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Proceedings Paper:Marsh, PD and Zaura, E (2017) Dental biofilm: ecological interactions in health and disease. In: Journal of Clinical Periodontology. 12th European Workshop on Periodontology, 06-09 Nov 2016, Segovia, Spain. Wiley: 12 months , S12-S22.

https://doi.org/10.1111/jcpe.12679

© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. This is the peer reviewed version of the following article: Marsh PD, Zaura E. Dental biofilm: ecological interactions in health and disease. J Clin Periodontol 2017; 44 (Suppl. 18): S12–S22.; which has been published in final form at https://doi.org/10.1111/jcpe.12679. This article may be used for non-commercial purposes in accordance with the Wiley Terms and Conditions for Self-Archiving.

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DentalBiofilm:EcologicalInteractionsinHealthandDiseaseなMarshP.D.1andZauraE.2に1DepartmentofOralBiology,SchoolofDentistry,UniversityofLeeds,Leeds,UKぬ2DepartmentofPreventiveDentistry,AcademicCentrefor DentistryAmsterdam,ねUniversityofAmsterdamandVrijeUniversiteitAmsterdam,TheNetherlandsの はRunningtitle:OralmicrobialinteractionsばKeywords:oralmicrobiome,interactions,ecology,metabolism,signalling,geneぱtransfer ひ 10Correspondingauthor:11E.Zaura12DepartmentofPreventiveDentistry13AcademicCentrefor DentistryAmsterdam14GustavMahlerlaan3004151081LAAmsterdam,TheNetherlands16Tel:+31-20-598068417e.zaura@acta.nl 18 19

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20Conflictofinterest:Theauthorshavestatedexplicitlythattherearenoconflicts21ofinterestinconnectionwiththisarticle22SourceofFunding:Nofundinghasbeenavailableother thanthatoftheauthor’s23institution24 25

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Abstract26Theoralmicrobiomeisdiverseandexistsasmulti-speciesmicrobial27communitiesonoralsurfacesinstructurally-andfunctionally-organised28biofilms.Aim.Todescribethenetworkofmicrobialinteractions(both29synergisticandantagonistic)occurringwithinthesebiofilms,andassesstheir 30roleinoral healthanddental disease.Methods.PubMeddatabasewassearched31for studiesonmicrobialecologicalinteractionsindentalbiofilms.Thesearch32resultsdidnot lendthemselvestosystematicreviewandhavebeensummarized33inanarrativereview instead.Results.547originalresearcharticlesand21234reviewswereidentified.Themajority(86%)ofresearcharticlesaddressed35bacterial-bacterialinteractions,whileinter-Kingdommicrobialinteractionswere36theleaststudied.Theinteractionsincludedphysicalandnutritionalsynergistic37associations,antagonism,cell-to-cellcommunicationandgenetransfer.38Conclusions.Oralmicrobialcommunitiesdisplayemergentpropertiesthat39cannotbeinferredfromstudiesofsinglespecies.Individualorganismsgrowin40environmentstheywouldnot tolerateinpureculture.Thenetworksofmultiple41synergisticandantagonisticinteractionsgeneratemicrobialinter-dependencies,42andgivebiofilmsaresiliencetominor environmentalperturbations,andthis43contributestooral health.Ifkeyenvironmentalpressuresexceedthresholds44associatedwithhealth,thenthecompetitivenessamongoralmicro-organismsis45alteredanddysbiosiscanoccur,increasingtheriskofdentaldisease.46Clinicalrelevance:47Scientific rationale:Micro-organisms persist in themouth asmulti-species biofilms48that deliver important benefits to the host. Microbes will interact because of their 49physicalproximity,andtheoutcomewillinfluenceoralbiofi lmcompositionandactivity.50Principal findings: A literature review confirmed that numerous synergistic and51antagonistic interactions occur among the resident microbes, resulting in tightly52integratedcommunities that are resilient againstminor environmentalperturbations,53whichcontributes tooralhealth.Practicalimplications: Treatment strategiesshould54alsoincludereducingenvironmentalpressuresthatdrivedysbiosissothat afavourable55ecologicalbalanceismaintained. 56

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Introduction57Themouth supports thegrowthofdiverse communitiesofmicro-organisms ┽58viruses,mycoplasmas,bacteria,Archaea┸fungiandprotozoa(Wade2013).These59communities persist on all surfaces as multi-species biofilms and form the60residentoralmicrobiome,whichgenerallyexists inharmonywiththehost,and61deliversimportantbenefitsthatcontributetooverallhealthandwell-being.The62micro-organisms foundwithin theseoralbiofilms live in closeproximitywith63oneanother,whichresultsinawiderangeofpotentialinteractions,whichcanbe64synergisticor antagonistic.Thecompositionofthemicrobiome is influencedby65theoralenvironment,andchanges in localconditionscanaffect themicrobial66interactionswithintheseoralcommunitiesanddetermine, inpart,whether the67relationship between the oral microbiome and the host is symbiotic or 68potentiallydamaging(dysbiotic),therebyincreasingtheriskofdiseasessuchas69cariesor periodontaldiseases(Marsh2003;Roberts┃Darveau2015).Our aim70was to review systematically the literatureonmicrobial interactions indental71biofilms in health and disease. However, the search strategy and outcomes,72presentedbelow,ledtoaconclusionthatthetopicistoobroadfor asystematic73report andso the resultsarepresentedas anarrative review,highlighting the74main microbial interactions in dental biofilms in health and introducing the75environmentaldriversfor ecologicaldysbiosistowardsdisease.76Literaturesearch77 A PubMed search procedure was performed on 19-07-2016. The query78combined four separatesearch items:1) ‘microbiota’, includingeither bacteria,79viruses,Archaea┸fungi,protozoaor mycoplasma;2)‘oral’,includingdistinctoral80niches; 3) interactions, including either ‘ecology’, ‘interaction’, ‘synergy’,81‘inhibition’, ‘co-occurrence’, ‘communication’, ‘metabolism’, ‘nutrients’, ‘gene82transfer’ or ‘quorum sensing’ and 4) ‘plaque’, ‘biofilm’, ‘community’ or83‘consortium’ (Supplementary Table S1). This resulted in 3758 hits. Of these,843593 passed theEnglish language filter.After the screening of the titles and85abstracts,theentriesthatdidnot relatetothetopicwereexcluded,leaving75986articles.Amongthesewere212reviews.87

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The vastmajority (86%) of the original research articles (N=547) addressed88bacterialinteractions(Table1).Theseincludedphysical(e.g.,co-aggregation,co-89adhesion)andnutritionalsynergisticinteractions,antagonisticinteractionssuch90as production of bacteriocins and other inhibitory substances, cell-to-cell91communicationandgene transfer.Thebacterial species involved ranged from92primarycolonizers to taxaassociatedwithcariesandperiodontaldisease.Only9345(8.2%)ofthestudiesinvolvedfungi,whileinteractionsinvolvingviruses(1894studies),Archaea (4 studies) andprotozoa (3 studies)were the least studied.95Inter-kingdom interactionswereaddressed in71studies,with themajorityof96thesefocusingonCandidaalbicansandoralstreptococci (Table1).97Duetothehighnumber ofarticlesincludedandthebroadrangeinthemethods98andtheoutcomesamongthestudiesfound, itwasnot possibletoreportonthe99results intheformofasystematicreview or meta-analysis.Instead,thearticles100thatwereidentifiedbythedescribedsearchprocedurewereusedasthebasisof101thenarrativereview below.102Microbialinteractionsinhealth103Theclosephysicalproximityofmicro-organismswithinoralbiofilms inevitably104increases theprobabilityof interactionsoccurring.Themostcommon typesof105interactionare listed inTable2,and canbe synergisticor antagonistic to the106participatingspecies (Diaz2012;Guoetal.2014;Hojoetal.2009;Huangetal.1072011; Jakubovics 2015a; Kolenbrander 2011; Ng et al. 2016; Nobbs and108Jenkinson2015).109110Synergisticinteractions111Physicalinteractionsandbiofilmarchitecture112Oralmicro-organismsmustattachtosurfacesi ftheyaretopersistinthemouth113andavoidbeinglostbyswallowing.Evidenceprimarilyderivedfromlaboratory114studies suggests that early colonisers adhere via specific adhesin-receptor 115mechanismstomolecules intheconditioningfilmsthatcoatoralsurfaces(Hojo116etal.2009),though,ultimately,microbialgrowthisthemajor contributor tothe117increase in biofilm biomass (Dige et al 2007). Oral micro-organisms have a118

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natural tendency toadhere toother microbesand thisprocess (co-adhesion ‒119theadherenceofplanktonic cells toalreadyattachedorganismson a surface)120facilitates the formation of multi-species biofilms (Kolenbrander 2011). In121addition toanchoring a cell to a surface, co-adhesionalsopromotesmicrobial122interactions by co-locating organisms next to physiologically-relevant partner 123species, thereby facilitating nutritional co-operation and food chains, gene124transfer and cell-cell signalling.Substantial changes in gene expression occur 125whencellsareincloseproximityor physicalcontactwithoneanother (Wrightet126al. 2013),while functional consequences can result, such as the protection of127obligatelyanaerobicbacteria inaerobicenvironmentsbyneighbouringspecies128that either consume oxygen (Bradshaw et al. 1994) or are oxygen-tolerating129(Diazetal.2002).Candidaalbicanscanalsoco-aggregatewithoral streptococci,130andcanformsynergisticpartnershipsinwhichtheyeastpromotesstreptococcal131biofilm formationwhilestreptococcienhance the invasivepropertyofCandida132(Diazetal.2012;Xuetal.2014).Thesephysicalandfunctionalassociationscan133manifest themselves in some of the complex multi-species arrangements134observedinoralbiofilmsformedinvivo┸suchas‘corncob’,‘test-tubebrush’and135‘hedgehog’ structures (Dige et al. 2014;MarkWelch et al. 2016; Zijnge et al.1362010).137 138Nutritionalinteractions139

The primary nutrients for oralmicro-organisms are host proteins and140glycoproteins, and these are obtained mainly from saliva for organisms in141supragingival plaque (for a review, see: Jakubovics 2015b) and from gingival142crevicular fluid(GCF)for thoselocatedinsubgingivalbiofilms(Weietal.1999).143Pure cultures of oral micro-organisms grow poorly or not at all on these144structurallycomplexsubstrates,andconsortiaofinteractingspeciesareneeded145for their catabolism. Proteins are broken down by the action ofmixtures of146proteases andpeptidases, but the catabolismofglycoproteins (consistingof a147proteinbackbonedecoratedwithlinear or branchedoligosaccharidesidechains) 148involves thesequential removalof terminalsugars fromside-chainsbefore the149protein backbone becomes accessible to proteolytic attack (Takahashi et al 1502015).Oralbacteriaexpressglycosidaseswithdifferentspecificitiessothat the151

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concertedactionofseveralspeciesisnecessaryfor thecompletedegradationof152host glycoproteins (Bradshaw et al. 1994).Similarly, combinations ofmutans153streptococci, Streptococcus oralis and Fusobacterium nucleatum degraded154albumin more effectively than any of the three species alone (Homer and155Beighton1992).Thebiofilmmatrix isanother potentialsource for carbonand156energyfor interactingconsortiaoforalbacteria.Fructansandsolubleglucansin157dental plaquecanbemetabolisedbycombinationsofbacteriathatproduceexo-158and/ or endo-hydrolyticenzymes (BergeronandBurne2001;Kooetal.2013).159Individualbacteriaaredependent,therefore,onthemetaboliccapabilityofother 160speciesfor accesstoessentialnutrients.161

Further complex nutritional inter-relationships develop in microbial162communities when the products of metabolism of one organism (primary163feeder) become themain source of nutr ients for another (secondary feeder),164resulting in the development of food-chains or foodwebs (Hojo et al. 2009)165(someexamplesare illustrated inFigure1). These foodwebscan result in the166completeandenergetically-efficient catabolismofcomplexhostmoleculestothe167simplestendproductsofmetabolism(e.g.CO2┸CH4┸H2S).Numeroussynergistic168metabolic interactionsoccur amongbacteria insubgingivalbiofilms inorder to169enablethemtodegradehostproteinsandglycoproteinsasnutrientsources(ter 170Steeg ┃ van der Hoeven 1989; ter Steeg et al 1987). These interactions are171discussed in more detail later in the section on ‘Ecological drivers towards172dysbiosisanddisease’.173

Nutritional inter-dependenciessuchasthosedescribedabovecontribute174to the temporalstabilityand resilienceoforalmicrobialcommunities,while a175consequenceoftherelianceofresidentoralbacteriaonthemetabolismofthese176complex substrates is that species avoid direct competition for individual177nutrients,andhenceareabletoco-existandmaintainastableequilibrium,also178termedmicrobial homeostasis (Alexander, 1971;Marsh, 1989).Thishas been179elegantly demonstrated in a computational study on KEGG pathway-based180metabolic distances between 11 oral bacteria that are known to interact181(Mazumdar et al. 2013).Metabolismwas amajor factor driving the order of182colonization,withspecificmetabolicpathwaysassociatedwithdifferentlayersin183thebiofilm,resultinginafunctionallystructuredcommunity.However,insucha184

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structured community, therewasanoptimal trade-offbetween their resource185sharingandfunctionalsynergy(Mazumdar etal.2013).186 187Cell-cellsignalling188Laboratory studies have shown thatmicrobial cells are able to communicate189with,andrespondto,neighbouringcellsinbiofilmsbymeansofsmall,diffusible,190effector molecules.Gram-positive cellsproducepeptides thatgenerallyhave a191narrowspectrumofactivity.InS.mutans┸twopeptides(competence-stimulating192peptide,CSP,andsigmaX-inducingpeptide,XIP)promotegeneticcompetencein193other cellsofS.mutans┹productionof thesepeptides is influencedby the local194pH(Guoetal.2014)andcarbohydratesource(Moyeetal.2014).CSP-mediated195quorum sensing has also been identified in S.gordonii andS. intermedius┻The196functionofCSPs istoalter genetranscriptionandproteinsynthesis involved in197biofilm formation, competence development, bacteriocin synthesis, stress198resistance, and autolysis (Guo et al. 2014;Senadheera andCvitkovitch 2008).199Somestreptococcican inactivateCSPs,andthereby inhibitbiofilmformationby200S.mutans(Wangetal.2011).CSPproducedbyS.gordoniicanalsoinhibitbiofilm201formation by C. albicans (Jack et al. 2015), so it is possible that a complex202networkof signalling interactionswill exist in amulti-species biofilm suchas203dental plaque.204

Autoinducer-2(AI-2)isproducedbyseveralgeneraoforalGram-positive205andGram-negativebacteria,andmaybea ‘universal language’for inter-species206and inter-kingdom communication in dental biofilms, and the efficiency of207signallingmight be enhanced by co-adhesion.Biofilm formationwith two co-208adheringspecies ┽S.oralisandActinomycesnaeslundii ┽was inhibitedwhenan209AI-2knockout ofS.oraliswasusedinsteadofthewildtype(Rickardetal.2006),210while AI-2 produced by Aggregatibacter actinomycetemcomitans inhibited211hyphaeformationandbiofilmformationbyC.albicans(Bachtiar etal.2014).AI-212 にproducedbyF.nucleatumhadadifferentialeffectonbiofilm formationwhen213culturedwith twodifferentspeciesoforalstreptococci;biofilm formationwas214enhancedwithS.gordoniibut reducedwithS.oralis (Jangetal.2013).Someof215theseresponsesaredependentontheconcentrationofthesignallingmolecules.216These cell–cell signalling strategies could enable cells to sense and adapt to217

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various environmental stresses and, thereby, regulate (and coordinate) the218expressionofgenesthatinfluencetheabilityofpathogenstocausedisease.219220Genetransfer 221Thecloseproximityofcells inbiofilmsprovides idealconditions for horizontal222gene transfer (HGT).HGT involveseither acquisitionofDNA from co-resident223speciesor fromexogenoussources(Petersenet al.2005;Roberts┃Kreth2014).224DNA can be transferred through: transduction by bacterial viruses225(bacteriophages), conjugation by bacterial pili, and transformation by DNA226uptake involvingnaturally competentbacteria; inaddition to themechanisms227above, DNA can also be transferred viamembrane vesicles inGram-negative228bacteria(Olsenetal.2013).HGTallowsoral bacteriatosamplefromanimmense229metagenome,andinthiswayincreasetheir adaptivepotentialtochangesinthe230oralenvironment (Roberts┃Kreth2014).For instance,metabolicadaptability231to carbohydrate-rich environments such as the oral cavity and gut has been232foundinaLactobacillussalivariusstraincarryingaplasmidwithgenesinvolved233inglycolysis(Roberts┃Kreth2014).HGTisthought tobethemainmechanism234inacquiringantibiotic resistancegenes (ARGs),whichare richlypresent in the235oralcavity(Sukumar etal.2016).236

Asdescribedearlier,signallingmoleculessuchascompetence-stimulating237peptide (CSP)markedly increase the ability of recipient cells to take upDNA238(SenadheeraandCvitkovitch2008).Extracellular DNA(eDNA)isacomponentof239thebiofilmmatrixandplaysacritical role inadhesionand inpossiblenutrient240storage and as a potential source of phosphate and other ions (Jakubovics ┃241Burgess 2015). eDNA release has been demonstrated in dual species242experiments with S. mutans and S. gordonii through S. mutans competence-243inducedbacteriocinproduction(Krethetal.2005);Gram-negativebacteriaalso244release eDNA, including Veillonella spp (Hannan et al. 2010), Porphyromonas245gingivalisandF.nucleatum(AliMohammedetal.2013).246

Evidence for horizontalgene transfer indentalbiofilmshas come from247the discovery that both resident (S. mitis, S. oralisょ and pathogenic (S.248pneumoniaeょ bacteria isolated from the naso-pharyngeal area possess genes249conferringpenicillinresistance thatdisplay acommonmosaicstructure (Chiet250

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al. 2007). Similar evidence suggests sharing of genes encoding for penicillin-251bindingproteinsamong residentoralandpathogenicNeisseriaspecies (Bowler 252et al. 1994), and IgA protease encoding genes among a range of oral 253streptococcalspecies(Poulsenetal.1998).254255Antagonisticinteractions256 Aconsiderablenumber ofstudiesaddressedantagonistic interactions involving257inter-species and inter-kingdom competition or “warfare”. The production of258antagonisticcompoundssuchasbacteriocins,hydrogenperoxide,organicacids,259different enzymes and release of lytic phages are just a few examples of260“weapons” that can give an organism a competitive advantage during261colonisationandwhencompetingwithother microbes(Table3).262

Bacteriocinsandbacteriocin-likesubstancesareproducedbybothGram-263positiveandGram-negativebacteria,with themost studiedoral speciesbeing264streptococci,andexamples includemutacinproducedbyS.mutans(Merrittand265Qi2012),sanguicinbyS.sanguinisandsalivaricinbyS.salivarius(Jakubovicset 266al.2014).Two typesofmutacinhavebeendetected; lantibiotics,whichhave a267broadspectrumofactivity,andthemorecommonnon-lantibiotics,whichhavea268narrower antimicrobial range (Merritt andQi2012).Lactobacillialsoproduce269bacteriocins,andarebeingevaluatedaspotentialoralprobiotics largelydueto270their antimicrobialproperties; for example, reuterin from Lactobacillusreuteri 271was active against selected periodontal and cariogenic bacteria (Kang et al.2722011).273 Bacterial “warfare” implies thatoneof the interactingpartnersbenefits274at theexpenseof theother.Thishasbeenshownwith two taxaoccupying the275same niche ┽ S. gordonii and S. mutans┸ where S. gordonii had a competitive276advantageover S.mutanswhenusingaminosugarsfromsalivaryglycoproteins277as an energy source: S. gordonii released hydrogen peroxide that inhibited278transcription of S. mutans genes responsible for the metabolism of these279compounds (Zengetal.2016). Indeed,hydrogenperoxide isoneof themost280studiedagentsproducedindentalbiofilmsbutitsimpactontheoralmicrobiota281is complex and difficult to predict. Under aerobic conditions (as could occur 282duringearlystagesofbiofilmformation),Streptococcussanguinisproduceshigh283

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concentrations ofhydrogen peroxide that are capable of inhibiting a range of284Gram-positive species (Holmberg ┃ Hallander 1972; Holmberg ┃ Hallander 2851973; Kreth et al. 2016); much lower concentrations are generated during286anaerobicgrowth.Streptococcusmutansissusceptibletohydrogenperoxide,but287strainsthatproducemutacinareableto inhibitother streptococci(Ashbyetal.2882009; Ryan ┃ Kleinberg 1995). Hydrogen peroxide production has been289proposed as a major mechanism for controlling the levels of putative290periodontopathicbacteria indentalplaque(Hillman┃Shivers1988;Hillmanet291al.1985).However,other bacteria in thesupragingivalbiofilms (e.g.Neisseria┸292Haemophilus and Actinomyces species) are also able to degrade hydrogen293peroxide,and little freeperoxidecanbedetected inplaque (Ryan┃Kleinberg2941995). Thus, there may be varying concentrations of hydrogen peroxide in295differentregionsofthebiofilm,andthebalancebetweensymbiosisanddysbiosis296maydependon thecomplex interplaybetweenmultipleantagonisticmicrobial297interactions.298

Counter-intuitively,antagonistic interactionsmightalsobebeneficial to299both partners involved andmight even stimulate the fitness of themicrobial300community (Stacy et al. 2014). In the presence of oxygen, A.301actinomycetemcomitansthat cross-feedswithlactateproducedbyS.gordonii ┸has302to survive high concentrations of hydrogen peroxide released by S. gordonii303(Figure 2).To ameliorate oxidative stress, A. actinomycetemcomitans not only304expresses catalase (H2O2-detoxifying enzyme), but also responds to elevated305H2Oにby induction of Dispersin B ‒ an enzyme that promotes dispersal of A.306actinomycetemcomitans biofilms, resulting in increased physical distance307between theA.actinomycetemcomitansand theH2O2-producingS.gordonii ┻On308the other hand, S. gordonii ┸ which does notmake its own catalase, is cross-309protectedbyA.actinomycetemcomitansfromself-inflictedoxidativestress.310 A highly diverse oral bacteriophage gene pool has been discovered311through ametagenomicsapproach (Dalmassoetal.2015;Edlundetal.2015a;312Naiduetal.2014;Prideetal.2012).Phagesarebacterialviruses thatmay lyse313competingcells.Theproductionofantagonisticfactorswillnot necessarily lead314to the complete exclusion of sensitive species as the presence of distinct 315microhabitatswithinabiofilmsuchasplaqueenablebacteria tosurviveunder 316

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conditionsthatwouldbe incompatibletothem inahomogeneousenvironment.317Noteworthy, although parasitic by their nature, phagesmight have beneficial 318role in theoral ecosystem: a recent comparisonof thebacteria-phagenetwork319revealed that phages supported a complexmicrobial community structure in320healththatwasabsent duringperiodontaldisease(Wangetal.2016).321

Antagonism will also be amechanism whereby exogenous species are322preventedfromcolonizingtheoralcavity(bacterialinterferenceor colonization323resistance).Oralstreptococcihavebeenshowntointerferewithcolonizationby324Pseudomonasaeruginosa throughnitrite-mediated interference (Scoffield┃Wu3252015; Scoffield ┃ Wu 2016), while a sophisticated colonization resistance326structure has been described in an invitromurine oralmicrobial community327with the ‘Sensor’ (Streptococcussaprophyticusょ sensing the intruding non-oral328Escherichia coli strain and producing diffusible signals to the ‘Mediator’329(Streptococcus infantisょ that de-represses the capacity of the ‘Killer ’330(Streptococcussanguinisょ toproducehydrogenperoxide, resulting in inhibition331oftheinvadingE.coli (Heetal.2014).332

333Ecologicaldriverstowardsdysbiosisanddisease334When theoralenvironment changes, theecologyof theecosystem isaffected.335This has an impact on the outcome of the interactions among the micro-336organisms in thebiofilms,whichwillaffect theproportionsof themembersof337thecommunity,andcan increase the riskofdisease (dysbiosis).Twoscenarios338willbedissectedbelow:oneleadingtowardsacariogenicandtheother towards339 aperiodontopathogenicecosystem.340 Dental caries isassociatedwithan increased frequencyofdietarysugar 341intake. Thesesugarsaremetabolised rapidly toacid (mainly lacticacid)anda342low pHisgeneratedwithinthebiofilm.LactatecanbeutilisedbyVeillonellaspp.,343andother species,e.g.Neisseria(Hoshino┃Araya1980)┸Haemophilus(Traudt┃344Kleinberg 1996), Aggregatibacter (Brown ┃ Whiteley 2007), Porphyromonas345(Lewisetal.2009),andActinomyces(Takahashi ┃Yamada,1996),andconverted346toweaker acids.Fewer carious lesionsand less lactate inplaquewasmeasured347in rats inoculated with S. mutans and Veillonella alcalescens than in animals348

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infectedwithS.mutansalone(vander Hoevenetal.1978).Higher proportionsof349Veillonella spp. have been detected in samples from caries lesions when350comparedtoplaquefromhealthyenamel(Grosset al.2012),perhapsbecauseof351the increased glycolytic activity and higher levels of lactate at these sites.352SymbiosisbetweenVeillonella andS.mutans hasbeendemonstrated inmixed353cultures: when Veillonella parvula was added to the pair of antagonists (S.354mutansandS.gordonii),itmitigatedtheinhibitoryeffectsofS.gordoniionsugar355metabolismandgrowthofS.mutans(Liuetal.2011).356 The frequentconditionsof lowpH inbiofilmsassociatedwithcariesare357inhibitorytothegrowthofmanyofthebacteriaassociatedwithenamelhealth,358resultingindecreasedmicrobialdiversity(Grossetal.2012;Jiangetal.2011;Li359et al. 2007; Peterson et al. 2013). Repeated conditions of low pH alter the360competitivenessofmembersof thebiofilmcommunityandselect for increased361proportions of acidogenic and acid-tolerating bacteria including mutans362streptococci, lactobacilli (Bradshaw et al. 1989), low-pH non-S. mutans363streptococci and bifidobacteria (Marsh 1994; Takahashi ┃ Nyvad 2008).364Sucrose-induceddysbiosis resultsnot only in reduced taxonomicdiversity,but365also in a changed metaproteome, as recently shown in microcosms where366proteinsinvolvedinacidtoleranceandacidproductiondominatedthedysbiotic367biofilms(Rudneyetal.2015).368 A counter mechanism against acidification of the ecosystem is alkali 369production by the members of the community, mainly through ammonia370production fromarginineandurea (Burne┃Marquis2000;Huangetal.2015;371Liu et al. 2012; Shu et al. 2003; Takahashi 2015). Recently, by applying a372metatranscriptomics andmetabolomics approach, amuch higher diversity in373alkali-generating pathwayswithin complex oral biofilms has been discovered,374including glutamate dehydrogenase, threonine and serine deaminase, and375upregulationinmembraneproteinsinvolvedinammoniagasconductionbesides376the urease activity and arginine deiminase system (Edlund et al. 2015b).377Additionally, this study revealed that Veillonella species are well adapted378towards acid stress by upregulating variouspathways that contributed to pH379recovery.380

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Thus, unlike health, dental caries is associated with a shift in the381composition of the biofilm to a community that is dominated by a strongly382saccharolyticandacid-tolerantmicrobiota leading to a lossofdiversity,and a383reduction in levelsandactivityofbeneficialbacteria(Grossetal.2012;Jianget384al.2011;Lietal.2007;Petersonetal.2013),althoughthediversitymayincrease385whenthelesionpenetratesdentine,perhapsreflectingimportantenvironmental386changes(Simón-Soroetal.2014).387 Incontrast,theaccumulationofmicrobialbiomassaroundthegingival388margininducesaninflammatoryresponse.Thisresultsinanincreasedflow of389GCF,whichdeliversnot onlycomponentsofthehostdefences(e.g┻390immunoglobulins,complement,neutrophils,cytokines,etcょ(Ebersole2003),but,391inadvertently,hostmoleculesthatcanactassubstratesfor proteolyticbacteria.392Someofthesehostmoleculesalsocontainhaemin(e.g┻haptoglobin,haemopexin,393haemoglobin),whichisanessentialcofactor for thegrowthofpotential394periodontopathogenssuchasP.gingivalis(Olczaketal.2005).Thechangein395localenvironmentalconditionsassociatedwithinflammationwill alter the396competitivenessandoutcomeofmultipleinteractionsamongthemicrobesthat397makeupthesubgingivalmicrobiota,leadingtosubstantialchangesinthe398microbialcompositionofthebiofilm.Althoughthereisagreementthatthereare399major changesintheproportionsofindividualspeciesinbiofilmsfrominflamed400sites(for examples,seereviewsbyDiazetal.,2016;Pérez-Chaparroetal.2014),401thereareconflictingreportsonwhether thediversityoftheresultantmicrobial402communitiesisaltered.Thediversitymayincreaseingingivitis(Kistler etal.,4032013;Schincagliaetal.,2016),buttheevidencefor chronicperiodontitisismore404contentious(Abuslemeetal.,2013;Hongetal.,2015;Kirstetal.,2015;Parket405al.,2015).406 The inflammatory response can influence the subgingival407microbiota in twoways: (1)via the impactofthehostdefences,and (2)by the408resultant changes to the environment. The innate defences will inhibit409susceptiblespecies,butanumber ofperiodontalpathogens,suchasP.gingivalis┸410can subvert the host response, for example, by degrading complement,411interferingwithneutrophilfunction,andblockingphagocytosis(for reviews,see412

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Hajishengallis┃Lamont,2014;Mysaketal,2014;Slaney┃Curtis,2008).Thus,413sensitive species will be eliminated (though somemay survive due to cross-414protection from neighbouring organisms), but those that can tolerate the415inflammatory response will flourish. It has been argued that the microbial416consortiathatareassociatedwithperiodontitisare‘inflammo-philic’inthatthey417have adapted to not only endure inflammation but also to exploit the altered418environmentalconditions (Hajishengallis,2014),suchassmall rises inpHand419temperature(Eggertetal.1991;Fedi┃Killoy1992;Haffajeeet al.1992;Nyako420et al. 2005). Such small changes to the local environment can alter gene421expression and increase the competitiveness of species such as P. gingivalis422withinmicrobialcommunities(Marshetal.,1993).However,amoresubstantial 423change to the inflamedpocket is thealterednutrient statusas a result of the424increased flow ofGCF. Inorder tostudy the impactof this, laboratorystudies425have been performed using serum as a surrogate for GCF, and complex426nutritional inter-relationshipsamongsubgingivally-derivedmicrobialconsortia427havebeenobserved (ter Steeg ┃vander Hoeven1989; ter Steegetal.1987).428When biofilms from patients with chronic periodontitis were inoculated into429pre-reduced (i.e. anaerobic) heat-inactivated human serum, the microbial 430compositionof the consortia changedover timeand these changes correlated431withdistinctstagesinglycoproteinbreakdowninvolvingbacteriawithdifferent432metabolic capabilities. Initially, carbohydrate side-chains were removed by433organismswithcomplementaryglycosidaseactivities; thiswas followedby the434hydrolysis of the protein core by obligately anaerobic bacteria leading to435extensive amino acid fermentation.Significantly, individual species grew only436poorlyinpurecultureonserum(ter Steeg┃vander Hoeven1989).437 Numerous nutritional inter-dependencies and physical438interactionswilldevelopamongthespeciescopingwiththearrayofnovelhost439factorsproducedduringtheinflammatoryresponse.For example,acomplexbut440symbioticmetabolicrelationshiphasbeendemonstratedinlaboratorystudiesof441P. gingivalis and T. denticola (Grenier, 1992; Tan et al., 2014). Early studies442demonstrated that isobutyric acid produced by P. gingivalis stimulated the443growthofT.denticola┸whilesuccinicacidgeneratedbyT.denticolaenhancedthe444

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growthofP.gingivalis(Grenier,1992).Morerecentstudieshaveshownthatthe445biomass is higher when both species are grown in co-culture, and glycine446producedbyP.gingivalis isutilisedby thespirochaete (Tanet al.,2014).Both447species respond to thepresenceof theother asseenbychanges inglobalgene448expression inboth species. Similarly, thegrowthof certain species thathave449been previously described as being ‘unculturable’ (e.g┻ Fretibacterium450fastidiosum┸PrevotellaHOT-376,TannerellaHOT-286)hasbeenshownrecently451to be due to their dependence on siderophores and to the close physical452proximityof ‘helper’strains (Vartoukianetal.2016a;Vartoukianetal.2016b).453Other studieshavedemonstrated the importanceofclosephysicalassociations454tobiofilmformationby interactingspeciesofGram-negativeanaerobicbacteria455(Sharmaetal.,2005;Okudaetal.,2012).456

Periodontaldiseasesmaybeanexampleof ‘pathogenicsynergism’ (van457Steenbergen et al. 1984), inwhich disease is a consequence of the combined458activity of an interacting consortium in which each member is only weakly459virulent.Differentspecieswouldundertake adistinct roleor function inorder 460for the consortium to persist, and cause disease. This is consistentwith the461recent concept of low abundance species (‘keystone pathogens’) having a462disproportionateeffectofthevirulenceofthewholecommunity(Hajishengallis463 ┃Lamont 2012;Hajishengallisetal.2011).Genetransfer canoccur withinthese464communities; this can include not only mobile elements that code for drug465resistancebutalso larger stretchesofDNAthateffectthevirulenceofrecipient466cells, for example,P.gingivalis possesses a ‘pathogenicity island’ (Curtis et al.4671999).468

Evidence for the role of the entire community and not just a few469pathogens in dysbiosis has recently been delivered by metatranscriptome470analysisofdentalbiofilmsfromsiteswithactiveperiodontaldisease(Yostetal.4712015): various streptococci, Veillonella parvula and Pseudomonas fluorescens472werehighlyactiveintranscribingputativevirulencefactorsbesidesperiodontal473pathogens such as Tannerella forsythia and P.gingivalis┻ The genes thatwere474over-represented at these sites were related to cell motility, lipid A and475peptidoglycanbiosynthesis,andtransportofiron,potassiumandaminoacids.476

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Microbial interactions in such complex consortia could influence477treatment outcomes. Although not advocated for routine use in periodontal478disease, antibioticsare frequentlyused as adjunctive treatment tomechanical479debr idement incaseswithsevereor recurrentdisease (Jepsen┃Jepsen2016).480However,careneeds tobe takenas,apart from theexistenceand inter-species481transfer of resistance genes within microbial communities, が-lactamase482producingbacteriaarecommonlypresentinsubgingivalbiofilmsandtheycould483protectneighbouringorganisms thatshouldbesusceptible to theactionof the484drug(Ramsetal.2013;vanWinkelhoffetal.1997;Walker etal.1987).485

Attempts have also been made to exploit antagonistic interactions to486resolve both periodontal disease and caries. For periodontal therapy, either 487bacterialinterferencehasbeenappliedbydeliberatelyimplantingbeneficial oral488bacteriaintoatreatedpocket (Teughelsetal.2013;vanEsscheetal.2013)or by489using predatory protozoa, such as Bdellovibrio species (Dashiff and Kadouri4902011;Loozenetal.2015;VanEsscheetal.2011),or bacteriophage[reviewedby491Allaker ┃ Douglas (2009)], while for caries prevention, different approaches492(e.g., lozenges, milk, yoghurt) with probiotic bacteria that are antagonistic493against S. mutans have been tr ied (Cagetti et al. 2013). A recent systematic494reviewontheuseofprobioticsinmanagingoraldiseasesconcludedthatthereis495sufficientevidence for supporting theuseofprobiotics in thecaseofgingivitis496andperiodontitisbutnot for caries(Gruner etal.2016),thoughthisisanareain497whichmoreresearchisrequired.498Conclusions499Microbialcommunities,suchasthosefoundindentalbiofilms,display‘emergent500properties’,i.e.their propertiesaremorethanthesumofthecomponent species,501andtheir characteristicscannotbeinferredfromstudiesofindividualorganisms502(Diazetal.2014).Themicrobiotaisstructurallyandfunctionallyorganised,and503it has been argued that such microbial communities could be considered as504primitivemulti-cellular organisms (Caldwelletal.1997;Ereshefsky┃Pedroso5052015). Inhealth,numerous interactionscontribute tostabilityandresilienceof506

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the ecosystem against environmental perturbations (Alexander, 1971;Marsh,5071989).508

Ifcertainkeyenvironmentalpressuresexceedthresholdsthatvaryfrom509patient to patient, then the competitiveness of certain bacteria is altered and510dysbiosis can occur, leading to caries or periodontal diseases. In caries and511periodontaldiseases,changesinthenutrient statusat thesiteduetoincreasesin512fermentable carbohydrates (and the resultant acidic conditions) and host513proteins (including haemin-containing molecules), respectively, disrupt the514microbial interactions thatcontrol thebalanceof themicrobialcommunities in515health. Effective prevention of dental disease will require interference with516thesefactorsthatdrivedysbiosis(Marsh2003),andagreater understandingof517microbialinteractionscouldleadtostrategiestoactivelypromoteoralhealth.518

Thecurrent literaturesearch ledustothefollowingconclusions:1)oral519microbial interactionsbelong to ahighlystudiedanddiverse topic,whichwas520toobroadfor asystematicreview;2)mostoralmicrobialinteractionshavebeen521investigated in laboratory systems, and occasionally animal models, and522thereforesomecautionshouldbeexercisedwhenextrapolatingthesefindingsto523events inhumans; 3) themajorityof the interactionsstudied involvebacteria524only, while other segments of the oral microbiota (fungi, Archaea┸ viruses,525protozoa) are understudied; 4) current technological advances (e.g┻526metagenomics, metatranscriptomics, metaproteomics, metabolomics, spectral527imagingfluorescenceinsituhybridization,etc)enablethestudyofmorecomplex528communitylevelinteractions,includingthoseamongmembersofthemicrobiota529fromdifferentkingdoms(Diazetal.2014)rather thanjusttheconventionaldual530speciesstudies; 5)bothsynergisticandantagonistic interactionscontribute to531theecologicalstabilityofthemicrobialcommunitythatcharacterisesoralhealth;532and6)moreattentionneedstobefocussedonwhatmicro-organismsaredoing533within these microbial communities (Takahashi 2015), rather than just534cataloguingwhichonesarepresent.Theoral microbiome inhealthanddisease535mightbebetter describedbyaseriesoffunctionsandinteractions,rather thanas536 a listof individualorganisms,as these functionsmightnot beprovidedby the537samemicrobesindifferentpeople(Lloyd-Priceetal.2016).538

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539References540Abusleme, L., Dupuy, A.K., Dutzan, N., Silva, N., Burleson, J.A., Strausbaugh, L.D., Gamonal, J.541

& Diaz, P.I. (2013). The subgingival microbiome in health and periodontitis and its542relationship with community biomass and inflammation. ISME J 7: 1016-1025.543

Alexander, M. (1971). Microbial Ecology, pp. 207-223. Wiley, London.544Ali Mohammed, M.M., Nerland, A.H., Al-Haroni, M. & Bakken, V. (2013). Characterization of545

extracellular polymeric matrix, and treatment of Fusobacterium nucleatum and546Porphyromonas gingivalis biofilms with DNase I and proteinase K. J Oral Microbiol 5:54710.3402/ jom.v3405i3400.20015.548

Allaker, R.P. & Douglas, C.W.I. (2009). Novel anti-microbial therapies for dental plaque-549related diseases. Int J Antimicrob Ag 33: 8-13.550

Ashby, M.T., Kreth, J., Soundarajan, M. & Sivuilu, L.S. (2009). Influence of a model human551defensive peroxidase system on oral streptococcal antagonism. Microbiol 155: 3691-5523700.553

Bachtiar, E.W., Bachtiar, B.M., Jarosz, L.M., Amir, L.R., Sunarto, H., Ganin, H., Meijler, M.M. &554Krom, B.P. (2014). Ai-2 of Aggregatibacter actinomycetemcomitans inhibitsCandida555albicans biofilm formation. Front Cell Infect Microbiol 4: 94.556

Bergeron, L.J. & Burne, R.A. (2001). Roles of fructosyltransferase and levanase-sucrase of557Actinomyces naeslundii in fructan and sucrose metabolism. Infect Immun 69: 5395-5585402.559

Bowler, L.D., Zhang, Q.Y., Riou, J.Y. & Spratt, B.G. (1994). Interspecies recombination560between the pena genes of Neisseria meningitidis and commensal Neisseria species561during the emergence of penicillin resistance in N. meningitidis: Natural events and562laboratory simulation. J Bacteriol 176: 333-337.563

Bradshaw, D.J., Homer, K.A., Marsh, P.D. & Beighton, D. (1994). Metabolic cooperation in564oral microbial communities during growth on mucin. Microbiol 140: 3407-3412.565

Bradshaw, D.J., McKee, A.S. & Marsh, P.D. (1989). Effects of carbohydrate pulses and pH on566population shifts within oral microbial communities in vitro. J Dent Res 68: 1298-5671302.568

Brown, S.A. & Whiteley, M. (2007). A novel exclusion mechanism for carbon resource569partitioning in Aggregatibacter actinomycetemcomitans. J Bacteriol 189: 6407-6414.570

Burne, R.A. & Marquis, R.E. (2000). Alkali production by oral bacteria and protection against571dental caries. FEMS Microbiol Lett 193: 1-6.572

Cagetti, M.G., Mastroberardino, S., Milia, E., Cocco, F., Lingstrom, P. & Campus, G. (2013).573The use of probiotic strains in caries prevention: A systematic review. Nutrients 5:5742530-2550.575

Caldwell, D.E., Wolfaardt, G.M., Korber, D.R. & Lawrence, J.R. (1997). Do bacterial576communities transcend darwinism? In: Advances in microbial ecology. Ed. J. G.577Jones. Boston, MA, Springer US: 105-191.578

Chi, F., Nolte, O., Bergmann, C., Ip, M. & Hakenbeck, R. (2007). Crossing the barrier:579Evolution and spread of a major class of mosaic pbp2x in Streptococcus pneumoniae,580S. mitis and S. oralis. Int J Med Microbiol 297: 503-512.581

Curtis, M.A., Hanley, S.A. & Aduse-Opoku, J. (1999). The rag locus of Porphyromonas582gingivalis: A novel pathogenicity island. J Periodontal Res 34: 400-405.583

Dalmasso, M., de Haas, E., Neve, H., Strain, R., Cousin, F.J., Stockdale, S.R., Ross, R.P. & Hill,584C. (2015). Isolation of a novel phage with activity against Streptococcus mutans585biofilms. PLoS ONE 10: e0138651.586

20

Dashiff, A. & Kadouri, D.E. (2011). Predation of oral pathogens by Bdellovibrio bacteriovorus587109j. Mol Oral Microbiol 26: 19-34.588

Diaz, P.I. (2012). Microbial diversity and interactions in subgingival biofilm communities.589Front Oral Biol 15: 17-40.590

Diaz, P.I., Hoare, A. & Hong, B.Y. (2016). Subgingival microbiome shifts and community591dynamics in periodontal diseases. J Calif Dent Assoc 44: 421-435.592

Diaz, P.I., Strausbaugh, L.D. & Dongari-Bagtzoglou, A. (2014). Fungal-bacterial interactions593and their relevance to oral health: Linking the clinic and the bench. Front Cell Infect594Microbiol 4.595

Diaz, P.I., Xie, Z., Sobue, T., Thompson, A., Biyikoglu, B., Ricker, A., Ikonomou, L. & Dongari-596Bagtzoglou, A. (2012). Synergistic interaction between Candida albicans and597commensal oral streptococci in a novel in vitro mucosal model. Infect immun 80:598620-632.599

Diaz, P.I., Zilm, P.S. & Rogers, A.H. (2002). Fusobacterium nucleatum supports the growth of600Porphyromonas gingivalis in oxygenated and carbon-dioxide-depleted601environments. Microbiol 148: 467-472.602

Dige I., Gronkjar, L. & Nyvad, B. (2014). Molecular studies of the structural ecology of natural603occlusal caries. Caries Res 48:451-460.604

Dige, I., Nilsson, H., Kilian, M. & Nyvad, B. (2007). In situ identification of streptococci and605other bacteria in initial dental biofilm by confocal laser scanning microscopy and606fluorescence in situ hybridization. Eur J Oral Sci 115: 459-467.607

Ebersole, J.L. (2003). Humoral immune responses in gingival crevice fluid: Local and systemic608implications. Periodontol 2000 31: 135-166.609

Edlund, A., Santiago-Rodriguez, T.M., Boehm, T.K. & Pride, D.T. (2015a). Bacteriophage and610their potential roles in the human oral cavity. J Oral Microbiol 7: 27423.611

Edlund, A., Yang, Y., Yooseph, S., Hall, A.P., Nguyen, D.D., Dorrestein, P.C., Nelson, K.E., He,612X., Lux, R., Shi, W. & McLean, J.S. (2015b). Meta-omics uncover temporal regulation613of pathways across oral microbiome genera during in vitro sugar metabolism. ISME J6149: 2605-2619.615

Eggert, F.M., Drewell, L., Bigelow, J.A., Speck, J.E. & Goldner, M. (1991). The pH of gingival616crevices and periodontal pockets in children, teenagers and adults. Arch Oral Biol 36:617233-238.618

Ereshefsky, M. & Pedroso, M. (2015). Rethinking evolutionary individuality. PNAS 112:61910126-10132.620

Fedi, P.F. & Killoy, W.J. (1992). Temperature differences at periodontal sites in health and621disease. J Periodontol 63: 24-27.622

Grenier, D. (1992). Nutritional interactions between two suspected periodontopathogens,623Treponema denticola and Porphyromonas gingivalis. Infect Immun 60: 5298-5301.624

Gross, E.L., Beall, C.J., Kutsch, S.R., Firestone, N.D., Leys, E.J. and Griffen, A.L. (2012). Beyond625Streptococcus mutans: Dental caries onset linked to multiple species by 16S rRNA626community analysis. PLoS ONE7: e47722.627

Gruner, D., Paris, S. & Schwendicke, F. (2016). Probiotics for managing caries and628periodontitis: Systematic review and meta-analysis. J Dent 48: 16-25.629

Guo, L., He, X. & Shi, W. (2014). Intercellular communications in multispecies oral microbial630communities. Front Microbiol 5: 328.631

Haffajee, A.D., Socransky, S.S. & Goodson, J.M. (1992). Subgingival temperature (i). Relation632to baseline clinical parameters. J Clin Periodontol 19: 401-408.633

Hajishengallis, G. (2014). Immunomicrobial pathogenesis of periodontitis: Keystones,634pathobionts, and host response. Trends Immunol 35: 3-11.635

Hajishengallis, G. & Lamont, R.J. (2014). Breaking bad: Manipulation of the host response by636porphyromonas gingivalis. Eur J Immunol 44: 328-338.637

21

Hajishengallis, G. & Lamont, R.J. (2012). Beyond the red complex and into more complexity:638The polymicrobial synergy and dysbiosis (PSD) model of periodontal disease639etiology. Mol Oral Microbiol 27: 409-419.640

Hajishengallis, G., Liang, S., Payne, M.A., Hashim, A., Jotwani, R., Eskan, M.A., McIntosh,641M.L., Alsam, A., Kirkwood, K.L., Lambris, J.D., Darveau, R.P. & Curtis, M.A. (2011).642Low-abundance biofilm species orchestrates inflammatory periodontal disease643through the commensal microbiota and complement. Cell Host Microbe 10: 497-644506.645

Hannan, S., Ready, D., Jasni, A.S., Rogers, M., Pratten, J. & Roberts, A.P. (2010). Transfer of646antibiotic resistance by transformation with eDNA within oral biofilms. FEMS647Immunol Med Microbiol 59: 345-349.648

He, X., McLean, J.S., Guo, L., Lux, R. & Shi, W. (2014). The social structure of microbial649community involved in colonization resistance. ISME J 8: 564-574.650

Hillman, J.D. & Shivers, M. (1988). Interaction between wild-type, mutant and revertant651forms of the bacterium Streptococcus sanguis and the bacterium Actinobacillus652actinomycetemcomitans in vitro and in the gnotobiotic rat. Arch Oral Biol 33: 395-653401.654

Hillman, J.D., Socransky, S.S. & Shivers, M. (1985). The relationships between streptococcal655species and periodontopathic bacteria in human dental plaque. Arch Oral Biol 30:656791-795.657

Hojo, K., Nagaoka, S., Ohshima, T. & Maeda, N. (2009). Bacterial interactions in dental658biofilm development. J Dent Res 88: 982-990.659

Holmberg, K. & Hallander, H.O. (1972). Interference between gram-positive microorganisms660in dental plaque. J Dent Res 51: 588-595.661

Holmberg, K. & Hallander, H.O. (1973). Production of bactericidal concentrations of662hydrogen peroxide by Streptococcus sanguis. Arch Oral Biol 18: 423-434.663

Homer, K.A. & Beighton, D. (1992). Synergistic degradation of bovine serum albumin by664mutans streptococci and other dental plaque bacteria. FEMS Microbiol Lett 69: 259-665262.666

Hong, B.-Y., Furtado Araujo, M.V., Strausbaugh, L.D., Terzi, E., Ioannidou, E. and Diaz, P.I.667(2015). Microbiome profiles in periodontitis in relation to host and disease668characteristics. PLoS ONE 10: e0127077.669

Hoshino, E. & Araya, A. (1980). Lactate degradation by polysaccharide-producing Neisseria670isolated from human dental plaque. Arch Oral Biol 25: 211-2.671

Huang, R., Li, M. & Gregory, R.L. (2011). Bacterial interactions in dental biofilm. Virulence 2:672435-444.673

Huang, X., Schulte, R.M., Burne, R.A. & Nascimento, M.M. (2015). Characterization of the674arginolytic microflora provides insights into ph homeostasis in human oral biofilms.675Caries Res 49: 165-176.676

Jack, A.A., Daniels, D.E., Jepson, M.A., Vickerman, M.M., Lamont, R.J., Jenkinson, H.F. &677Nobbs, A.H. (2015). Streptococcus gordonii comCDE (competence) operon678modulates biofilm formation with Candida albicans. Microbiol 161: 411-421.679

Jakubovics, N.S. (2015a). Intermicrobial interactions as a driver for community composition680and stratification of oral biofilms. J Mol Biol 427: 3662-3675.681

Jakubovics, N.S. (2015b). Saliva as the sole nutritional source in the development of682multispecies communities in dental plaque. Microbiol Spectr 3.683

Jakubovics, N.S. & Burgess, J.G. (2015). Extracellular DNA in oral microbial biofilms. Microbes684Infect / Institut Pasteur 17: 531-537.685

Jakubovics, N.S., Yassin, S.A. & Rickard, A.H. (2014). Community interactions of oral686streptococci. Adv Appl Microbiol 87: 43-110.687

22

Jang, Y.-J., Sim, J., Jun, H.-K. & Choi, B.-K. (2013). Differential effect of autoinducer 2 of688Fusobacterium nucleatum on oral streptococci. Arch Oral Biol 58: 1594-1602.689

Jepsen, K. & Jepsen, S. (2016). Antibiotics/antimicrobials: Systemic and local administration690in the therapy of mild to moderately advanced periodontitis. Periodontol 2000 71:69182-112.692

Jiang, W., Jiang, Y., Li, C. & Liang, J. (2011). Investigation of supragingival plaque microbiota693in different caries status of chinese preschool children by denaturing gradient gel694electrophoresis. Microb Ecol 61: 342-352.695

Kang, M.-S., Oh, J.-S., Lee, H.-C., Lim, H.-S., Lee, S.-W., Yang, K.-H., Choi, N.-K. & Kim, S.-M.696(2011). Inhibitory effect of lactobacillus reuteri on periodontopathic and cariogenic697bacteria. J Microbiol 49: 193-199.698

Kirst, M.E., Li, E.C., Alfant, B., Chi, Y.-Y., Walker, C., Magnusson, I. and Wang, G.P. (2015).699Dysbiosis and alterations in predicted functions of the subgingival microbiome in700chronic periodontitis. Appl Environ Microbiol 81: 783-793.701

Kistler, J.O., Booth, V., Bradshaw, D.J. & Wade, W.G. (2013). Bacterial community702development in experimental gingivitis. PLoS ONE 8: e71227.703

Kolenbrander, P.E. (2011). Multispecies communities: Interspecies interactions influence704growth on saliva as sole nutritional source. Int J Oral Sci 3: 49-54.705

Koo, H., Falsetta, M.L. & Klein, M.I. (2013). The exopolysaccharide matrix: A virulence706determinant of cariogenic biofilm. J Dent Res 92: 1065-1073.707

Kreth, J., Giacaman, R.A., Raghavan, R. & Merritt, J. (2016). The road less traveled – defining708molecular commensalism with Streptococcus sanguinis. Mol Oral Microbiol in press709

Kreth, J., Merritt, J., Shi, W. & Qi, F. (2005). Co-ordinated bacteriocin production and710competence development: A possible mechanism for taking up DNA from711neighbouring species. Mol Microbiol 57: 392-404.712

Lewis, J.P., Iyer, D. & Anaya-Bergman, C. (2009). Adaptation of Porphyromonas gingivalis to713microaerophilic conditions involves increased consumption of formate and reduced714utilization of lactate. Microbiol 155: 3758-3774.715

Li, Y., Ge, Y., Saxena, D. & Caufield, P.W. (2007). Genetic profiling of the oral microbiota716associated with severe early-childhood caries. J Clin Microbiol 45: 81-87.717

Liu, J., Wu, C., Huang, I.-H., Merritt, J. & Qi, F. (2011). Differential response of Streptococcus718mutans towards friend and foe in mixed-species cultures. Microbiol 157: 2433-2444.719

Liu, Y.-L., Nascimento, M. & Burne, R.A. (2012). Progress toward understanding the720contribution of alkali generation in dental biofilms to inhibition of dental caries. Int J721Oral Sci 4: 135-140.722

Lloyd-Price, J., Abu-Ali, G. & Huttenhower, C. (2016). The healthy human microbiome.723Genome Med 8: 51.724

Loozen, G., Boon, N., Pauwels, M., Slomka, V., Rodrigues Herrero, E., Quirynen, M. and725Teughels, W. (2015). Effect of Bdellovibrio bacteriovorus hd100 on multispecies oral726communities. Anaerobe 35: 45-53.727

Mark Welch, J.L., Rossetti, B.J., Rieken, C.W., Dewhirst, F.E. & Borisy, G.G. (2016).728Biogeography of a human oral microbiome at the micron scale. PNAS 113: E791-800.729

Marsh, P. (1989). Host defenses and microbial homeostasis: Role of microbial interactions. J730Dent Res 68: 1567-1575.731

Marsh, P.D. (1994). Microbial ecology of dental plaque and its significance in health and732disease. Adv Dent Res 8: 263-271.733

Marsh, P.D. (2003). Are dental diseases examples of ecological catastrophes? Microbiol 149:734279-294.735

Marsh, P.D., McKee, A.S. & Mothershaw, A.S. (1993). Continuous culture studies of736Porphyromonas gingivalis. In: Biology of the Species Porphyromonas gingivalis. H.N.737Shah, D. Mayrand and R.J. Genco (Eds). Boca Raton, CRC Press, pp. 105-123.738

23

Mazumdar, V., Amar, S. & Segrè, D. (2013). Metabolic proximity in the order of colonization739of a microbial community. PLoS ONE 8: e77617.740

Merritt, J. & Qi, F. (2012). The mutacins of Streptococcus mutans: Regulation and ecology.741Mol Oral Microbiol 27: 57-69.742

Moye, Z.D., Zeng, L. & Burne, R.A. (2014). Fueling the caries process: Carbohydrate743metabolism and gene regulation by Streptococcus mutans. J. Oral Microbiol. 6.744

Mysak, J., Podzimek, S., Sommerova, P., Lyuya-Mi, Y., Bartova, J., Janatova, T., Prochazkova,745J. & Duskova, J. (2014). Porphyromonas gingivalis: Major periodontopathic pathogen746overview. J Immunol Res 2014: 476068.747

Naidu, M., Robles-Sikisaka, R., Abeles, S.R., Boehm, T.K. & Pride, D.T. (2014).748Characterization of bacteriophage communities and crispr profiles from dental749plaque. BMC Microbiol 14: 175.750

Ng, H.M., Kin, L.X., Dashper, S.G., Slakeski, N., Butler, C.A. & Reynolds, E.C. (2016). Bacterial751interactions in pathogenic subgingival plaque. Microb Pathog 94: 60-69.752

Nobbs, A.H. & Jenkinson, H.F. (2015). Interkingdom networking within the oral microbiome.753Microbes Infect 17: 484-492.754

Nyako, E.A., Watson, C.J. & Preston, A.J. (2005). Determination of the ph of peri-implant755crevicular fluid in successful and failing dental implant sites: A pilot study. Arch Oral756Biol 50: 1055-1059.757

Okuda, T., Kokubu, E., Kawana, T., Saito, A., Okuda, K. & Ishihara, K. (2012). Synergy in758biofilm formation between Fusobacterium nucleatum and Prevotella species.759Anaerobe 18: 110-116.760

Olczak, T., Simpson, W., Liu, X. & Genco, C.A. (2005). Iron and heme utilization in761Porphyromonas gingivalis. FEMS Microbiol Rev 29: 119-144.762

Olsen, I., Tribble, G.D., Fiehn, N.-E. & Wang, B.-Y. (2013). Bacterial sex in dental plaque. J763Oral Microbiol 3.764

Park, O.-J., Yi, H., Jeon, J.H., Kang, S.-S., Koo, K.-T., Kum, K.-Y., Chun, J., Yun, C.-H. & Han, S.H.765(2015). Pyrosequencing analysis of subgingival microbiota in distinct periodontal766conditions. J Dent Res 94: 921-927.767

Pérez-Chaparro, P.J., Gonçalves, C., Figueiredo, L.C., Faveri, M., Lobão, E., Tamashiro, N.,768Duarte, P. & Feres, M. (2014). Newly identified pathogens associated with769periodontitis: A systematic review. J Dent Res 93: 846-858.770

Petersen, F.C., Tao, L. & Scheie, A.A. (2005). DNA binding-uptake system: A link between cell-771to-cell communication and biofilm formation. J Bacteriol 187: 4392-4400.772

Poulsen, K., Reinholdt, J., Jespersgaard, C., Boye, K., Brown, T.A., Hauge, M. & Kilian, M.773(1998). A comprehensive genetic study of streptococcal immunoglobulin A1774proteases: Evidence for recombination within and between species. Infect Immun77566: 181-190.776

Pride, D.T., Salzman, J., Haynes, M., Rohwer, F., Davis-Long, C., White, R.A., III, Loomer, P.,777Armitage, G.C. & Relman, D.A. (2012). Evidence of a robust resident bacteriophage778population revealed through analysis of the human salivary virome. ISME J 6: 915-779926.780

Rams, T.E., Degener, J.E. & van Winkelhoff, A.J. (2013). Prevalence of ɴ-lactamase-producing781bacteria in human periodontitis. J Periodontal Res 48: 493-499.782

Rickard, A.H., Palmer, R.J., Blehert, D.S., Campagna, S.R., Semmelhack, M.F., Egland, P.G.,783Bassler, B.L. & Kolenbrander, P.E. (2006). Autoinducer 2: A concentration-dependent784signal for mutualistic bacterial biofilm growth. Mol Microbiol 60: 1446-1456.785

Roberts, F.A. & Darveau, R.P. (2015). Microbial protection and virulence in periodontal tissue786as a function of polymicrobial communities: Symbiosis and dysbiosis. Periodontol7872000 69: 18-27.788

24

Roberts, A.P. & Kreth, J. (2014). The impact of horizontal gene transfer on the adaptive789ability of the human oral microbiome. Front Cell Infect Microbiol 4: 124.790

Rudney, J.D., Jagtap, P.D., Reilly, C.S., Chen, R., Markowski, T.W., Higgins, L., Johnson, J.E. &791Griffin, T.J. (2015). Protein relative abundance patterns associated with sucrose-792induced dysbiosis are conserved across taxonomically diverse oral microcosm793biofilm models of dental caries. Microbiome 3: 69.794

Ryan, C.S. & Kleinberg, I. (1995). Bacteria in human mouths involved in the production and795utilization of hydrogen peroxide. Arch Oral Biol 40: 753-763.796

Schincaglia, G.P., Hong, B.Y., Rosania, A., Barasz, J., Thompson, A., Sobue, T., Panagakos, F.,797Burleson, J.A., Dongari-Bagtzoglou, A. & Diaz, P.I. (2016). Clinical, immune, and798microbiome traits of gingivitis and peri-implant mucositis. J Dent Res in press799

Scoffield, J.A. & Wu, H. (2015). Oral streptococci and nitrite-mediated interference of800Pseudomonas aeruginosa. Infect Immun 83: 101-107.801

Scoffield, J.A. & Wu, H. (2016). Nitrite reductase is critical for Pseudomonas aeruginosa802survival during co-infection with the oral commensal Streptococcus parasanguinis.803Microbiol 162: 376-383.804

Senadheera, D. & Cvitkovitch, D.G. (2008). Quorum sensing and biofilm formation by805Streptococcus mutans. Adv Exp Med Biol 631: 178-188.806

Shu, M., Browngardt, C.M., Chen, Y.-Y.M. & Burne, R.A. (2003). Role of urease enzymes in807stability of a 10-species oral biofilm consortium cultivated in a constant-depth film808fermenter. Infect Immun 71: 7188-7192.809

Sharma, A., Inagaki, S., Sigurdson, W. & Kuramitsu, H.K. (2005). Synergy between tannerella810forsythia and fusobacterium nucleatum in biofilm formation. Oral Microbiol Immun81120: 39-42.812

Simón-Soro, A., Guillen-Navarro, M. & Mira, A. (2014). Metatranscriptomics reveals overall813active bacterial composition in caries lesions. J Oral Microbiol 6:81410.3402/ jom.v3406.25443.815

Slaney, J.M. & Curtis, M.A. (2008). Mechanisms of evasion of complement by816Porphyromonas gingivalis. Front Biosci 13: 188-196.817

Stacy, A., Everett, J., Jorth, P., Trivedi, U., Rumbaugh, K.P. and Whiteley, M. (2014). Bacterial818fight-and-flight responses enhance virulence in a polymicrobial infection. PNAS111:8197819-7824.820

Sukumar, S., Roberts, A.P., Martin, F.E. & Adler, C.J. (2016). Metagenomic insights into821transferable antibiotic resistance in oral bacteria. J Dent Res 95: 969-976.822

Takahashi, N. (2015). Oral microbiome metabolism: From "who are they?" To "what are they823doing?". J Dent Res 94: 1628-1637.824

Takahashi, N. & Nyvad, B. (2008). Caries ecology revisited: Microbial dynamics and the caries825process. Caries Res 42: 409-418.826

Takahashi, N. & Yamada, T. (1996). Catabolic pathway for aerobic degradation of lactate by827Actinomyces naeslundii. Oral Microbiol Immun 11: 193-198.828

Tan, K.H., Seers, C.A., Dashper, S.G., Mitchell, H.L., Pyke, J.S., Meuric, V., Slakeski, N., Cleal,829S.M., Chambers, J.L., McConville, M.J. & Reynolds, E.C. (2014). Porphyromonas830gingivalis and Treponema denticola exhibit metabolic symbioses. PLoS Path 10:831e1003955.832

ter Steeg, P.F. & van der Hoeven, J.S. (1989). Development of periodontal microflora on833human serum. Microb Ecol Health Dis2: 1-10.834

ter Steeg, P.F., van der Hoeven, J.S., de Jong, M.H., van Munster, P.J. & Jansen, M.J. (1987).835Enrichment of subgingival microflora on human serum leading to accumulation of836Bacteroides species, peptostreptococci and Fusobacteria. Antonie Van Leeuwenhoek83753: 261-272.838

25

Teughels, W., Durukan, A., Ozcelik, O., Pauwels, M., Quirynen, M. & Haytac, M.C. (2013).839Clinical and microbiological effects of Lactobacillus reuteri probiotics in the840treatment of chronic periodontitis: A randomized placebo-controlled study. J Clin841Periodontol 40: 1025-1035.842

Traudt, M. & Kleinberg, I. (1996). Stoichiometry of oxygen consumption and sugar, organic843acid and amino acid utilization in salivary sediment and pure cultures of oral844bacteria. Arch Oral Biol 41: 965-978.845

van der Hoeven, J.S., Toorop, A.I. & Mikx, R.H. (1978). Symbiotic relationship of Veillonella846alcalescens and Streptococcus mutans in dental plaque in gnotobiotic rats. Caries847Res 12: 142-147.848

van Essche, M., Loozen, G., Godts, C., Boon, N., Pauwels, M., Quirynen, M. & Teughels, W.849(2013). Bacterial antagonism against periodontopathogens. J Periodontol 84: 801-850811.851

van Essche, M., Quirynen, M., Sliepen, I., Loozen, G., Boon, N., van Eldere, J. & Teughels, W.852(2011). Killing of anaerobic pathogens by predatory bacteria. Mol Oral Microbiol 26:85352-61.854

van Steenbergen, T.J., van Winkelhoff, A.J. & de Graaff, J. (1984). Pathogenic synergy: Mixed855infections in the oral cavity. Antonie Van Leeuwenhoek 50: 789-798.856

van Winkelhoff, A.J., Winkel, E.G., Barendregt, D., Dellemijn-Kippuw, N., Stijne, A. & van der857Velden, U. (1997). Ȳ-lactamase producing bacteria in adult periodontitis. J Clin858Periodontol 24: 538-543.859

Vartoukian, S.R., Adamowska, A., Lawlor, M., Moazzez, R., Dewhirst, F.E. & Wade, W.G.860(2016a). Cultivation of ‘unculturable’ oral bacteria, facilitated by community culture861and media supplementation with siderophores. PLoS ONE 11: e0146926.862

Vartoukian, S.R., Moazzez, R.V., Paster, B.J., Dewhirst, F.E. & Wade, W.G. (2016b). First863cultivation of health-associated Tannerella sp. Hot-286 (BU063). J Dent Resdoi:86410.1177/0022034516651078865

Wade, W.G. (2013). The oral microbiome in health and disease. Pharmacol Res 69: 137-143.866Walker, C.B., Tyler, K.Z., Low, S.B. & King, C.J. (1987). Penicillin-degrading enzymes in sites867

associated with adult periodontitis. Oral Microbiol Immun 2: 129-131.868Wang, B.-Y., Deutch, A., Hong, J. & Kuramitsu, H.K. (2011). Proteases of an early colonizer869

can hinder Streptococcus mutans colonization in vitro. J Dent Res 90: 501-505.870Wang, J., Gao, Y. & Zhao, F. (2016). Phage-bacteria interaction network in human oral871

microbiome. Environ Microbiol 18: 2143-2158.872Wei, G.X., van der Hoeven, J.S., Smalley, J.W., Mikx, F.H. & Fan, M.W. (1999). Proteolysis and873

utilization of albumin by enrichment cultures of subgingival microbiota. Oral874Microbiol Immun 14: 348-351.875

Wright, C.J., Burns, L.H., Jack, A.A., Back, C.R., Dutton, L.C., Nobbs, A.H., Lamont, R.J. &876Jenkinson, H.F. (2013). Microbial interactions in building of communities. Mol Oral877Microbiol 28: 83-101.878

Xu, H., Jenkinson, H.F. & Dongari-Bagtzoglou, A. (2014). Innocent until proven guilty:879Mechanisms and roles of streptococcus-Candida interactions in oral health and880disease. Mol Oral Microbiol 29: 99-116.881

Yost, S., Duran-Pinedo, A.E., Teles, R., Krishnan, K. & Frias-Lopez, J. (2015). Functional882signatures of oral dysbiosis during periodontitis progression revealed by microbial883metatranscriptome analysis. Genome Med 7: 27.884

Zeng, L., Farivar, T. & Burne, R.A. (2016). Amino sugars enhance the competitiveness of885beneficial commensals with Streptococcus mutans through multiple mechanisms.886Appl Environ Microbiol 82: 3671-3682.887

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Zijnge, V., van Leeuwen, M.B.M., Degener, J.E., Abbas, F., Thurnheer, T., Gmür, R. &888Harmsen, H.J.M. (2010). Oral biofilm architecture on natural teeth. PLoS ONE 5:889e9321.890

891Acknowledgements892We are thankful to medical information specialist Ilse Jansma at VUmc893Amsterdamfor her adviceonthesearchstrategy.894

895

27

896

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Table1.Detailsonoriginalresearcharticles(N=547)obtainedinPubMed897searchdescribedinSupplementarytableS1.898Membersoftheinteraction(s)

Details

Bacteria-Bacteria(N=473)

- Oralhealth:N=205;Cariespathogen(CP):N=107;Periodontalpathogen(PP):N=149;CP┃PP:N=6;Oralvsnon-oralspecies:N=6

- Antagonism(A):N=116;Synergy(S):N=214;A┃S:N=3;Metabolism:N=98;Communication:N=32;Genetransfer:N=10

Bacteria-Fungi(N=45) - Candidaalbicans:N=40;C.albicansandotherCandidaspecies:N=3;

undefinedCandidaspp.:N=2- Bacteriainvolved:Streptococcusmutans┺N=11;Streptococcusgordonii:

N=10;other oralstreptococci:N=9;Actinomyces┺N=5;StaphylococcusaureusN=2;Aggregatibacteractinomycetemcomitans┸Enterococcusfaecalis┸FusobacteriumnucleatumN=1each;probioticlactobacilli:N=1;microbialconsortiaor microcosm:N=8

- Antagonism:N=11;Synergy:N=33;Communication:N=5Bacteria-Viruses(N=18) - Bacteriophages:N=6;Herpesviruses:N=7;virome:N=3;CRISPR:N=3Bacteria-Archaea(N=4) - Viannaetal(2008;2009),Horzetal(2012;2015):Metanogenic

archaea┃periodontalpathogensFungi-Fungi(N=7) - differentCandidaspecies:N=6;Pichiavsopportunisticfungi

(Mukherjeeetal.,2014)Fungi-Viruses(N=1) - Plotkinetal(2016):HSVenhancesC.albicansadherenceBacteria-Protozoa(N=3) - Dashiff┃Kadouri (2011);vanEsscheetal(2011);Loozenetal(2015):

Bdellovibriobacteriovorus‒bacterialpredator 899

29

Table2.Typesofsynergisticandantagonisticmicrobialinteractionsthatoccur 900amongoralmicro-organismsgrowingindentalplaquebiofilms.901Interactions:Synergistic AntagonisticEnzymecomplementation【enzymesharing BacteriocinproductionFoodchains(foodwebs) HydrogenperoxideproductionCo-adhesion Organicacidproduction/ generationof

inhibitorypHconditionsCell-cellsignalling BacteriophagereleaseGenetransfer Competitionfor essentialnutrientsEnvironmentalmodification Predation 902

30

Figurelegends:903Figure1.Examplesofnutritionalinteractionsamongoralmicro-organisms904(FiguremodifiedfromFigureぬinHojoetal,2009).905Figure2.Modelfor S.gordoniiandA.actinomycetemcomitansinteractions:906hydrogenperoxideproductionbyS.gordonii(Sg)supportslactateconsumption907byA.actinomycetemcomitans(Aa)(FigureS8fromStacyetal2014).A.908actinomycetemcomitansexpressesH2O2-detoxifyingenzymecatalase(KatA),909whichalsoprotectsS.gordoniifromself-inflictedoxidativestress.DispersinB910(DspB)isanenzymethatpromotesdispersalofA.actinomycetemcomitans911biofilmsandresultsinincreaseddistancebetweentheA.actinomycetemcomitans912andtheH2O2-producingS.gordonii ┻Thethreezones(Peroxidekillingzone,913SynergyzoneandCarbonstarvationzone)correspondtodifferent914concentrationsinoxygen,hydrogenperoxideandlactateinthebiofilm,as915indicatedwiththerespectivetr iangles.916Supplementarymaterial:917SupplementaryTableS1.PubMedquerysearchtermsandresults.918

PubMedQuery19-07-2016

Itemsfound/

included

(((("Microbiota"[Mesh] OR"Metagenome"[Mesh] OR"Bacteria"[Mesh] OR"Archaea"[Mesh] ORMicrobiot*[tiab] ORMetagenom*[tiab] ORBacteria*[tiab] OReubacteria*[tiab] ORmicrobiom*[tiab] ORmicroorganism*[tiab] ORmicroorganism*[tiab] ORcommensal*[tiab] ORflora[tiab] ORfloras[tiab] ORmicroflora*[tiab] ORcolonisati*[tiab] ORcolonizati*[tiab] ORmicrobial*[tiab] OR"Viruses"[Mesh] ORVirus*[tiab] ORviral[tiab] OR"Archaea"[Mesh] ORArchaea*[tiab] ORArchaeobacteria*[tiab] ORArchebacteria*[tiab] ORArchaebacteria*[tiab] ORArchaeon[tiab] OR"Fungi"[Mesh] ORFung*[tiab] ORmold*[tiab] ORcandida*[tiab]ORprotozoa*[tiab]ORmycoplasma[tiab]))AND("Mouth"[Mesh] ORMouth*[tiab] ORoral[tiab] OR"cavitasoris"[tiab] ORsaliva*[tiab] ORtongue*[tiab] ORdental[tiab] ORdentition[tiab] ORteeth[tiab] ORtooth[tiab] ORgum[tiab] ORpalat*[tiab] ORlip[tiab] ORlips[tiab] ORgingiva*[tiab] ORperiodont*[tiab]ORuvula*[tiab] OR"Cheek"[Mesh] ORcheek*[tiab] ORbucca*[tiab] OR"PalatineTonsil"[Mesh] ORtonsil*[tiab] OR"Waldeyer ring"[tiab] ORcrevic*[tiab] ORperiodontalpocket*[tiab]))AND("Ecology"[Mesh] ORecolog*[tiab] ORinteraction*[tiab] ORsynerg*[tiab] ORco-occurren*[tiab] ORinhibition[tiab] ORcommunicat*[tiab]ORmetabol*[tiab] ORmetabolism[tiab] ORmetabolic[tiab] OR"metabolism"[Mesh] ORnutrient[tiab] ORgenetransfer[Mesh] ORquorumsensing[tiab]))AND(plaqueORbiofilmORcommunityORconsortium)

3758

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