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This is a repository copy of Dental biofilm: ecological interactions in health and disease..
White Rose Research Online URL for this paper:http://eprints.whiterose.ac.uk/113767/
Version: Accepted Version
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.
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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
17
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
18
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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891Acknowledgements892We are thankful to medical information specialist Ilse Jansma at VUmc893Amsterdamfor her adviceonthesearchstrategy.894