemplateDNAwasaddedtoeachreactionandthevolumeineachtubewasadjustedwithddH2Ouptoafinalvolumeof20µl.Positiveandnegativecontrols(listedintable4)wereincludedineachPCRset-up.ThePCRconditionswereasfollows:1cycleofinitialdenaturationat95˚Cfor2min,30cyclesofdenaturationat95˚Cfor30sandannealingat60˚Cfor30sandelongationat72˚Cfor40s,onecycleoffinalelongationat72˚Cfor5min.PrimersandcontrolsarelistedinTable3and4.ThePCRproductswererunon1%SeaKem®LEAgaroseSeaKem agarose-gels (Lonza) stained with Gelred (Invirtogen). Quantitative DNA Markerɸx174 BsuRI (HaeIII) Marker 9 (Thermo Sscientific) was used to show the proteins sizes.VisualizationwasperformedusingGelDocTMXR170-8170 (BioRad)withQuantityOne1-Danalysissoftwareversion4.5.2.

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PCRamplificationofnptIIandnptIIIgenesintheKmRisolatesandenvironmentalsamplesTo determine if nptII and/or nptIII genes were present in the total DNA from theenvironmental samples and the kanamycin resistant bacterial colonies, two primer pairs,amplifyingoneshortandonelongfragmentofDNA,wereusedforbothnptIIandnptIII(Table3).Thereactionswereperformedinatotalvolumeof20µlcontainingthefollowing:1µlofeachprimerat10µMconcentration,10µlmastermix (DreamTaqPCRMastermix,ThermoFisher),5µlwaterand50/100ngtemplateDNA.Maximum3µltemplateDNAwasaddedtoeachreaction,andthefinalvolumewasadjustedupto20µlwithddH2O.PrimersandcontrolsarelistedinTable3and4,respectively.For theamplificationofnptII (longandshort)andnptIII short, thePCRconditionswereasfollows:1cycleofinitialdenaturationat95˚Cfor2min;30cyclesofdenaturationat95˚Cfor30 s, annealing at 60 ˚C for 30s and elongation at 72 ˚C for 40 s; and one cycle of finalelongationat72˚Cfor5min.FornptIIIlongthefollowingpcrprogrammewasused:1cycleofinitialdenaturationfor3minat94˚C;35cyclesofdenaturingat94˚Cfor40s,annealingat55˚Cfor40sandelongationat72˚Cfor40sandafinalextensionstepat72˚Cfor2min.ThelongPCRproductswererunon1%SeaKem®LEAgarose-gels (Lonza)stainedwithGelred,while the shorter fragmentswere run on 2% agarose gels. Theɸx174 bsuri ladder fromThermoscientificwasused.VisualizationwasperformedusingGelDocwithQuantityOnesoftware.Table3.PCRprimers(Eurogentech)Target Name Size Primersequence(5ʹ-3ʹdirection) Reference16SrRNA 16S_F16S_R 571bp TGGAGAGTTTGATCMTGGCTCAG

CTTTACGCCCARTRAWTCC(Woegerbaueretal.2014)

nptIIshort NptII_FNptII_R

129BP GATCTCCTGTCATCTCACCTTGCTTCGCTCGATGCGATGTTTC

(Woegerbaueretal.2014)

nptIIlong NptII:Full_FNptII:Full_R

795bp ATGATTGAACAAGATGGATTGCTCAGAAGAACTCGTCAAGAAGG

(Woegerbaueretal2014)

nptIIIshort NptIII_FNptIII_R

82bp ACATATCGGATTGTCCCTATACGAATCGGCCAGATCGTTATTCAGTA

(Woegerbaueretal.2014)

nptIIIlong NptIII_Full_FNptIII_Full_R

523bp GGCTAAAATGAGAATATCACCGGCTTTAAAAAATCATACAGCTCGCG

(Vakulenkoetal.2003)

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Controlsusedinthisstudy Table 4. Bacterial strains used as controls in this study Strain Relevance Control

A.baylyiADP1200Com+(1200)

Km(S) Negativecontrol (48)

A.baylyiADP1200Com+KmR Km(R),nptII Positivecontrol (48)

A.baylyiBD413JV28-KmR Km(R),nptII Positivecontrol (49)

A.baylyiADP1200Com+Km+ Km(R),nptIII Positivecontrol (48)

Water DNAse/RNAsefreewater(Sigma)

Negativecontrol -

*allourbacterialstrainscontrolswerekindlyprovidedfromtheDepartmentofPharmacy,UiTTheArcticUniversityofNorway

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