Aldrichwww.ccc64.com

RESEARCHOpenAccessTheadultnasopharyngealmicrobiomeasadeterminantofpneumococcalacquisitionAmeliekeJHCremers1,AldertLZomer1,2*,JennaFGritzfeld3,GerbenFerwerda1,SachaAFTvanHijum2,4,DanielaMFerreira3,JoshuaRShak5,KeithPKlugman5,JosBoekhorst2,4,HarroMTimmerman4,MarienIdeJonge1,StephenBGordon3andPeterWMHermans1,6AbstractBackground:SeveralcohortstudieshaveindicatedassociationsbetweenS.
pneumoniaeandothermicrobesinthenasopharynx.
Tostudycausalrelationshipsbetweenthenasopharyngealmicrobiomeandpneumococcalcarriage,weemployedanexperimentalhumanpneumococcalcarriagemodel.
Healthyadultvolunteerswereassessedforpneumococcalcarriagebycultureofnasalwashsamples(NWS).
Thosewithoutnaturalpneumococcalcarriagereceivedanintranasalpneumococcalinoculationwithserotype6Bor23F.
Thecompositionofthenasopharyngealmicrobiomewaslongitudinallystudiedby16SrDNApyrosequencingonNWScollectedbeforeandafterchallenge.
Results:Among40selectedvolunteers,10werenaturalcarriersand30wereexperimentallychallenged.
Atbaseline,fivedistinctnasopharyngealmicrobiomeprofileswereidentified.
Thephylogeneticdistancebetweenmicrobiomesofnaturalpneumococcalcarrierswasparticularlylargecomparedtonon-carriers.
Amorediversemicrobiomepriortoinoculationwasassociatedwiththeestablishmentofpneumococcalcarriage.
Perturbationofmicrobiomediversityuponpneumococcalchallengewasstrainspecific.
Shiftsinmicrobiomeprofileoccurredafterpneumococcalexposure,andthosevolunteerswhoacquiredcarriagemoreoftendivertedfromtheiroriginalprofile.
S.
pneumoniaewaslittleprominentinthemicrobiomeofpneumococcalcarriers.
Conclusion:Pneumococcalacquisitioninhealthyadultsismorelikelytooccurinadiversemicrobiomeandappearstopromotemicrobialheterogeneity.
Keywords:Experimentalhumanmodel,Microbiome,Nasopharyngealcarriage,StreptococcuspneumoniaeBackgroundStreptococcuspneumoniae(thepneumococcus)isoneoftheorganismsthatcommensallyresideinthehumannasopharynx.
Recently,anexperimentalhumanpneumo-coccalcarriagemodelwasestablished[1]whichenablesthecontrolledstudyofpneumococcalcarriageepisodesinthehumannasopharyngealniche.
Anepisodeofpneumo-coccalcarriageisusuallyabeneficialimmunogenicevent[2],butitisalsothepotentialinitiationofinvasivepneumococcalinfections[3].
Pneumococcalcarriageismostprevalentinchildrenunder5,rangingfrom10%upto90%dependingonspecificagegroupandsetting[4,5],andisgenerallylessprevalentinadults[6-12].
Theinci-denceofinvasivepneumococcaldisease(IPD)is10–20/100,000/yearindevelopedcountries[13-16]andisesti-matedtobeevenhigherindevelopingcountries[17],withthoseunder5andover50yearsoldmostatrisk.
Annu-ally,pneumoniacausesanestimated1.
3millionchildhooddeathsworldwide[18].
TheburdenofIPDmaybere-ducedbylimitingthedegreeofpneumococcalcarriage.
Insightintothedeterminantsofpneumococcalcarriagewillprovidebetterunderstandingofhowinterferencewiththisphenomenoncouldinfluenceitsprevalence.
Knowndeterminantsofpneumococcalcarriagearehosttraitsre-latedtoexposureandimmunity[4,19,20],antibioticuse,andpneumococcalvaccination[12].
Anadditionalelem-entthatappearstobeassociatedwithpneumococcalcar-riageinchildrenistheendogenousmicrobiotapresentin*Correspondence:aldert.
zomer@radboudumc.
nlEqualcontributors1LaboratoryofPediatricInfectiousDiseases,DepartmentofPediatrics,Radboudumc,6500HBNijmegen,TheNetherlands2CentreforMolecularandBiomolecularInformatics(CMBI)BacterialGenomics,Radboudumc,6500HBNijmegen,TheNetherlandsFulllistofauthorinformationisavailableattheendofthearticle2014Cremersetal.
;licenseeBioMedCentral.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/4.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycredited.
TheCreativeCommonsPublicDomainDedicationwaiver(http://creativecommons.
org/publicdomain/zero/1.
0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
Cremersetal.
Microbiome2014,2:44http://www.
microbiomejournal.
com/content/2/1/44thenasopharyngealcavity.
Forinstance,ithasbeenreportedthatinchildren,nasopharyngealcarriageofS.
pneumoniaeisinverselycorrelatedwithcarriageofS.
aureus[21-24].
Furthermore,investigationofthepediatricnasopharyngealnichebya16SrDNAsequencingapproachhasrevealedassociationsbetweenpneumococcalcarriageandspecificbacterialgenera[25].
However,asthesestudiesarecross-sectional,itisunknownhowtheen-dogenousnasopharyngealmicrobiotamodulatestheestab-lishmentofpneumococcalcarriageandwhatchangesarebroughtaboutuponexposuretopneumococci.
Inthisstudy,wecomparedthecompositionofthenasopharyn-gealmicrobiomewithandwithoutnaturallyacquiredpneumococcalcarriageinhealthyadults.
Inaddition,auniqueexperimentalhumanpneumococcalcarriagemodelwasemployedtostudywhetherspecificcompositionsofthemicrobiotaareassociatedwithsubsequentestablish-mentofpneumococcalcarriageandwhethertheadultnasopharyngealmicrobiomeisperturbedbyexposuretoS.
pneumoniae.
ResultsSelectedvolunteersandqualityofsamplesAmongthe40volunteersselectedformicrobiomeanalysis,10werenaturalcarriers.
Theremaining30non-carrierswereexperimentallychallenged.
Pneumococcalcarriageasdetectedbyculturewasestablishedin14ofthe26selectedvolunteerswhowereinoculatedwithserotype6B,andin2ofthe4challengedwithserotype23F.
Fromthese40volunteers,117sampleswereeligibleformicrobiomedataanalysis(Additionalfile1:TableS1).
Thecomposi-tionsoftheindividual117adultnasopharyngealmicro-biomesaredisplayedinAdditionalfile2:FigureS1.
Themeasuredreadcount,richness,anddiversitypersamplewerenotinfluencedbythe16SrDNAconcentrationintheextractedDNAsamples(Additionalfile3:FigureS2).
DistinctmetagenomicprofilesexistintheadultnasopharyngealmicrobiomeThegeneramostfrequentlydetectedintheadultnaso-pharyngealmicrobiomeatbaselinewereCorynebacter-ium,Dolosigranulum,Staphylococcus,andStreptococcus(Additionalfile4:TableS2).
Startingfromthesemostabundantgenera,usinghierarchicalclusteringandprin-cipalcomponentanalysis(PCA)onmicrobialdensitiesof155operationaltaxonomicunits(OTUs)presentintheindividualadultnasopharyngealcommunities,fivedis-tinctmicrobiomeprofileswereidentifiedandappointedasprofileAtoE(Figure1).
CorrespondingPCAplotsandprincipalcoordinateanalysis(PCoA)plotsaredisplayedinAdditionalfile5:FigureS3.
Therandomforestanalysisshowedalow(~15%)overalloutofbagerrorbetweentheclassassignmentsofthesamples,supportingthecluster-inganalysis.
Inaddition,thePearsondistanceswithinprofileswerelowercomparedwiththedistancebetweensamplesfromdifferentprofiles(median(IQR):0.
25(0.
07–0.
45)and0.
79(0.
51–0.
95),respectively,p30%inadults)andadecreasedabundanceofProteobacteria(64%inchildren,<15%inadults)[31,32].
Whereaspneumococ-calcarriageinchildrenhaspreviouslybeenassociatedwithahighlevelofStreptococciinthenasopharynxandalowdiverseconcurrentmicrobiome[25,33],inouradultvolun-teers,naturalpneumococcalcarriagewasassociatedwithlowproportionsofstreptococcalreadsnexttonormallydiverseandphylogeneticallyheterogeneousmicrobiomecompositions.
Thismayindicatethat,incontrasttochil-dren,inadults,thepneumococcusgenerallydoesnotFigure2Distributionofnaturalpneumococcalcarriersoverbaselinemicrobiomeprofiles.
**:p<0.
01.
Cremersetal.
Microbiome2014,2:44Page4of10http://www.
microbiomejournal.
com/content/2/1/44predominateinthenasopharyngealmicrobialcommunityuponacquisition.
Weobservedthatamorediversemicrobiomepriortochallengewasassociatedwiththeestablishmentofpneumo-coccalcarriagewhencombiningthedatafromboth23F-and6B-inoculatedvolunteers.
Wehavenotedthattheremaybestrain-specificeffectsonthemicro-biomecompositionfollowingsuccessfulcolonization,andtherefore,wecannotexcludestrain-specificmicro-biomediversityrequirementsforsuccessfulcolonization.
However,thedecreaseinpvaluewhilecombiningthe23Fand6Bdatasuggeststhatthisisnotthecase,mak-ingpoolingavalidapproach.
Althoughinotherhost-associatedmicrobialcommunitiesamorediversemicrobiotawasfoundtobeassociatedwithresistancetopathogens,wedidnotobservethisphenomenoninourstudy.
Here,amorediversenasopharyngealmicro-biomeappearstofacilitatepneumococcalcarriage.
AsFigure4Microbiomediversitypriortoinoculationinvolunteerswithandwithoutestablishmentofpneumococcalcarriage.
Microbiomerichness(A)diversity(Shannonindex)(B)andPDwholetree,(C).
*:p<0.
05.
Figure3Within-groupphylogeneticdistanceofbaselinemicrobiomesinvolunteerswithorwithoutnaturalpneumococcalcarriage.
UnweightedUniFrac(A)andweightedUniFrac(B)p<0.
0001.
Cremersetal.
Microbiome2014,2:44Page5of10http://www.
microbiomejournal.
com/content/2/1/44pneumococcalcarriageisproposedtobeanimmunizingevent[2],wespeculatethatshort-termlow-levelpneumo-coccalcarriageactuallypromoteshealthasthiswillboostprotectiveimmunityagainstthepneumococcus.
NoneofthevolunteerswiththeStaphylococcus-dominatedmicrobiomeprofileCacquiredpneumococcalcarriage,andnoneofthepneumococcalcarriersturnedtoprofileC.
ThisobservationisinlinewiththefrequentlydescribedinversecorrelationbetweencarriageofS.
pneumoniaeandS.
aureusinbothchildrenandadults[11,21-24],althoughwecannotexcludethatadifferentStaphylococcusspeciesisresponsiblefortheobservedphenomenoninourcase.
Afterpneumo-coccalchallenge,weobservedcontinuousshiftsinmicro-biomeprofiles,althoughvolunteerswithoutpneumococcalacquisitionmoreoftenreturnedtotheiroriginalpro-file.
StabilityintheupperrespiratorytractmicrobiomehasbeennotedbeforebyCharlsonetal.
,wherethenaso-pharyngealcommunitycompositionwaslessrobustoverFigure5Perturbationsinthenasopharyngealmicrobiomeafterpneumococcalchallenge.
Microbiomerichness(A)anddiversity(B)anditsphylogeneticdivergencefromthemicrobiomepriortochallenge(C).
6Binoculatedwithserotype6B,23Finoculatedwithserotype23F;*:p<0.
05.
Cremersetal.
Microbiome2014,2:44Page6of10http://www.
microbiomejournal.
com/content/2/1/44timecomparedtothemicrobiomeintheoropharynx,butremainedrelativelystable[34].
Furthermore,whereaschallengewithserotype6Bwasnotassociatedwithspe-cificchangesinmicrobiomediversity,challengewithsero-type23Fwas.
Asthe23F-inoculatedvolunteerswereprimarilystaphylococcalcarriers,thismayhaveinfluencedthechangesobserved.
However,theincreasedvariabilityafterchallengewith23Fwasnotobservedinothervolun-teerswithStaphylococcus-dominatedprofileswhowerechallengedwith6B.
Therefore,wesuggestthatperturba-tionsinmicrobiomediversityafterpneumococcalexpos-uremaybestraindependent.
ConclusionsInhealthyadults,thenasopharyngealmicrobiomecanbesubdividedinatleastfivebacterialcommunityclasseshavingatleastfourdistinctprofiles,withprofilesB–EdominatedbyStreptococcusspp.
,Staphylococcusspp.
,Corynebacteriumspp.
,oracombinationofCorynebac-teriumspp.
andDolosigranulum,whereasprofileAismorediverse.
ThereisevidenceofaninversecorrelationbetweencarriageofS.
pneumoniaeandS.
aureus.
Inourstudy,amorediversemicrobiomewasassociatedwiththeestablishmentofpneumococcalcarriage.
Further-more,S.
pneumoniaewasgenerallylittleprominentincarriersanditsacquisitionappearstopromotemicrobialheterogeneity.
Whetherpneumococcalcarriageinhealthyadultsisaprosperouseventbesidesonerelatedtodiseaseremainstobeelucidated.
MethodsExperimentalstudymodelWeperformedtheexperimentalhumanpneumococcalcarriagemodelaspreviouslydescribed[1].
Inshort,healthyadultvolunteerswererecruitedintheUnitedKingdomaccordingtoinclusionandexclusioncriteriadescribed[1].
Atbaseline,wecollectedanasalwashsample(NWS)andassessedpneumococcalcarriagebycultureoftheNWSspecimen.
Ofthe40volunteersin-cludedformicrobiomeanalysis,10werenaturalcarriers.
Thosevolunteerswhowerenotnaturalpneumococcalcarrierswereinoculatedwitheitheraserotype6Bstrain(BHN418[35])or23Fstrain(P833[36])at60,000-to320,000-colony-forming-unit(CFU)S.
pneumoniaeasthesegenerallyhavealowinvasivenessrateandhavebeensuccessfullyusedinpreviouscarriagestudies[1,35].
Follow-upNWSwerecollectedat2,7,and14daysafterchallenge.
Acquisitionofpneumococcalcarriagewiththeinoculatedstrainwasdeterminedbycultureandconfirmedbylatexagglutinationserotypingofrecoveredstrain.
Thisstudyhasbeenconductedac-cordingtotheDeclarationofHelsinkiprinciples,andethicalapprovalwasobtainedfromtheNationalHealthServiceResearchEthicsCommittee,Sefton,Liverpool(11/NW/0592).
Writteninformedconsentwasreceivedfromallvolunteerspriortoinclusioninthestudy.
BacterialDNAextractionDirectlyafterNWScollection,wemixed2mlofthesamplewith4mlRNAprotectBacteriaReagent(cat.
no.
76506,Qiagen,Venlo,TheNetherlands)toprecipitateandprotectnucleicacidsinthesample.
After5minofincubationatroomtemperature(RT),thesamplewasstoredat80°Cuntilfurtheruse.
Thethawed6-mlsus-pensionwaspelletedina2-mltubebythreecentrifuga-tionsteps,eachfor20minat13,200rpmat4°Cinamicrocentrifuge(cat.
no.
5415R,Eppendorf,Hamburg,Germany).
Tothepellet,weadded0.
3mllysisbufferwithprotease(AgowaMagminiDNAextractionkit,cat.
no.
NAP40401,LGCGenomics,Berlin,Germany),50mgsterilizedzirconia/silicabeads(diameter0.
1mm,cat.
no.
11079101z,BioSpecProducts,Bartlesville,OK,USA),and0.
3mlphenol(PhenolBioUltra,cat.
no.
77607,Sigma-Aldrich,St.
Louis,MO,USA).
Thesamplewasmechanic-allydisruptedbybeadbeatinginaTissueLyserLT(cat.
no.
85600,Qiagen,Venlo,TheNetherlands)fortwotimes2minat50Hz,coolingthesampleoniceaftereachstep.
Wecentrifugedthehomogenatefor10minat10,000rpmatRTandtransferredtheaqueousphasetoa1.
5-mltube.
Afteradditionofabindingbuffer(twicetheaqueousphasevolume)and10μlofmagneticbeads,thesamplewasincubatedfor30minatRTinamixingmachine.
Wewashedthemagneticbeadswith200μlofeachwashbuf-fer1and2andelutedtheDNAwith63μlelutionbufferaccordingtothemanufacturer'sinstructions.
BacterialDNAquantificationandsampleselectionWedeterminedthebacterialDNAconcentrationineacheluatebyqPCRonthe16SrDNAgene.
Theprimerandprobesequenceswereasfollows:forwardprimer5′-CGAAAGCGTGGGGAGCAAA-3′;reverseprimer5′-GTTCGTACTCCCCAGGCGG-3′;probe5′-(FAM)-ATTAGATACCCTGGTAGTCCA-(MGB)-3′aspreviouslypublishedbyBogaertetal.
[32].
The25-μlPCRmixwas1*TaqManUniversalPCRMasterMix,10μMofeachpri-mer(1μl),5μMprobe(1μl),6.
5μlDNA-freewater,and3μltemplateDNA.
ThermalcyclingwasperformedinaABI7500FastReal-TimePCRSystem(cat.
no.
4351107,LifeTechnologies,Carlsbad,CA,USA),withthefollowingcyclingconditions:2min50°C,10min95°C,and50cyclesof15sat95°Cand1minat65°C.
The16SrDNAstandardcurveconsistedofa10-folddilutionseriesofamixofgen-omicDNAextractedfromthreebacteriacommontotherespiratorytract:Streptococcuspneumoniae(TIGR4),Moraxellacatarrhalis(RH4),andHaemophilusinfluenzae(1521062).
WeextractedgenomicDNAwiththeQiagenGenomic-tip20/GKit(cat.
no.
10223,Qiagen,Venlo,TheNetherlands)andquantifieditbyaspectrophotometerCremersetal.
Microbiome2014,2:44Page7of10http://www.
microbiomejournal.
com/content/2/1/44(NanoDropND-1000,ThermoFisherScientific,Wilmington,DE,USA).
ThosevolunteerswhoseextractedDNAsam-plesallcontainedatleast1pgbacterialDNA/μl[37]wereconsideredeligibleformicrobiomeanalysis.
Toavoidfalse-positiveresults,bothDNAextractionandamplifica-tionprocedureswereaccompaniedbynegativecontrols.
16SrDNApyrosequencingandhandlingofDNAsequencesWeamplifiedtheV3-V6regionofthe16SrDNAgenewithforwardprimer5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGNNNNNNACTCCTACGGGAGGCAGCAG-3′(italicizedsequence:454LifeSciencesprimerA;boldsequence:broadlyconservedbacterialprimer338F;NNNNNN:thesample-specificsix-basebarcodeusedtotageachPCRproduct)andreversepri-mer5′-CCTATCCCCTGTGTGCCTTGGCAGTCTCAGCRRCACGAGCTGACGAC-3′(italicizedse-quence:454LifeSciencesprimerB;boldsequence:broadlyconservedbacterialprimer1061R).
WepurifiedtheampliconsfromthePCRproductusingtwokitscon-secutivelyaccordingtotheirmanufacturer'sinstructions:theMSBSpinPCRapaceKit(cat.
no.
1020220400,Isogen,DeMeern,TheNetherlands)andthePureLinkQuickPCRPurificationKit(cat.
no.
K310002,LifeTechnologies,Bleiswijk,TheNetherlands)usingbindingbuffer3Bandanelutionvolumeof40μl.
Acompositesampleforpyro-sequencingwaspreparedbypooling100ngpurifiedPCRproductfromeachsample.
Fiftymicrolitersoftheampli-conlibrary(concentration14.
5ng/μl)wassubmittedforpyrosequencingonthe454LifeSciencesGS-FLX+platformusingTitaniumsequencingchemistry(bothRoche,Germany)atGATCBiotech,Konstanz,Germany.
Onehundredseventeensampleswereanalyzed(Additionalfile1:TableS1).
WeanalyzedthepyrosequencingdatawithaworkflowbasedonQIIMEv1.
2[38],usingsettingsasrecom-mendedintheQIIME1.
2tutorial,withthefollowingex-ceptions:readswerefilteredforchimericsequencesusingChimeraSlayer[39],andOTUclusteringwasper-formedwithsettingsasrecommendedintheQIIMEnewsletter2010[40].
TheRDPclassifierversion2.
2wasperformedfortaxonomicclassification[41].
Sampleswithreadcountsbelow500wereexcludedfromfurtheranalysis.
Sequencedataandsubjectcharacteristicsareavailableathttp://www.
cmbi.
ru.
nl/bamics/supplementary/cremers_2014_ehpc/index.
htm.
Diversityestimates,correlation,andclusteringanalysisTocorrectfordifferencesinreadcountwhilecalculatingdiversityestimates,individualsampledataweredown-sampledtothelowestreadcountincludedinthestudy.
Wemeasuredalphadiversitywithinsamplesbyrichness(numberofOTUs)andtwodiversityestimates:theShannonindexthatincreaseswithOTUnumberandwithequalityofOTUabundances,andthePDwholetreewhichaccentuatesphylogeneticallydistantOTUs.
WemeasuredbetadiversitybetweensamplesbytheUniFracdistancethatestimatesthefractionofasample'sphylogenetictreethatdiffersfromanothersample,with(weighted)orwithout(unweighted)emphasisonthemostabundantOTUs.
Startingfromthemostabundantgenerapresentinthenasopharynxatbaseline,westud-iedthepresenceofmicrobiomeprofilesusinghierarch-icalclusteringbyPearsoncorrelationsandPCAonmicrobialdensityexpressedasthepercentageofreadsinasamplethatisassignedtoaspecificOTU,whereanOTUshouldaccountforatleast1%ofthereadsinonesample.
PCAonOTUabundancedataandPCoAonweightedandunweightedUniFracdistanceswereper-formedinRusingprcompandlabDSV[42],respectively,withdefaultsettings.
Randomforestanalysiswithmicro-biomeprofilesasclassesandthemicrobialdensitydataasclassifierswasperformedusingtherandomForestpackageinBioconductor[43].
Inaddition,wecomparedPearsondistanceswithinprofilestothosebetweenprofiles.
Themicrobialcommunitiesobservedinsamplescollectedafterinoculationwereallocatedtoamicrobiomeprofileusingrandomforestanalysishandlingmicrobialdensitydataasdescribedabove.
CorrelationanalysisbetweenOTUswasperformedbypairwiseSpearman'scorrelations.
StatisticalanalysesDifferencesindichotomousvariables(pneumococcalcarriage)werestatisticallytestedbyaFisher'sexacttest.
Normalityofthedistributionofcontinuousvari-ableswastestedbyShapiro-Wilktest.
Differencesinmicrobiomecharacteristicsbynormallydistributedvariables(Shannonindex,PDwholetree)werestatisti-callytestedbyanunpairedt-testfortwoindependentgroupsoraone-wayanalysisofvariance(ANOVA)formultipleindependentgroupsandbyapairedt-testorrepeated-measuresone-wayANOVAforperturbationsinthemicrobiomeovertime.
Differencesinnotnormallydistributedvariables(numberofOTUs,Pearsondistance,UniFrac)werestatisticallytestedbyaMann-WhitneyUtestfortwoindependentgroupsoraKruskal-Wallistestformultipleindependentgroups.
Totestperturbationsinthemicrobiomeovertime,aWilcoxonmatched-pairssignedranktestorFriedmantestwasapplied.
Forallana-lyses,thesignificancelevelwassetat0.
05.
MultipletestingcorrectionwasperformedbytheBenjamini-Hochbergfalsediscoveryrateprocedure[44].
AvailabilityofsupportingdataSequencedataandsubjectcharacteristicsareavail-ableathttp://www.
cmbi.
ru.
nl/bamics/supplementary/cremers_2014_ehpc/index.
htm.
Cremersetal.
Microbiome2014,2:44Page8of10http://www.
microbiomejournal.
com/content/2/1/44AdditionalfilesAdditionalfile1:TableS1.
SamplesselectedformicrobiomeanalysisandtheirbacterialDNAconcentrationsby16SrDNAqPCR(pg/μl).
Additionalfile2:FigureS1.
Microbiomecompositionofthe117individualnasopharyngealsamplesrepresentedatgenuslevel.
Positivepneumococcalcultureresultsaredisplayedontheleft(green:naturalcarrier,red:carriageofinoculationstrain)andtheirindividualdiversitymetricsontheright.
Additionalfile3:FigureS2.
Correlationbetween16SrDNAquantityandreadcount,richness,anddiversity.
The16SrDNAconcentrationintheextractedDNAsampleshasnotinfluencedthesequencingyieldintermsofthenumberofreads(p=0.
074)(panelA),thenumberofOTUs(p=0.
64)(panelB),ortheShannonindex(p=0.
14)(panelC)persample.
Additionalfile4:TableS2.
Mostprevalentbacterialgenerapresentintheadultnasopharynxatbaselinebynaturalcarriagestatus.
Additionalfile5:FigureS3.
PCAandPCoAplotsoftheindividualnasopharyngealmicrobialcommunitiesatbaseline.
PCAprincipalcomponentanalysis,PCoAprincipalcoordinateanalysis.
Individualvolunteersaredisplayedasdotscoloredaccordingtomicrobiomeprofile,intwo-dimensionalgraphswithcombinationsofthethreePCAcomponentsontheaxes(panelA).
PCoAwasperformedonunweightedandweightedUniFracdistances(panelB).
Additionalfile6:FigureS4.
ThepercentageofreadsfromeachOTUthatdifferentiatesbetweenthemicrobiomeprofilesperNWS.
NWSnasalwashsample.
Additionalfile7:FigureS5.
Distributionofexperimentalpneumococcalcarriersoverthefivenasopharyngealmicrobiomeprofilesatbaseline.
Volunteerswhoreceivedapneumococcalchallengewithserotype6Baredisplayed.
Additionalfile8:FigureS6.
OTUabundancesatbaselineinvolunteerswithorwithoutacquisitionofexperimentalpneumococcalcarriage.
Volunteerswhoreceivedapneumococcalchallengewithserotype6Baredisplayed.
Nodesrepresenttaxa,andedgeslinkthedifferenttaxonomiclevels.
Thefoldincreaseiscalculatedasthelog2oftheratiooftherelativeabundanceinpre-existingmicrobiomecompositionsofvolunteerswithoutandwithestablishmentofpneumococcalcarriageafterchallenge(0=nodifferencebetweenthosewhodidanddidnotestablishcarriage,1=twiceasabundantinthosewhoestablishedcarriage,andsoon).
ThesignificanceisexpressedasthepvalueofaMann-WhitneyUtestofthebaselinesamplesfromallchallengedvolunteers.
Notethattherelationbetweennodesizeandtotalabundanceisnon-linear.
Additionalfile9:FigureS7.
Distributionofpneumococcalculture-positivesamplesafterchallengewithserotype6Boverthemicrobiomeprofiles.
Additionalfile10:FigureS8.
ChangesfrombaselineOTUabundances2daysafterpneumococcalchallengeamongserotype6B-challengedvolunteers.
Nodesrepresenttaxa,andedgeslinkthedifferenttaxonomiclevels.
Thefoldincreaseiscalculatedasthelog2oftheratiooftherelativeabundanceinsamplesbeforeand2daysafterpneumococcalchallenge(0=nodifferencebetweenbeforeandafterchallenge,1=twiceasabundantafterchallenge,andsoon).
ThesignificanceisexpressedasthepvalueofaMann-WhitneyUtestofthesamplesbeforeand2daysafterchallenge.
Notethattherelationbetweennodesizeandtotalabundanceisnon-linear.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Authors'contributionsAC,AZ,JG,GF,JB,HT,MJ,SG,andPHwereinvolvedinthedesignofthemicrobiomestudy;JG,DF,andSGdesignedandconductedtheexperimentalmodel;AC,JG,andJSprocessedthesamples;AC,AZ,andJBanalyzedthedata;ACandAZwrotethemanuscript;andallauthorsassistedindatainterpretationandmadesuggestionsforimprovementofthemanuscript.
Allauthorsreadandapprovedthefinalmanuscript.
AcknowledgementsThisstudywasfundedbytheBillandMelindaGatesFoundation(ExperimentalHumanPneumococcalCarriagemodel;OPP1035281),theEuropeanRegionalDevelopmentFund2007–2013(Gelderland-Overijssel,ImmunoForceproject2011-013287),andtheNIHRComprehensiveLocalResearchNetwork(CLRN).
WesincerelythankDr.
DebbyBogaertforsharinghermethodologicalexpertiseonnasopharyngealmicrobiomestudiesandallvolunteerswhoparticipatedinourstudy.
Authordetails1LaboratoryofPediatricInfectiousDiseases,DepartmentofPediatrics,Radboudumc,6500HBNijmegen,TheNetherlands.
2CentreforMolecularandBiomolecularInformatics(CMBI)BacterialGenomics,Radboudumc,6500HBNijmegen,TheNetherlands.
3DepartmentofClinicalSciences,LiverpoolSchoolofTropicalMedicine,L35QALiverpool,UK.
4NIZOfoodresearchB.
V.
,6710BAEde,TheNetherlands.
5HubertDepartmentofGlobalHealth,RollinsSchoolofPublicHealth,EmoryUniversity,Atlanta,GA30322,USA.
6Currentaddress:Crucell–Johnson&Johnson,Leiden,TheNetherlands.
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