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UnencapsulatedStreptococcuspneumoniaefromconjunctivitisencodevarianttraitsandbelongtoadistinctphylogeneticcluster
NatCommun.AuthormanuscriptavailableinPMC2015May12.
Publishedinfinaleditedformas:
PMCID:PMC4231546
NIHMSID:NIHMS632227
NatCommun.20145:5411.
Publishedonline2014Nov12.doi:10.1038/ncomms6411
UnencapsulatedStreptococcuspneumoniaefromconjunctivitisencode
varianttraitsandbelongtoadistinctphylogeneticcluster
MichaelD.Valentino, 1,2,3AbigailMansonMcGuire, 3JasonW.Rosch, 4PauloJ.M.Bispo, 1,2CorinnaBurnham, 4ChristineM.
Sanfilippo, 5,RobertA.Carter, 4MichaelE.Zegans, 6BernardBeall, 7AshleeM.Earl, 3ElaineI.Tuomanen, 4TimothyW.
Morris, 5,WolfgangHaas, 1,2andMichaelS.Gilmore1,2,3,*
1
DepartmentofOphthalmology,MassachusettsEyeandEarInfirmary,243CharlesSt.C703,Boston,MA02114,USA
2
DepartmentofMicrobiologyandImmunobiology,HarvardMedicalSchool,77AveLouisPasteur,Boston,MA02115,USA
3
TheBroadInstituteofMITandHarvard,320CharlesSt.Cambridge,MA02141,USA
4
DepartmentofInfectiousDiseases,St.JudeChildrensResearchHospital,262DannyThomasPl.,Memphis,TN38105,USA
5
BauschandLomb,Inc.,1400GoodmanSt.N.,Rochester,NY14609,USA
6
DepartmentofMicrobiologyandImmunology,GeiselSchoolofMedicineatDartmouth,1RopeFerryRd.,Hanover,NH03755,USA
7
StreptococcusLaboratory,CentersforDiseaseControlandPrevention,1600CliftonRd.,Atlanta,GA30333,USA
*
th
Towhomcorrespondenceshouldbeaddressed:MassachusettsEyeandEarInfirmary,243CharlesStreet,7 Floor,ConnectorBuilding,Boston,MA
02114.Phone:(617)5733845.Fax:(617)5734290.Email:michael_gilmore@meei.harvard.edu
Presentaddress:UpstateStemCellcGMPFacility,UniversityofRochester,601ElmwoodAve.,Rochester,NY,14642,USA
Presentaddress:ActelionClinicalResearch,1820ChapelAve.,CherryHill,NJ,08002,USA
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Abstract
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Streptococcuspneumoniae,aninhabitantoftheupperrespiratorymucosa,causesrespiratoryandinvasive
infectionsaswellasconjunctivitis.Strainsthatlackthecapsule,amainvirulencefactorandthetargetofcurrent
vaccines,areoftenisolatedfromconjunctivitiscases.Hereweperformacomparativegenomicanalysisof271
strainsofconjunctivitiscausingS.pneumoniaefrom72postalcodesintheUS.Wefindthatthevastmajorityof
conjunctivitisstrainsaremembersofadistinctclusterofcloselyrelatedunencapsulatedstrains.Thesestrains
possessdivergentformsofpneumococcalvirulencefactors(suchasCbpAandneuraminidases)thatarenotshared
withotherunencapsulatednasopharyngealS.pneumoniae.Theyalsopossessputativeadhesinsthathavenotbeen
describedinencapsulatedpneumococci.Thesefindingssuggestthattheunencapsulatedstrainscapableofcausing
conjunctivitisutilizeapathogenesisstrategysubstantiallydifferentfromthatdescribedforS.pneumoniaeatother
infectionsites.
Keywords:Streptococcuspneumoniae,conjunctivitis,nontypeable,unencapsulated,comparativegenomics
Introduction
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Streptococcuspneumoniaeisaleadingcauseofinvasiveinfectionsincludingpneumonia,meningitis,andsepsis,as
wellasnoninvasiveinfectionsincludingpharyngitisandotitismedia.Thepolysaccharidecapsule,akeyvirulence
13
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4231546/
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factor,isthetargetofcurrentvaccines13.Vaccinationhassubstantiallyreducedmorbidityandmortality3,buthas
hadlimitedimpactonconjunctivitis,infectionofthemucousmembranecoveringtheeyeandliningtheeyelids4.
Werecentlycollected271S.pneumoniaeisolatesduringthecourseofclinicaltrialsforthetreatmentofbacterial
conjunctivitis57,andfoundthatover90%wereunencapsulated8,andhenceunaffectedbycurrentvaccinedesign.
UnencapsulatedS.pneumoniaestrainshavecausedlargeconjunctivitisoutbreaksinschoolsandcolleges913,
militarytrainingfacilitiesintheUS14,andatotherlocationsworldwide15.Recentoutbreakshaveinvolvedone
multilocussequencetype(MLST)inparticular,ST44813.However,apreviousstudyofepidemiologically
unrelatedconjunctivitiscasesfoundthatmostcaseswerecausedbyencapsulatedstrains4.Thatstudyexamined
isolatespriortothewidespreaduseofthePCV7vaccineintroducedin20004.
Withaviewtowardassessingtheimpactofthevaccineandimprovedvaccinedesign,andtobetterunderstandthe
diversityofstrainsandgeneticbasisforS.pneumoniaepathogenesisinconjunctivitis,herewedescribetheresults
ofanextensivecomparativeanalysisofS.pneumoniaecurrentlyassociatedwithconjunctivitis.
Results
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Epidemiologyofconjunctivitis
TodeterminethediversityofS.pneumoniaecausingconjunctivitis,271strains58werecharacterizedbyMLST16(
Fig.1,SupplementaryData1).SequencetypeST44817,18wasfoundtocausethemajorityofinfections(67.2%).
Thenextmostcommontypescausedsubstantiallyfewer:ST344(8.9%),ST1186(4.8%),ST2315(4.4%).
Together,10differentsequencetypesofunencapsulatedS.pneumoniaeaccountedfor90.8%ofconjunctivitis
cases.AdiversesetofstrainsofS.pneumoniaefromothertypesofinfections,forwhichclosedgenomesare
availableinGenbank,wereincludedforcomparison(SupplementaryData2).Adistinct,deeplyrootedclusterofS.
pneumoniaewasformedthatincluded11unencapsulatedMLSTtypesencompassing89.3%ofconjunctivitis
isolates(Fig.2).Only1sequencetypethatisencapsulated,ST199,causedmorethan2cases.Thisshowsthat
conjunctivitisintheUSismainlycausedbyacloselyrelatedgroupofunencapsulatedS.pneumoniaesequence
types,althoughotherstrainscancauseconjunctivitis,mostlikelyasanextensionofupperrespiratoryinfection.
Figure1
LocationandMLSTprofileofconjunctivitisisolates
Figure2
MLSTbasedphylogeneticrelationshipsamongconjunctivitisstrains
Traitsoftheunencapsulatedconjunctivitiscluster
TodeterminewhetherstrainsfromconjunctivitisthatoccurwithinthedistinctbranchofS.pneumoniaepossess
novelgenecontent,atotalof21genomesofrepresentativesofthemajorconjunctivitisassociatedsequencetypes
weresequenced(SupplementaryData3).Diversitywasmaximizedbyselectingvaryingdatesofisolationandsites
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oforigin.Additionally,genomesofselectencapsulatedconjunctivitisstrainswerealsosequenced,includingST199
(whichcaused5cases)andstrainsofsequencetypesST632,ST667,ST180.
Genesencodingatotalof4,433proteinorthogroupswereidentifiedbyOrthoMCL,1,160ofwhichwerepresentin
singlecopyinallgenomes.Thesecoreorthogroupgeneswereusedtogenerateasinglenucleotidepolymorphism
(SNP)basedphylogenetictree(Fig.3).AsforMLST,theSNPbasedcoregenometreeshowedthatstrainsisolated
fromepidemicconjunctivitisbelongtoadistinct,deeplyresolvedgroupthatincludesST448,ST1186,ST344,
ST1270andST2315.LineageswithinthisgroupweretermedtheEpidemicConjunctivitisCluster(ECC),since
theirgenomesarehighlyrelatedandtheseSTs(ST448,ST344,ST1186)areassociatedwithepidemic
conjunctivitisoutbreaks9,10,14,17,18.Croucherandcolleaguesrecentlynotedonegroupofunencapsulatedstrains
(denotedSequenceCluster12[SC12]),wasthemostdivergentclusterfromthemainpopulationintheirstudy19.
SC12includesSTsST448andST344associatedwithconjunctivitis.Thephylogenydeterminedherewas
unchangedafterfilteringrecombinogenicregionsofDNAusingBRATNextGen20,showingthatrecombination
wasnotthemaindriverforthispopulationstructure.Encapsulatedstrainsthatarerarercausesofconjunctivitis
(ST632,ST667,ST180andST199)areinterspersedamongstrainsthatcauseinfectionatothersites.Theextentof
divergenceofsharedgeneswithinECCgenomesfromthoseofothersitesofinfectionwasquantified21
(SupplementaryFig.1).ECCgenomescomparedtoeachotherexhibitanaveragenucleotideidentity(ANI)value
of99.0%+/0.4,highlightingtheverycloserelationshipamongECClineages.ECCstrainsaresignificantlymore
distantlyrelatedtothosefromothersitesofinfection(97.9%+/0.11ANI,p<0.001,Studentsttest).
Figure3
ECCstrainsbelongtoawellresolvedgroupofthespeciesS.
pneumoniae
ECCstrainspossessadistinctgenerepertoire
ClusteringofgenomesbasedonsimilaritiesingenecontentalsoplacesECCstrainsintoawellresolvedgroup,
independentlyrecapitulatingphylogenicstructure(SupplementaryFig.2)andsupportingthehypothesisthatthe
peculiaroculartropismofECCstrainsstemsatleastinpartfromnovelgenecontent.AsintheSNPbased
phylogeny,strainsthatarerarercausesofconjunctivitisareinterspersedamongnonocularS.pneumoniae.To
identifygenesthatdistinguishtheECCfromotherstrains,becausehorizontalgeneflowcancomplicatethe
analysis,wearbitrarilysetgenepresencein80%orgreaterofECCgenomes,and<20%ofthenonECC
comparatorstrains(orviceversa)asthecutoff.Wefound230genesthatfulfillthisenrichmentcriteria
(SupplementaryData4).Ofthese,103genesareinallECCgenomesandabsentfromallcomparators.Conversely,
70genesaremissingfromECCthatarepresentin80100%ofnonocularS.pneumoniaecomparatorgenomes
(SupplementaryData5).Ofthose,29werefoundinallnonoculargenomesandnoECCstrains.Inpatternsof
genepresenceandabsence,encapsulatedconjunctivitisstrainswerefoundtobemostcloselyassociatedtothose
fromothertypesofinfection.
ThecomparativelylargeproportionofconjunctivitiscausedbyST448suggeststhatitsgenomemaybeespecially
refinedtocausethisdisease(oralternatively,thatamongECClineages,ST448ismorewidelydistributedand
abundantinnature).SeventeenorthogroupsareuniquetoST448(SupplementaryData6),includingahypothetical
mobileelementwithclosestrelativeinS.mitis.NogenesarespecificallymissingfromST448thatinallotherECC
genomes.
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Evidenceforlargescalesurfaceremodeling
Inplaceofacapsuleoperon,allECCstrainsweinvestigatedpossesstheatypicallocusthatincludesaliC
(X231_0947)andaliD(X231_0948),butnottheoftenassociatedpspKgene22,23.However,intheabsenceofa
capsule,alargenumberofnovelsurfacefeatureswerefound.ExclusivetoECCaretwodifferentAntigenI/II
familyadhesins(X231_1085andX231_1187)(SupplementaryData4),thatappeartooriginatefromStreptococcus
macedonicusandStreptococcusmitisrespectively(Fig.4A).OwingtothepresenceofmultipleSspBdomains
withintheseproteins,wetermedthemSspBC1(X231_1085)andSspBC2(X231_1187).SspBdomaincontaining
proteinshavebeenshowntobindthehumanscavengerproteingp340,whichcontributestobacterial
aggregation24.Totestforthisfunctionality,arepresentativeECCstrain(ST448),andanonECCencapsulated
conjunctivitisstrainthatlacksSspBC1andSspBC2(ST199),wereincubatedwithgradedconcentrationsofgp
340.AsshowninFigure4B,theST448strainexhibitedgp340concentrationdependentaggregations.Wealso
identifiedauniquegeneinferredtoencodeasurfaceprotein(X231_1186)termedherePspO.Thissurfaceprotein
geneisdirectlyadjacenttothatencodingSspBC2,suggestingapotentialvirulenceisland.PspOincludesaC
terminalglucanbindingdomainandasurfaceexclusiondomain.
Figure4
HomologsofagglutininreceptorsinECC
Anothergenepredictedtoaffectthehost/pathogeninterface,thatoccursexclusivelyinECCstrains,encodesanew
divergentputativezincmetalloprotease(X231_0594),ZmpC2(SupplementaryFig.3A).Theclosestorthologisin
S.pseudopneumoniaeIS7493,anditshares31%aminoacidsequenceidentitywiththeknownZmpCofS.
pneumoniae,mainlyinthePeptidase_M26_Cdomain(SupplementaryFig.3A).Recently,adifferent,structurally
related,atypicalzincmetalloproteaseC(zmpC,nowtermedZmpC1)wasidentifiedinaS.pneumoniae
conjunctivitisisolate,andwasshowntocleavemucinsfromcornealepithelialcells25,26.ZmpC1(X231_0222)also
occursin100%ofECCand0%ofcomparators.
Additionalsurfacerelatedfunctionsofpotentialrelevancetoconjunctivitis,includeaputativesialidase
(X231_0534),nowtermedNanO1.Itshares88%aminoacidsequenceidentitywithsialidaseA(neuraminidaseA)
ofS.pseudopneumoniae.ThetypicalNanAofS.pneumoniae,whichiscarriedbyallnonocularreferencestrains,
hasbeendisplacedbyNanO1inECC(SupplementaryFig.3B).Closerexaminationofthesequencesurrounding
nanO1identifiesasecondgene,alsoannotatedasencodingasialidase(referredtoasNanO2,X231_0533),
suggestingNanO1andNanO2fromS.pseudopneumoniaerecombinationallydisplacedwildtypeNanA
(SupplementaryFig.3C).Additionally,theneuraminidasealleleNanC,foundinapproximately51%ofS.
pneumoniaeisolatesfromnonocularsites27,wasnotfoundwithinanyECCgenome.
Cholinebindingproteins(Cbp)areimportantvirulencefactorsthatcontributetoS.pneumoniaeadhesionand
transcytosis28.AllECCgenomesencodeanovel,divergentCbp(BM49_0273),CbpI1,thatismostcloselyrelated
toavariantinS.pseudopneumoniae.AllECCstrainsalsoencodeasecondCbpvariant(X231_0220,CbpI2)that
israreoutsidethiscluster,occurringin3nonocularcomparatorstrains(AP200,G54,TIGR4)28.CbpI1andCbpI2
shareastructurallyrelatedcysteinerichsecretorydomainandaCterminalcholinebindingdomain,and48.3%
overallaminoacidsequenceidentity.Interestingly,CbpI2andZmpC1areadjacenttooneanotherwithintheECC
genomes.
S.pneumoniaegeneswithknownrolesincolonizationandvirulenceincludecbpA,nanB,bgaA,strH,ply,hyl,
plpA(aliA),psaABC,prtA,choP,pdgA,adr,spxB,amiAamiF,msrA/B2,andthetwoPilusIslets2931.Choline
bindingproteinA(CbpA),amainadhesininrespiratoryinfectionsandotherwisehighlyconservedinnonocular
strains31,32,issubstantiallydivergentinallECCgenomes.TwopolymorphicformsofCbpAwerefound,CbpAC1
inST448andST1186genomes,andCbpAC2inST344,ST1270,andST2315(Fig.5).PortionsofCbpAC1and
33
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CbpAC2showlittleresemblancetoCbpA,insteadbeingcloselyrelatedtothebetaantigenofS.agalactiae33.The
absenceofCbpAfromECCstrainswasverifiedbyWesternblotusingthreedifferentCbpAspecificmonoclonal
antibodies34,indicatingthatthevariantCbpApossessesasubstantiallydifferentstructureintheotherwise
conservedkeyepitopesprobed(SupplementaryFig.4).TheregionofcanonicalCbpAthatmediatesbindingtothe
eukaryoticpolymericIgreceptor(pIgR)35,hasbeenreplacedbyS.agalactiaedomainsthatbindtheFcportionof
IgAdirectly33,36.ThedifferentvariationsinthedivergenceofCbpAC1andCbpAC2fromCbpAsuggestthat
variationsofthehybridCbpAlocusevolvedindependently.Interestingly,inECCthetwocomponentsystemthat
regulatesCbpAexpression37,alsoexhibitsgreaternucleotidesequenceidentitytoitscounterpartinS.agalacticae
(SupplementaryFig.4D).DifferencesinnucleotidesequenceoneithersideofthetwovariantCbpAClocisupport
separateevolutionofthesedeterminantswithinECCstrains.
Figure5
ECCgenomesencodeanatypicalCbpA
TwoPilusIsletshavebeendescribedthatcontributingtoS.pneumoniaeepithelialcelladhesion29,30.Neitherpilus
isletoccurredinanyECCstrain.ExoglycosidaseBgaAisabsentfromallECCisolates,asisathreegenePTS
system(SP_0645toSP_647)thatoccursimmediatelyadjacentbgaAinTIGR4,displacedbyapproximately1kb
ofsequencewithhighidentity(>91%)tosequencesinS.mitisandS.pseudopneumoniae.Otherwise,allother
virulenceassociatedgenes,includingnanB,strH,ply,hyl,plpA(aliA),psaABC,prtA,choP,pdgA,adr,spxB,
amiAamiF,msrA/B2arepresentinallECCstrains,andarehighlyconserved(99.6%+/0.3inferredaminoacid
sequenceidentitytoTIGR4).
Metabolicdifferences
AllECCstrainsencodeaputativephosphoenolpyruvatedihydroxyacetonephosphotransferasesystem
(X231_1297toX231_1300,SupplementaryData4).UniformlyabsentfromECCstrainsareoperonsforarginine
metabolism(SP_2148toSP_2151),andafucosebinding,uptake,andcatabolicpathway(SP_2158toSP_2170)
(SupplementaryData5).ThisblockofmetabolicfunctionshasbeendisplacedinECCgenomeswitha12.7kb
sequencethatencodes,amongotherthings,ZmpC2.SomeECC(ST448,ST344,ST1270)lackthepiaoperon
mediatingironuptake,whichinotherstrainshasbeenlinkedtovirulenceinmousemodelsofpulmonaryand
systemicinfection38.Fiveothergeneswithputativeannotationsasaminoacidtransporters(SP_0111,SP_0112,
andSP_0709toSP_0711)arepresentin100%ofcomparators,butuniformlyabsentinECC,suggestinga
substantiallyalterednutrientprofileintheconjunctivalmucosa(SupplementaryData5).
RecombinationandHGT
Theoccurrenceofmultigeneblocksofdifferencesuggeststhatmovementofpathogenicityislandsorothermobile
elementswereinvolvedintheevolutionofECC.Ofthe230orthogroupsenrichedinECC,180genesoccurin15
clusters(Table1).TheaverageG+Ccontent(36.8%+/3.8)islowerthantherestofthegenome(39.7%,p<0.01,
Studentsttest),whichiscommonformobileelements39.TwoclustersexclusivetoECC,an18kbpredicted
phage(cluster9)and13kbencodingcoregenes(VirD/VirB/TrsE)ofaTypeIVsecretionsystem(cluster11),are
adjacent.Interestingly,thecluster9/cluster11elementoccursatdifferentlocationswithinECCSTs,suggesting
eitherindependentacquisition,orinternalmovement.Thatitismobileandpresumablycouldbelostifnotfor
selection,yetisretained,suggeststhatitmayhavearoleinmediatingthepeculiaroculartropismofECC.
Table1
GeneclustersenrichedinECCgenomes.
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Themajority(75%)ofECCcarryresistanceelements(SupplementaryFig.5)consistentwithantibiotic
susceptibility40.Macrolideresistanceisthemostcommon,andconferredbytheMacrolideEffluxGenetic
Assembly(MEGA)cassetteinST448andST1186,andbyaTn916likeintegrativeconjugativeelement(ICE)in
ST344andST1270(SupplementaryFig.6).ST2315wastheonlyECCisolateresistanttophenicols,whichwas
conferredbyanSpn11930likeICEelement.
Comparisontostrainsidentifiedinasymptomaticcarriage
Asymptomaticnasopharyngealcarriagegenerallyprecedesdisease.TodeterminewhetherECCstrainswere
representedinlargedatasetsfromasymptomaticcarriage,welookedfortheiroccurrenceintworecentlyreported
studies19,41.ThisexpandedanalysissubstantiatesthedeeplyrootedandwellclusteredgroupingoftheECC
strains,butimportantly,showsthatECCSTsaredistributedamongadditionalcloselyrelated,unencapsulated
strainsisolatedfromthenasopharyngesthathavenotyetbeenassociatedwithconjunctivitis(Fig.6).Itwasthusof
interesttocomparethetraitsofECCstrainsisolatedfromconjunctivitistothoseisolatedfromthenasopharynxby
investigatingthepresenceorabsenceofaselectionofnewlyidentifiedECCgeneswithaputativecontributionto
conjunctivitispathogenesis.Fromtheasymptomaticcarriagedatasets,weselectedgenomesequencesof96strains
thatwereofsequencetypescloselyrelatedtothosethatconstitutedtheECCgroupandalsodiversestrainsspread
acrossthephylogenetictreerepresentingthemostprevalentSTsassociatedwithnasopharyngealcarriageregardless
oftheirencapsulationstatus(Fig.6,SupplementalFig.7andSupplementalData7).Allgenesfoundtobeenriched
inECCstrainsisolatedfromconjunctivitiswerealsofoundtobepresentwithinnasopharyngealisolatesofST448,
ST2315,ST344genomes,indicatingthesestrainsarehighlysimilartothoseisolatedfromconjunctivitis,and
supportinganinfectionmodelwhereasymptomaticcarriageinthenasopharynxprecedesocularinfection.Ofthe
cumulative3,701nasopharyngealisolatesrepresentedinthetwonasopharyngealsurveys,norepresentativesof
ST1186orST1270wereobserved,incontrasttotheiroccurrenceatratesof13/271(4.8%)and3/271(1.1%)
respectivelyinconjunctivitiscases,indicatingtheirrarityamongthecirculatingpopulationdespitetheirenrichment
incasesofconjunctivitis.
Figure6
PresenceofECCgenesingenomesofnasopharyngealisolates
GenesweidentifiedasenrichedinECCstrainsisolatedfromconjunctivitis,cbpAC1,cbpAC2,andnanO1/nanO2
wereonlyfoundtooccuramongasymptomaticcarriagestrainsofthesamesequencetypes.Othergeneswefound
enrichedinECC(sspBC1,sspBC2,zmpC1,zmpC2)occurredalsoinunencapsulatedlineagesthathavenotyet
beenobservedinconjunctivitis,andthemajorityoftheselineagesarecloselyrelatedphylogeneticallytoECC
strains(Fig.6).Interestingly,somesequencetypesphylogeneticallycloselyrelatedtoECCstrains(ST5126,
ST4142,ST4139)werefoundtolackallECCgenesthatwereinvestigated.AstheseSTswerenotidentified
amongconjunctivitisstrains,theirabilitytocausethisdiseaseremainsunknown.
Discussion
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WithaviewtowardunderstandingstraindiversityandthemolecularbasisforvirulenceofS.pneumoniaein
conjunctivitis,andtoimprovingvaccinedesigntocoverthisdisease,wecharacterizedrecentisolatesfromacross
theUS.Wefoundthatnearly90%ofconjunctivitiscaseswerecausedbyfiverelatedSTs(ST448,ST344,
ST1186,ST1270,ST2315)thatoccurwithinadistinctclusteroftheS.pneumoniaespecies(seeFig.6and
19,41
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SupplementalFig.7),apopulationstructuresupportedbytworecentreports19,41.Thisdivergenceischaracterized
bynovelgenecontentconstitutingapproximately10%ofthegenome.AlthoughECCstrainspossessalarge
amountofnovelgenes,sharedgenesexhibitanaveragenucleotideidentityof97.9%+/0.11ANIwithstrains
fromothertypesofinfection,andthereforeECCstrainsdonotconstituteanewspecies(ANI<95%)bythis
definition21,42,19.
WefoundgenescbpAC1,cbpAC2,nanO1,nanO2wereonlycarriedbySTsthatareassociatedwithconjunctivitis
(seeFig.6andFig.7).OthergenesenrichedinECC,includingthesspBCagglutinins,zmpC1,zmpC2,andthe
triosemetaboliccassette,werefoundtobesharedamongafewcloselyrelatedunencapsulatedSTs(ST6153,
ST6691,ST6729,ST2996,ST1054,ST449)thathavenotbeenidentifiedincasesofconjunctivitis,whichmay
stemfromthepaucityofstudiesthathaveidentifiedMLSTtypesofS.pneumoniaecausingconjunctivitis.These
additionalgenesarelargelyabsentinencapsulatedandmoredistantlyrelatedunencapsulatedgenomesunrelatedto
conjunctivitishowever(seeFig.6andFig.7).ThesefindingssuggestthatsomeofthegenesenrichedinECCare
fundamentaltotheformationofthelargerunencapsulatedlineagetowhichECCmembersbelong(seeFig.6and
SupplementalFig.7).
Figure7
VirulencefactordifferencesbetweeninvasiveandECCstrains
Typifyingtheconjunctivitisassociatedstrainsisalackofcapsule,renderingthemunaffectedbycurrentpolyvalent
pneumococcalcapsulevaccines.Aswouldbepredictedforalineagethatprofessionallylacksthepolyanionic
capsulethroughwhichsurfaceproteinsmustfoldandextend,thesestrainshavesubstantiallydifferentsurface
features,includingthoseknowntocontributetovirulence(Fig.7).Novelfeatures,specifictoECCSTsinclude
substantiallyalteredformsofCbpA,CbpAC1andCbpAC2.Interestingly,thesenolongerpossessthekeydomain
thatmediatesbindingtohostpolymericimmunoglobulinreceptor(pIgR),whichS.pneumoniaeusetofacilitate
transcytosisfromnasopharyngealepitheliaintothebloodstream35.Instead,bothCbpAC1andCbpAC2appearto
haveindependentlyswappedthatdomainforonethatmediatesdirectbindingtosecretoryIgA(sIgA)33,36,43.The
implicationisthatECCstrainsbindsIgAinasubtly,butimportantlydifferentway,possiblycoatingthemselves
withIgA,inamanneranalogoustothatmediatedbyproteinAofS.aureus44.Alternatively,theseCbpAvariants
mayactasadhesinsforattachmenttosurfacescoatedwithantibodies,assuggestedforimmunoglobulinreceptors
inStreptococcuspyogenes45.Thatthischangeappearstohaveoccurredtwice,andthatonlyvariantsofCbpA
occurinunencapsulatedSTsassociatedwithconjunctivitis,suggeststhatitisimportantfortheoculartropism.In
additiontothevariantCbpA,allECCgenomesalsoencodetwonovel,divergentcholinebindingproteinCbpIs,
heretermedCbpI1andCbpI2.NofunctionhasyettobeascribedtoCbpI,CbpI1orCbpI2.
OtherfactorsuniquetoSTsassociatedwithconjunctivitisthatcouldaffectthehost/microbeinterfaceincludethe
displacementofNanAwithtwovariantsialidases,NanO1andNanO2.Thisrecombinationeventisseenonlyin
ECCmembersandisabsentineventheclosestnonECCrelatives.Sincesialicacidresiduesexhibitvariation
amonghostcelltypesithasbeensuggestedthattheyaremediatorsoftissuetropism46.Thisisofpotential
relevancetoconjunctivitis,sinceproteinsfoundattheocularsurfacearedecoratedbycovalentlyboundsialic
acids47,whichhavebeenshowntomediateocularsurfacebindingofPseudomonasaeruginosaandEscherichia
coli48,49.
Arecurringmotifwasthereplacementofsurfacefeaturesoptimizedforfunctioninthepresenceofacapsule,with
surfacefeaturesderivedfromunencapsulatedoralstreptococci(e.g.S.mitis).OthershavenotedthatS.mitis
appearstobeareservoirforgeneticdiversity50.AdditionalnovelsurfacefeaturesofECCandcloselyrelated
unencapsulatedstrainslikelyinvolvedincolonization,andlikelyoriginatinginoralstreptococci,includetwo
AntigenI/II(AgI/II)familyofadhesins/agglutininsencodedinseparategenomicislands,SspBC1andSspBC2.
Notably,sspBC2isrestrictedtoECCandcloselyrelatedstrainspotentiallyimplicatingitsinvolvementinthe
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unusualocularsurfacetropism,whereassspBC1isalsofoundwithinmoredistantlyunencapsulatedgenomes,
implicatingitspossibleinvolvementincolonizationofthenasopharynxandadnexa(seeFig.6).TheAgI/IIfamily
ofadhesinsarecentraltocolonizationandbiofilmformationbycommensalandpathogenicspeciesof
Streptococcus24,51.ApreviousreviewnotedtheirubiquitouspresenceamongstreptococciexceptforS.
pneumoniae51.TheobservationherethatthesegenesoccurinunencapsulatedstrainsofS.pneumoniae,suggests
eitherincompatibilityorfunctionalredundancywiththepneumococcalcapsule.Intermsofcolonization
(nasopharyngealand/orocular)andconjunctivitispathogenesis,SspBdomainsofAgI/IIproteinsmediatebinding
tohumanscavengerproteingp34024,whichoccursintearsandontheocularsurface52.
AllSTsassociatedwithconjunctivitisencodedanovelphageelement(cluster9/cluster11).Thiselementwasalso
seenincloselyrelatedunencapsulatednasopharyngealstrains,whereasitwasrarelyfoundinmoredistantlyrelated
unencapsulatedstrains.Interestingly,thiselementislocatedatdifferentsitesinthegenomesoftheconjunctivitis
associatedsequencetypes,butisalwaysconsistentwithinanST.Thissuggeststhatitwaslackingfromacommon
ancestorandhasbeenacquiredindependently,orthatitisinternallymobile.Therearenoobviousadhesinsor
virulencetraitsencodedwithinthiselement,butitmaycontributetobiofilmformationasdescribedforotherphage
elementsinS.pneumoniae53.ItspresencewithinnumerousnasopharyngealSTssuggestsitmayplayabasic
functionincolonizationforunencapsulatedvarietiesofS.pneumoniae.
AuniquemetabolicfeatureprofilewasfoundamongECCandcloselyrelatedstrains,suggestingthattheir
colonizationabilityislikelynutritionallydistinctfromthatofstrainsassociatedwithinvasiveinfection.A
phosphoenolpyruvatedihydroxyacetonePTSgeneclusteroccurringinECCandcloselyrelatednasopharyngeal
strainsandonly1distantlyrelatedstrainfromotitismedia(Hungary19A6),(cluster8),alsofoundinS.mitisandS.
pseudopneumoniae,suggeststhattheabilitytometabolizeDhaisimportantformucosalsurfacecolonization.DhaP
wasdetectedamongthephosphorylatedintermediatemetabolitespresentontheocularscleraandcornealtissues54.
Inadditiontothegainofputativemetaboliccapabilitiesdescribedabove,allSTgenomesassociatedwith
conjunctivitislackedtheabilitytometabolizefucose,asugarthatdecoratesocularglycanspresentintheglycocalyx
ofcornealepithelialcells49.Fucosylatedglycanscoatingmucinsareknowntopromotebacterialcolonizationinthe
gut,servingasbothadherencetargets,aswellasacarbonsource55,56.Specificallyattheocularsurface,fucose
residueshavebeenimplicatedintheattachmentofP.aeruginosaandE.colitoocularepithelialcells49.Moreover,
applicationofexogenousfucosewasshowntosuppressinflammationinrabbitcornealandexplantedhuman
corneamodelsofwoundhealing57.NasopharyngealSTscloselyrelatedtoECCmembers(ST6691,ST6729,
ST1054,ST449,ST2996,ST6153)werealsofoundtolacktheelementstometabolizefucose.Thesefindings
suggesttheinabilitytometabolizefucoseisirrelevantforcolonizingthenasopharynx,butmayconferanadvantage
attheocularsurface,potentiallybypromotinganantiinflammatoryenvironmentand/orbypreservinganimportant
bacterialligand.
Whileasymptomaticcarriageinthenasopharyngealcavityislikelytobeaprecursortoinfection,
ST4489,12,13,15,18andrelatedSTscommonlyisolatedconjunctivitiswerenothighlyprevalentinrecentlargescale
surveysofasymptomaticS.pneumoniaecarriageinthenasopharynx19,41.WhereasST448wasfoundtobebyfar
theleadingcauseofconjunctivitisinthisstudyaswellasinothers15,18,itrepresentedonly1.43%of3,084isolates
foundtobeasymptomaticallycarriedbyChewapreechaetal.41,and1.14%ofnasopharyngealisolatesexamined
byCroucheretal.19.Indeed,therearefourotherunencapsulatedSTsfoundatsimilarorhigherprevalencewithin
thenasopharynges(ST4133,ST4395,ST4965,ST4136rangingfrom1.43to2.92%),themostprevalentofwhich,
ST4133,hasnotbeenreportedasacauseofconjunctivitis,isnotcloselyrelatedtotheSTsmostcommonly
associatedwithconjunctivitisanddoesnotencodethegenesenrichedinECCthatweresearched,highlightingthe
pointthatitisnotthesimplelackofcapsulethatpredisposesthesestrainstocauseconjunctivitis.Thefourmost
commonSTs(ST4414,ST802,ST315,ST4209)innasopharyngealcarriageareallencapsulated(acumulative
19.98%of3,084isolates),andlackallECCassociatedgenes(exceptforaphage(cluster1)sharedonlyin
ST4414)andwerenotfoundamongourcollectionof271conjunctivitisisolates.Similarfindingswereseenby
19
st
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19
Croucheretal. ,with1.14%ofnasopharyngealisolatesbeingST448(21stmostcommonST),inthiscase
representingthemostcommonunencapsulatedSTrecoveredintheirstudy.Takentogether,thesefindingshighlight
thatprevalenceinthenasopharyngesdoesnotdirectlycorrelatewithconjunctivalinfection,infurthersupportofthe
hypothesisthatgenesuniquetoECCgenomesarecriticalforconjunctivalinfection.
ItisunlikelythattheunencapsulatedclustercontainingECCmembersaroseduetovaccineuse,ashasbeen
speculated8,basedupontheextentofdivergencebetweenECClineagesinvestigatedhereinandnonocular
lineages(anaverage27,754+/1,831SNPs).BasedonarecentdeterminationofS.pneumoniaemutationrate58
(andassumingthismeasureistrueforECCmembersaswell),thebifurcationbetweenECCandthemainbranch
ofthespeciestookplaceapproximately8,400yearsago(8,385+/553years).Thattherateofdivergence
measuredforotherS.pneumoniaealsoappliestoECCratesofchangestemsfromacomparisonofthedistance
betweenstrainsisolatedfromgeographicallyandtemporallyrelatedoutbreaksinMaineandNewHampshire.With
epidemiologiccentersabout7monthsapart,strainsfromtheNewHampshireoutbreak(ECC_1854,ECC_1910)
differfromthosefromtheMaineoutbreak(ECC_0072,ECC_0083)4.67+/2.1SNPs,amutationrateof1.43
106substitutionspersiteperyear,inagreementwithpreviouscalculations1.57106substitutionspersiteper
year58.ThisdatingissimilartoestimatesofcladedivergenceinE.faecium59,andS.aureus60,bothofwhichwere
attributedtoincreasingurbanization.Thissuggeststhatincontrasttotheancestralline,thereisanespecially
importantroleforpersontopersontransmissioninthepropagationofeitherthislineage,orthebranchassociated
withrespiratoryinfection.
Insummary,wefoundthat5STscommonlyassociatedwithconjunctivitis(whichaccountedfor90%ofS.
pneumoniaeconjunctivitiscasesstudied)belongtoadistinctclusterofunencapsulatedstrainswithintheS.
pneumoniaespecies.Thesestrainsaretypifiedbysubstantiallydifferentfeaturesincludingelementsexclusiveto
strainsassociatedwithconjunctivitis(CbpAC1,CbpAC2,NanO1,NanO2)thatmaycontributetotheirocular
tropism.Additionalfeaturesweresharedwithonlycloselyrelatedunencapsulatedvarieties(e.g.ZmpC,SspBC2),
orsporadicallyamongdistantlyrelatedunencapsulatedstrains(e.g.SspBC1).Currently90%oftheS.pneumoniae
strainsassociatedwithconjunctivitisarenotcoveredbyexistingvaccines.Furthermore,becauseoftheextensive
variationobserved,vaccinesunderdevelopmentthattargetconventionalS.pneumoniaevirulencetraits(e.g.CbpA)
mayormaynotprovidecoverageforpreventingconjunctivitis.Thisknowledgeofconservedandvariantfeatures
occurringintheECCmemberswillbecriticalforfuturevaccinedesignstrategies.
Methods
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Bacterialstrains
Alargeandcomprehensivecollectionof280S.pneumoniaeconjunctivitisstrainswereassembledfromacrossthe
USA,including271isolatesobtainedfrom72differentzipcodes,aswellasoneisolatefromNewDelhi,Indiaas
partofanational,multicenter,passivesurveillancestudyofbacterialconjunctivitis57.Alsoincludedweretwo
conjunctivitisisolates(6intotal)fromeachlargeoutbreakoccurringatDartmouthCollege10,12,anelementary
schoolinMaine13,17,andasuburbofMinnesota9,aswellasthreeotherconjunctivitisisolatesofunknownorigin
wereobtainedfromtheCDCStreptococcusLaboratory(SupplementaryData1).Strainswereculturedon5%
sheepbloodagarplates(BDBiosciences,SanJose,CA)orinToddHewittbrothsupplementedwith5%yeast
extract,andincubatedat37Cin5%CO2.BacterialisolateswereconfirmedasS.pneumoniaebasedontheir
hemolysisphenotypeonbloodagarplates,bilesolubility,andsusceptibilitytooptochinwhengrownina5%CO2
atmosphere8.
OcularisolatecharacterizationbyMLSTandcapsuletyping
Multilocussequencetypingandcapsuletypingwereperformedonthe271strainscollectedfromthelargeUS
multicentertrial57(Fig.1B).Briefly,sequencetypesweredeterminedbasedonsequencesforaroE,gdh,gki,recP,
spi,xptandddlgenes61.ThepresenceofcapsulewasdeterminedwithbothOMNIserum,aswellasthecapsule
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typespecificPneumotestLatexserum,bothobtainedfromtheStatensSerumInstitut(MiraVistaDiagnostics,
Indianapolis,IN).Initialcapsuletypingresultsfor50selectedisolateswereconfirmedbytheStatensSerumInstitut
inDenmark.ApreliminaryreportofthedistributionofMLSTtypeshasbeenpresentedpreviously62.
GenomeSequencing
StrainsisolatedfromthreewelldocumentedmajorUSoutbreaksinMaine,NewHampshireandMinnesota,as
wellasstrainsthatwererepresentativeoftheMLSTsequencetypesmostprevalentamongthe271strainscollected
fromthemulticenterUSstudy,wereselectedforgenomesequencing(SupplementaryData1).Briefly,totalDNA
waspurifiedfroma5mLovernightcultureusingtheDNeasyDNAextractionkit(Qiagen,Valencia,CA).Library
preparationforIlluminasequencingbyIlluminawascarriedoutusingtheNexteraDNASamplePreparationkit
(Illumina,SanDiego,CA),accordingtomanufacturersspecifications.DNAqualitywasverifiedonaBioTek
Synergy2microplatereader(Winooski,VT)priortoquantificationusingaQubitfluorometeranddsDNAHigh
Sensitivityassaykit(Invitrogen,Carlsbad,CA).Atransposomewasusedtosimultaneouslyfragmentandappend
adaptersequencesto50ngofDNApersample,followedbyadditionofdualindexsequencesinalimitedcycle
PCRstep.QualityandquantityofeachsamplelibrarywasmeasuredonanAgilentTechnologies2100Bioanalyzer
(SantaClara,CA),withatargetfragmentsize300bp.ThegenomesofstrainsECC_0072,ECC_0083,
ECC_1854,ECC_1910,SC_0381,andSC_0391weresequencedattheSt.JudeChildrensHospitalHartwell
CenterforBioinformaticsandBiotechnology,Memphis,TNonanIlluminaGAXIIsequencer,accordingto
manufacturesspecifications.Forallothergenomes,librarieswerenormalizedto2nM,multiplexedandsubjected
toeither150,200or250bppairedendsequencingonanIlluminaMiSeqPersonalSequencerattheMass.Eyeand
EarInfirmaryOcularGenomicsInstitute(Boston,MA),accordingtomanufacturersspecifications.
Genomeassembliesandannotation
SequencereadswereassembleddenovoutilizingCLCGenomicsWorkbenchv4.9(CLCBio,Cambridge,MA)
(SupplementaryData3).Onaverage,3.7millionhighqualitypairedendreadswerecollectedforeachstrain,
representing>240foldcoverageofthe2.1Mbgenomes.Sequencereadsbelowaqualityscoreof25atany
positionwereexcludedfromfurtheranalyses.Allgenomescomparedinthisstudy(SupplementaryData2)were
annotatedusingtheRapidAnnotationusingSubsystemTechnology(RAST)server63,andGlimmerv.364,with
comparisontofamilyprofilesintheFIGfam(proteinfamiliesgeneratedbytheFellowshipforInterpretationof
Genomes(FIG))release63database.Whereverpossible,manualsearchofthePFAM65databasewasusedto
assignfunctionstogenesannotatedashypothetical.
Orthogroupsandgenefamilies
OrthogroupswerecalculatedacrossallofthegenomesinourdatasetusingOrthoMC66,withaBLASTevalueof
105andaninflationindexof2.5.Orthogroupscontainorthologs,whichareverticallyinheritedgenesthatlikely
havethesamefunction,andalsopossiblyparalogs,whichareduplicatedgenesthatmayhavedifferentfunction.
PhylogenicandANIanalyses
SNPbasedphylogenybaseduponMLSTallelesequenceandsinglecopycorealignmentwasgeneratedusing
PhyMLandstatisticswerecalculatedfor1000bootstrapreplicates67.TogenerateaMLSTbasedtree,DNA
sequencesforthesevenMLSTlociwereconcatenatedandalignedforeachofthe31sequencetypesrepresentedin
theconjunctivitisisolates(Fig.2)andthe26nonocularreferencegenomes(SupplementaryData2).
Aphylogenetictreeofallgenomesinourdataset,includingthe21genomesnewlysequencedaswellas26
referencegenomes(SupplementaryData2),wasgeneratedusingall1160singlecopycoreorthogroups,including
StreptococcusmitisstrainB6asanoutgroup.BRATNextGenanalysiswasconductedonthe1160singlecopy
coreorthogroupalignmentofthe47S.pneumoniaegenomestoidentifyfilteroutrecombinogenicregions19,68.
Percentaveragenucleotideidentity(ANI)wascalculatedbydividingthenumberofidenticalnucleotideresiduesin
21
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sharedgenesbythetotalnumberofnucleotidesinsharedgenes21.Sharedgenecontentbetweenstrainsinpairwise
genomecomparisonswasgeneratedbysearchingtheCDSpredictionsfromonegenomeannotationagainstthe
annotationsofthesecondgenomeandconservedgeneswereidentifiedbyBLASTmatching>60%overall
sequenceidentity21.
Identificationofantibioticresistancegenes
TheResfinderdatabasewasusedtoidentifycandidateantibioticresistancegenesasdescribedpreviously69.Fora
subsetoftheisolates,susceptibilitywastestedinmicrotiterplatesandminimuminhibitoryconcentrations(MICs)
weredeterminedbybrothmicrodilutionaccordingtotheprocedurerecommendedbytheClinicalandLaboratory
StandardsInstitute(CLSI).
WesternBlot
Logarithmicallygrowingcells(OD600=0.5)werepelletedbycentrifugationandsubjectedtolysisin0.1%Triton
X100.Toensureequalloading,proteinconcentrationwasdeterminedforeachlysateviaabsorbanceat280nm
andloadedaccordingly.DuplicategelsstainedwithCoomasiewereusedtoconfirmequivalentloading.Lysates
wererunon10%NuPAGEBisTrisgels(Invitrogen).ProteinsweresubsequentlytransferredtoPVDFmembranes
byWesternBlot.CbpAwasdetectedusing3monoclonalantibodies(1:5000)inPBST/5%nonfatdrymilk
(NFDM).Thethreemonoclonalantibodies(14A3,3G12,and3H11)recognizethehighlyconservedloopregions
intheR2domainofCbpAandweregeneratedaspreviouslydescribed34.Pneumolysinwasdetectedusingrabbit
polyclonalserumgeneratedagainstrecombinantpneumolysin.SecondaryHRPconjugatedantibodies(Biorad,
Hercules,CA)wereusedat1:5000inPBST/5%NFDM.ImagesofthecompleteWesternblotsareprovidedin
SupplementaryFigure8.
Aggregationassayswithgp340
Bacterialisolates(ST448,ECC_3540andST199,SC_3526)wereculturedovernightinToddHewittbroth,
pelletedaftercentrifugation(5000gfor10minutes),washedtwiceinPBSandresuspendedtoanopticaldensity
atOD600=0.6inPBS.Bacterialsuspensions(300l)wereincubatedin5mlculturetubesinanorbitalshakerat
300RPMfor1hat37Cwith0,0.5and1.0g/mlofpurifiedgp340(DMBT1recombinanthumanprotein,Life
Technologies).Tubeswerethenrestedfor1hat37Ctoallowbacterialaggregatestosettle.Gramstainingwas
performedforeachreactiontodemonstratebacterialaggregationandrepresentativeimageswereacquiredusingan
OlympusBX60microscope.
CharacterizationofECCgenesinnasopharyngealgenomes
Additionalgenomesofnasopharyngealisolateswereanalyzedforgenesfoundinouroriginaldatasettobespecific
toECCgenomesincluding:(a)29additionalrepresentativesofstrainsknowntobeassociatedwithECC(23
ST448,4SLV448,1ST344,1ST2315),(b)19representativesofSTscloselyrelatedtothoseassociatedwith
ECC,(c)44unencapsulatedSTsnotcloselyrelatedtoECCmemberswereanalyzed(SupplementaryData7),(d)4
encapsulatedSTsthatweremostprevalentinChewapreechaetal.41.Thisincludedallunencapsulated
nasopharyngealgenomesinCroucheretal.19(16genomes)and8draftgenomesofnasopharyngealisolates
currentlyavailablefromeithertheNCBIGenBankorEuropeanNucleotideArchive(ENA),including5genomes
newlydepositedtoNCBIGenBank70.Whenavailable,wemaximizedthediversityofthissetbydownloading
severalrepresentatives,spanningvariousdatesofisolation,orwhenadditionalinformationonstraindiversitywas
available(e.g.BayesianAnalysisofPopulationStructure41).GenomesweredownloadedfromtheENAread
archiveandassembledusingCLCGenomicsWorkbenchasdescribedabove.Alltogetheranadditional96
genomesofnasopharyngealoriginwereselectedtoserveasalocalBLASTdatabase,whichwasusedtosearch
(>80%querycoverage,>80%nucleotideidentity)forthepresenceofgenesidentifiedasspecifictotheECCgenes
inouroriginaldataset(SupplementalData7).
SupplementaryMaterial
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SupplementaryMaterial
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Clickheretoview.(201K,xlsx)
Clickheretoview.(1.9M,pdf)
Acknowledgements
Goto:
PortionsofthisprojectweresupportedbyNIHgrantsEY024285,MolecularBasisforOcularSurfaceTropismin
Conjunctivitis,andbytheHarvardwideProgramonAntibioticResistance,AI083214.HHSN272200900018C
supportedtheinvolvementofAMM,AMM.AdditionalsupportforthisprojectwasobtainedfromtheALSAC
organizationofSt.JudeChildrensResearchHospital(JWR,CB,RAC,EIT).Additionally,portionsofthisproject
weresupportedbyBauschandLomb,Inc.(CMS,WH,TWM,MSG).MDVwassupportedinpartbyNIH
fellowshipthroughEY007145.WegratefullythankDrs.F.Lebreton,D.VanTyneandM.Martinforhelpful
commentsthroughoutthecourseofthisworkandduringmanuscriptpreparation.Fundingagencieshadnorolein
studydesign,dataanalysis,decisiontopublishorpreparationofthemanuscript.PJMBwassupportedbyagrant
fromtheCoordenaodeAperfeioamentodePessoaldeNvelSuperior,Brazil(CAPES#9775137).
Footnotes
Goto:
Authorscontributions.MSGandMDVconceivedanddesignedthestudy.MDV,AMM,PJMB,CB,CMS,RACperformed
theexperiments.MDV,AMM,JWR,MSGcontributedtotheanalysisandinterpretationoftheresults.Themanuscriptwas
writtenbyMDVandMSG.MEZ,BB,TWM,WHprovidedstrainsfromconjunctivitis.AME,EITprovidedadditionalguidance
duringmanuscriptpreparation.
Accessioncodes.DraftgenomesequenceshavebeendepositedintheDDBJ/EMBL/GenBankdatabasesunderBioProject
PRJNA22902.SeeSupplementaryData2forindividualaccessioncodesforthedraftgenomesequences.
SupplementaryInformationaccompaniesthispaper.
Competingfinancialinterests:Theauthorsdeclarenocompetingfinancialinterests.
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