Nutitional Influences

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DOI: 10.1177/08959374970110012101

1997 11: 81ADRG.H.W. Bowden and Y.H. Li

Nutritional Influences on Biofilm Development

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N U T R I T I O N A L I N F L U E N C E S O N B I O F I L MD E V E L O P M E N T

G H W B O W D E N *Y.H. Li

Department of Oral BiologyFaculty of DentistryUniversity of Manitoba780 Bannatyne AvenueW innipeg, Manitoba, Canada R3E 0W 2*To whom correspondence and reprint requests should beaddressedAdv Dent Res ll(l):81-99, April, 1997

A b s t r a c tThe amounts and types of nut r ients in theenvironment influence the development and final bacterialand chemical composi t ion of b iof i lms. In o l igot rophicenvironments , organisms respond to nut r ient s t ress byalterations in their cell morphology and cell surfaces, whichenhance adherence. Little is known of the responses to stressby bacteria in the animal oral cavity. The environment in theoral cavity is less extreme, and saliva provides a constantsource of nutrients. Catabolic cooperation among oral bacteriaallows carbon and nitrogen from salivary glycoproteins to beutilized. Modification of growth environments of oral bacteriacan influence their cell surfaces and adhesion. Studies inexperimental animals have shown that feeding either glucoseor sucrose diets or fasting has little effect on the initial stagesof development of oral biofilms. However, diet can influencethe proportions of different bacterial species later in biofilmdevelopment. Studies of competition among populations incommunities of oral bacteria in vitro and in vivo have shownthe significance of carbon limitation and excess and changesin environmental pH. Relatively few studies have been madeof the role of a nitrogen metabolism in bacterial competitionin biofilms. In keeping with biofilms in nature, oral biofilmsprovide a sequestered habitat, where organisms are protectedfrom removal by saliva and where interactions among cellsgenerate a biofilm environment, distinct from that of saliva.Oral biofilms are an essential component in the etiologies ofcaries and periodontal disease, and understanding the biologyof oral biofilms has aided and will continue to aid in theprevention and treatment of these diseases.Key words: biofilms, nutrition, planktonic communities,ecology.Presented at the 14th International Conference on OralBiology, Biofilms on Oral Surfaces: Implications for Healthand Disease , held March 1 8-20, 1996, in Monterey,California, organized by the International Assoc iation forDental Research and supported by Unilever Dental Research

Black (1886) described isolation and cultivation oforal bacteria and in 1889 (Black, 1889), in adiscussion of reviews of his ideas, made severalinteresting observations: D r. W illiams showed thepresence of these organisms in the gelatinous microbicplaques upon teeth, together with the effects upon the enam elbelow ; that the one thing necess ary to the beginnin g ofcaries is the formation of such a gelatinous microbic plaquein a secluded position where its acids may act without toofrequent disturbance ; and Later I have become able to somanipulate the culture-medium as to cause the gelatine to beformed or not formed at will, and by hardening with formalinhave been able to cut sections of it . These observations byBlack were among the earliest descriptions of a detailedstudy of oral bacteria in biofilms and even included theconcept of manipulation of the culture environment to

encourage polymer formation.Intensive study of oral biofilms has been made for the past40 years. The studies have been wide-ranging, includingplaque structure (Frank and Houver, 1970; Schroeder and deB oever , 1970; N yvad and Fejersko v, 198 9) , bacter ia lcomposition (Bowden et al, 1975; B owden, 1991; Mooreand Moore, 1994) , mechanisms involved in bacter ia ladherence (Gibbons, 1989; Ofek and Doyle, 1994), immuneresponses (Brandtzaeg, 1988; Ebersole, 1990; Michalek andChilders, 1990; Smith and Taubman, 1992), and the conceptsof competition and responses to the environment among oralbacteria (Ritz, 1967, 1969; van der Hoeven and de Jong,1 9 8 4 ; Ham ilton and B uckley , 1991). The sum of thesestudies has been to allow oral microbiologists to apply theconcepts of microbial ecology to plaque biology (Bowden etal , 1978, 1979; Theilade, 1990) and dental disease (Marsh,1 9 8 9 ; B owd en, 1991). The study of the biology of dentalplaque has been paralleled by studies of biofilms elsewherein nature, medicine, and industry (Costerton et al., 1981,1 9 8 7 1995; Characklis and Marshall, 1990; Fletcher, 1991;Gilbertet al, 1993).

Dental plaque which develops on tooth surfaces above thegingival margin (supragingival dental plaque) is an essentialcomponent in the etiology of caries. Supragingival plaquehas been the subject of most of the studies of oral biofilms.Although biofilms of bacteria also develop on tooth surfacesbelow the gingival margin (subgingival dental plaque),relatively little is known of their characteristics, although thebacteria which are found in subgingival habitats associatedwith subgingival plaque have been well-described (Mooreand Moore, 1994).Coupled to the signif icance of bacteria in the cariesprocess has been the recognition of the importance of hostnutrition in promoting caries risk (Burt and Ism ail, 1986). Asmight be expected, this known interrelationship has resultedin considerable interest in the impact of host nutr i t ion,

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82 BOWDEN& Li ADV DENT RE S APRIL1997par t icular ly the in take of sucrose and other ref inedcarbohydrates, on the development and composition of dentalplaque in humans (Carlsson and Egelberg, 1965; Krasse etal , 1967; de Stoppelaaret al, 1970) and animals (Van Houteet al, 1976a,b; B eckers and van der Hoeven, 1982, 1984;Van der Hoeven et al, 1985a; B eighton and Hay day, 1986;De Jonget al, 1986).In vitro studies of nutritional influenceson the adherence of oral bacteria (Rosan et al, 1982; Knoxe tal., 1985) and bacterial competition (van der Hoeven and deJong, 1984; de Jonget aL, 1986; McDermidet al, 1986; vander Hoeven and Gottschal, 1989; Marsh, 1995) have alsobeen made.The impact of nutrient on the development and survival offree bacteria and those in biofilms in nature has also receiveda t t e n t i o n ( P o i n d e x t e r , 1 9 8 1 ; K j e l l e b e r g et al, 1 9 8 5 ;Charackliset al, 1990; Fletcher, 19 91). How ever, the naturalenvironments considered in many of these s tudies areoligotrophic (Poindexter, 1981) and low in nutrient comparedwith the oral cavity. Consequently, different aspects ofnutrition, such as starvation (Kjelleberg, 1993), may assumemore significance. N evertheless , bacteria in oral biofilmsu n d e r g o p e r i o d s o f n u t r i e n t l i m i t a t i o n ( C a r l s s o n a n dJohansson, 1973; de Jong et al, 1984; van der Hoeven andCamp, 1991), and responses by bacteria in oral biofilms andthe associated planktonic phases to ensure survival representan area of significant interest. In the following review, wewill try to draw attention to aspects of nutrition that caninfluence the development and composition of biofilms oforal bacteria but also, to some extent, those in nature.

NUTRITIONAL INFLUENCESIN THE ENV IRONMENTW hen the impact of nut r i t ion on the developme nt andc o m p o s i t i o n o f b i o f i l m s i n a g i v e n e n v i r o n m e n t i sconsidered, several possibilities for allogenic and autogeniceffects might be proposed: (1) a direct effect, where a specificnutrient provides an important energy, nitrogen, or carbonsource for one or more of the micro-organisms in theenvironment; (2) an effect where the products of metabolismof an organism provide a signif icant nutr ient for otherorganisms; (3) an indirect effect, where the products ofbacterial metabolism of a nutrient alter the environment in away which influences the resident microflora; and (4) aneffect whereby a substrate is provided for the production ofspecific polymers by biofilm cells. In the oral cavity, dietarysucrose could be seen as potentially playing these rolesproviding an energy source when present in limiting amounts,promoting the production of lactic acid as a substrate forVeillonella, lowe r ing the env irom ental pH when it i savailable in excess, and acting as a substrate for streptococcalglucos yl t ransfera ses to produc e soluble and insolubleglucans.

If comparisons are drawn between the environments ofbiofilms accumulating in nature and those of the animal oralcavity, some differences can be identified. Among the most-studied aspects of nutrition which can influence biofilms innature are those associated wi th aquat ic environments

(Poindexter, 1981; Kjelleberg et al, 1983; Gottschall, 1992;Smith, 1993a,b; Morita, 1993). The levels of nutrients inthese oligotrophic environments are low relative to those int h e o r a l c a v i t i e s o f a n i m a l s , w h i c h i n c l u d e c o m p l e xsecretions l ike saliva and which could be described aseutrophic or copiotrophic. Moreover, the oral cavity isprovided with components of the host's diet at more or lessregular intervals, supplementing the substrates available fromsaliva. Only in some animals which hibernate are there verylong per iods where the oral bacter ia survive wi thoutadditional nutrients, and although the oral flora of animalshas been studied (Dent and Marsh, 1981), nothing is knownof the oral flora during hibernation. However, it has beenshown that patients receiving their nutrients via stomach tubemaintain an oral microflora, albeit distinct from that of thoseeating normally (Littleton et al, 1967), suggesting that salivacontains sufficient nutrients to sustain oral biofilms. Also,s tudies in exper imental animals (Beckers and van derHoeven, 1984; Beighton and Hayday, 1986; Smith andBeighton, 1986) have shown that bacterial biofilms developon tooth surfaces during periods of fasting for up to 36 hrs.

In contrast, although changes in nutrient can occur inaquatic environments, regular input of rich substrates (feastand famine) , which comes as food among humans indeveloped countries, may not be common. Thus, whilebacteria forming biofilms in nature often do so underconditions of starvation or severe nutrient stress, this may beless common among oral bacteria.Another significant difference between the oral cavity andaquatic environments is the extent of the planktonic phase.W hile saliva flows over oral surfaces as a 0.1-mm -thick film,the volume of planktonic phases in aquatic environments maybe almost infinite compared with the biofilm, and this hasimplications in terms of nutrient supply and the removal ofmetabolites produced by biofilm cells. The oral cavity isperhaps m ost like a very shallow, slow-moving stream, wherebiofilms are barely covered by water.Although the nutritional features of the environments inthe oral cavity and in nature are subject to change, bacterialb iof i lms may also develop in environments which arecontrolled and essentially nutrient-stable. This occurs in someindustrial processes and also in association with in-dwellingprosthetic devices or damaged tissue surfaces in humans. Inthe latter case, the environment is usually relatively rich innutrients, and therefore nutrient limitation and fluctuationmay not be a significant feature of biofilm development.

STAGES IN THE DEVELOPM ENTOF BIOFILMSMany researchers have shown that development of biofilmsfollows a series of similar stages in a wide variety ofenvironments in nature and also in vitro (Costerton et al,1987; Characklis, 1990; Gilbert et al, 1993). B riefly, thesestages are: (1) adherence of cells to a conditioned surface; (2)rapid division and growth of adherent cells; and (3) a plateauof accumulation. These stages may vary between imperviousand porous substrata. Impervious substrata may affect the



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VOLll(l) NUTRITION AND BIOFILM DEVELOPMENTTABLE 1

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O U TLIN E O F TH E P O TE N TIA L E F F E C TS O F N U TR IE N TS O N TH E S TAG E S O F D E V E LO P M E N T O F A B I O F ILMEnvironment/Structure Influence of NutrientDirect Indirect Selected References

Planktonic phaseConditioning film

Local environment

Bacterial communityProportion of bacterialpopulations

Adherence

CompositionSaliva in oral cavity

Nutrients interactdifferently withbacterial communities.Low-pH food can pro-duce transient change.

Competition for nutrient.? growth in salivaryplanktonic phasein the mouth

Starvation, modificationof cell morphology andsurfaces; not knownin the mouth.

Inclusion of productsof bacteria,e.g.,gluco-syltransferases; glucans

Modification of environ-ment through metabolicproducts,e.g.,acid.

Competition under alteredenvironmental conditions,e.g.,p H.

Cells respond to alteredenvironment, modify cellmorphology and surfaces.

Bradshawetal. (1995)Kjelleberg ef a/. (1985)Marshall KC (1988)(1983a,b)

Brighton ef a/. (1988)B owden and Hamilton (1989)Ligtenberget al. (1991)McDermid ef a/. (1986)van der Hoevenet al.(1985a)Kjelleberg (1993)Knox and W icken (1984)Morriset al. (1985)

BiofilmInitial stages of development

Established biofilm

Bacterial competitionon surface. Salivaprovides adequatenutrient.

Biofilm environmentCompetitionMatrix formation

Competition under alteredenvironmental conditions.

Biofilm environmentCompetitionMatrix formation

Beckers and van der Hoeven(1982)B eighton and Hay day (1986)Li and Bowden (1994b)Marsh** al. (1994)Beightonetal. (1986)Costertonet al (1981, 1994)Edgar and Higham (1990)Fletcher (1991)Marsh efa l (1995)Margolis and Moreno (1994)van der Hoevenet al (1985b)

biofilm, e.g., by liberation of active agents such as fluoride(Li and Bowden, 1994b), or the conditioning film couldprovide a source of nut r ient (Kjel leberg et al, 1 9 8 3 ;Marshall, 1988). Porous substrata, on the other hand, mayprovide a constant nutrient source to biofilm cells (Gilbert etal , 1993). Superim posed on accum ulation of biofilm cellsduring development is the loss of cells through the action ofshear forces and the shedding of daughter cells, together withincreased accumulat ion resul t ing f rom adherence (co-aggregation) of bacteria from the planktonic community to

biof i lm cel ls . In environments which suppor t a mixedplanktonic flora, the final composition of the biofilm willreflect the outcome of bacterial succession, resulting fromcompetition among adherent bacteria.The type and availability of nutrient influence the biofilmthroughout developm ent, from the initial conditioning film onthe surface, through adherence of pioneer bacteria and theproduction of matrix, to bacterial competition in the laterstages of accumulation. An outline of the potential effects ofnutrient on the development of a biofilm is shown in Table 1.



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84 BOWDEN & Li ADV DENT RE S APRIL1997EVENTS IN THE PLANKTONIC PHASEASSOCIATED WITH SURFACES

Formation of surface-conditioning filmsIn most environments, exposed surfaces adsorb moleculeswhich form a conditioning film (Fletcher, 1991) to whichbacteria will adhere. In the oral cavity, this conditioning filmis called the acquired tooth pellicle, and it includes a varietyof salivary and other molecules which can provide specificreceptor sites to facilitate the adherence of different bacterialspecies (Scannapieco, 1994). The com ponents of the acquiredpellicle are derived mainly from molecules present in salivaand other physiological fluids, such as antibodies, present inthe oral cavity (Rolla et aL, 1983a; Scannapieco, 1994).However, the composition of saliva may be influenced bydiet (Tabak and Bowen, 1989), potentially influencing thenature of the conditioning film and, through this, bacterialadherence. In addition, the products of bacterial metabolismand specif ic bacterial enzymes present in saliva can beincorporated into the pellicle, and these may promote theadherence of specific bacterial species. For example, it isknown that glucosyltransferase levels in saliva are increasedfollowing sucrose rinses. These enzymes can be found bothfree in saliva and incorporated into the acquired tooth pellicle(Rolla et aL, 1983b), where they have the potential to produceglucans from dietary sucrose and influence the adherence ofS. mutans an d S. sobrinus via glucan-binding proteins( S c h i l l i n g et aL, 1989 ) . The inf luenc e of d i f ferentconditioning films on the early events in biofilm developmenthas also been described by Bradshaw et aL (1995). Crudeglucosyltransferase preparations from S. mutans an d S.sanguis influenced the final proportions of populations in thecomm unity. W hole and parotid saliva also produced somedifferences in one-day biofilms, but these differences werenot apparent after 4 days of accumulation. The latter resultcould reflect the outcome of competition among the biofilmcells, reducing the effects of initial adherence.

As mentioned above, in addition to a role in facilitatingspecific bacterial adherence, conditioning films may act as anutrient source. This latter point has not been given a greatdeal of attention in oral microbiology. However, electronmicroscopic examination of plaque structure has revealeda r e a s w h e r e t h e a c q u i r e d p e l l i c l e a p p e a r s d e g r a d e d(Schroeder and de Boever, 1970), and this may represent theresults of microbial activity (Leach and Saxton, 1966;Armstrong and H ay ward, 1968; Hay ward and Arms trong,1970).The planktonic bacterial communityBacteria from fluid phases associated with surfaces are thesource of cells which comprise the biofilm. The presence ofcells in the fluid phase could be the result of contaminationfrom other habitats and/or cell shedding from communities inthe immediate environment. Thus, biofilm communitiesthemselves can serve as a source of cells which are liberatedinto the fluid phase, and these may subsequently recolonizefresh surfaces. Contamination from other habitats canaccount for the bacteria found early in life in the human oral

cavity, when organisms from care-givers colonize infants.The levels and types of nutrients available can influencethe state of the cells in the planktonic populations, either bystimulation of growth, or indirectly through alteration of thecharacteristics of the environment. The nature and am ounts ofsubstrates present may influence the morphology of bacteria,their surfaces, adherence, and, through competit ion, thepropor t ions of d i f ferent populat ions in the p lanktonicenvironment.In the oral cavity, saliva serves as the basis for the fluidphase associated with tooth surfaces. Saliva flows as a film0.1 mm or less in thickness over oral surfaces (Dawes et aL,1989), and the rate of flow mm/min of unstimulated salivaranges from 0.8-7.6, which increases to 1.3-ca. 350, forstimulated saliva. In addition, it is likely that, during foodconsumption, nutrients are not distributed evenly throughoutthe mouth (Dawes and Macpherson, 1993).The bacterial flora of saliva is complex, and the organismspresent probably are shed from oral biofilms. Although salivais an effective nutrient for selected oral bacteria, it also servesto remove organisms from the mouth (Scannapieco, 1994).Generally, it is thought that bacteria cannot divide rapidlyenough to survive against the flow of saliva, although theyprobably metabolize substrate and respond to environmentalstress.In some retention areas of the mouth where saliva mayflow slowly or form pools, organisms may grow. If growthoccurs, then competition for nutrients among organisms inthe salivary film above oral biofilms becomes a possibility.Saliva as a nutrientIt is not surprising that much of the interest of competitionbetween oral bacteria in suspended culture has concernedtheir activities in the presence of limiting carbon, which is anatural state, and carbohydrate excess, which is associatedwith dental caries. However, for long periods, particularly atnight, the oral cavity will be free of dietary carbohydrate, andonly saliva will be present to support the survival orcompetition among oral bacteria in the oral cavity. Evenwhen dietary carbohydrates are present, saliva will provide asource of other substrates to support growth. The nutritionalvalue of saliva for oral bacteria becomes a most significantfactor, therefore, both in the survival and growth of oralbacteria during periods when diet is absent and also in thepresence of dietary carbohydrate. Bibby (1976) suggestedthat the bacterial population of the mouth and of plaques isthe product of an evolutionary process. I t is apparentlydepe nde nt to a much gre ater extent upon the naturalenvironment provided by the mouth than by the transientpresence of foodstuffs therein .

There are now-considerable data confirming the ability ofsaliva to support the growth of certain oral bacteria. Oralbacteria can degrade mucin and salivary glycoproteins toprovide both carbon and nitrogen sources (Beighton andSmith, 1986; Smith and Beighton, 1986; de Jong and van derHoeven, 1987; Beighton et aL, 1988; van der Hoeven et aL,1989; Bradshaw et aL, 1994). It has also been shown thatdifferent bacteria become dominant in anaerobic enrichmentcultures in saliva from different glands (van der Hoeven et



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VOLll(l) NUTRITION AND BIOFILM DEVELOPMENT 85al , 1989). For exam ple, Prevotella oralis was dominant inc u l t u r e s i n p a r o t i d a n d Eu bacterium lentum insubmandibular-sublingual saliva. Van der Hoeven et al(1989) proposed that saliva from different glands could act asa nutritional selective pressure on oral bacteria. In a laterstudy (Ligtenberg et al, 1991), enrichm ent culture in salivafrom different glands showed that, in contrast to the flora ofanaerobic enrichments, Streptococcus species were dominantin aerobic enrichment cultures.Influence of nutrient on bacteria in the planktonic phaseA well-documented nutritional influence on bacterial cells isthei r response to s tarvat ion (Mor i ta , 1993) . In aquat icenvironments, oligotrophic bacteria colonize surfaces andmay express increased adherence, coupled to an increase insurface hydrophobicity, in response to starvation (Kjelleberget al, 1983, 1985; Kjel leberg and Herm ansson, 1984) .Similar starvation effects have been shown on bacteria in vivoin fish and mice (Conway et al, 1986). Although starvationof cells in the planktonic phase may promote adherence,reduction in nutrients has also been shown to enhance therelease of cells from pre-formed biofilms (Delaquis eta/.,1989).

Little is known of any starvation responses among oralbacteria, or whether such responses are stimulated in the oralenvironment. Extremely low levels of nutrient similar tothose found in aquatic environments in nature rarely, if ever,occur in the mouth. However, there is reason to believe thatnutrient limitation of bacteria is a characteristic of the oralcavity (Carlsson and Johansson, 1973; van der Hoeven, 1976;de Jong et al., 1984; van der Hoeven and Camp, 1991).Consequently, the effect of l imitation of substrates onbacteria could occur among members of the oral planktoniccommunity, which, in turn, may influence their adherence.

Despite the lack of information on starvation among oralb a c t e r i a , c o n s i d e r a b l e d a t a a r e a v a i l a b l e o n t h ehydrophobicity of their cell surfaces (Ofek and Doyle, 1994),which has been shown to be related to cell adherence andinfluenced by cultural conditions. A standard test commonlyused to measure the adherence of oral bacteria is their degreeof adsorption to saliva-coated hydroxyapatite. Studies ofdifferent oral bacteria in this model (Gibbons and Etherden,1983; Clark et al, 1985; Fives-Taylor and Thom pson, 1985;Morriset al, 1985) have shown that the degree of adsorptioncan be directly related to surface hydrophobicity; however,th is may not a lways be the case (Knox et al, 1 9 8 5 ) .Examination of non-adherent mutants and enzyme-treatedbacteria indicates that surface hydrophobicity is often areflection of the presence of cell-surface proteins (Gibbonsetal , 1983; Fives-Taylor and Thom pson, 1985; Morris et al,1985). Loss of surface protein can lead to a less-adherent cell,which is also more hydrophilic.

Since growth conditions, including the availability ofnutrient, can influence the composition of bacterial cell wallsand cell-surface proteins (Knox and W icken, 1984; Brow nand W illiams, 1985; B owdenet al, 1991), it is to be expectedthat they could affect cell adherence. Orstavik and Orstavik(1982) drew attention to the loss of the degree of adherence

of oral streptococci during laboratory subculture. This wasconf irmed by W estergren and Olsson (1983 ) , who alsodemonstrated a reduction in hydrophobicity and suggestedthat these phenomena were related to changes in the cell wall.Chemostat culture of a strain of Streptococcus sanguis(Knox et al, 1985) confirm ed that varia tions in carbonsource, sodium and potassium levels , and growth ra te

influenced the ability of the strain to adhere to saliva-treatedhydroxy apatite. How ever, there w as no direct correlationbetween cell hydrophobicity and adherence, and the authorsst ressed that speci f ic b inding by cel l - sur face prote ins(Gibbons, 1989) was probably a more significant feature ofadherence. Similarly, they found that the amounts and natureof extracellular proteins released into the medium could notbe related directly to adherence. It is important to note thatthe changes in ext racel lu lar prote in prof i les ref lectedliberated proteins and not those remaining associated with thecell walls, which might affect adherence more directly.Increased amounts of an outer membrane protein of

Prevotella intermedia genotype II (P. nigrescens) was foundby Bowden et al (1991) to be associated with biofilm cellsaccumulating on the wall of a chemostat vessel. Increases ingrowth rate and reduction in culture pH caused cells in theplanktonic phase to exhibit increased amounts of a 40-kDaprotein, which was also found in high concentrations in theouter membranes of associated biofilm cells. It is possiblethat increased expression of this protein, although not thedirect result of changes in the type of nutrient, was stimulatedby nutrient stress introduced by high growth rates and low pHand was also linked to biofilm formation.An indirect effect of carbohydrate on the degree ofadherence of cel l s to hydroxyapat i te sur faces i s the

environmental pH. Rosan et al (1982), Cam pbell et al(1983), and Li and Bowden (1994b) reported a reduction inthe adherence of cells of oral bacteria grown at low pH. Themore detailed study by Knox et al (1985) also showed aneffect of environmental pH. Cells of S. sanguis grown inc h e m o s t a t s w i t h g l u c o s e o r f r u c t o s e w i t h i n c r e a s e dconcentrations of sodium or potassium were measured foradherence to hydroxyapatite in a competitive adsorption test.Cells from the fructose medium with high potassium at anenvironmental pH of 5 .5 gave the h ighest values foradherence.The results presented above for oral bacteria confirm that,

in common with other environments in nature, the cellswhich comprise the planktonic community may respond tochanges in the environment, which can influence their abilityto adhere to surfaces and to detach from pre-formed biofilms(see below). Therefore, increases in fermentable carbohydratein the diet or the ingestion of low-pH food or drinks canlower the pH of the planktonic environment and potentiallymodify adherence of cells from the planktonic community tooral surfaces. However, an important aspect of adherence oforganisms from the planktonic phase to surfaces in the oralcavity is the interaction between salivary components andbacterial surfaces, which may also influence the ability ofcells to adhere (Scannapieco, 1994). Consequently, althoughone can propose that nutrient and other conditions in theby guest on August 11, 2011 For personal use only. No other uses without permission.adr.sagepub.comDownloaded from


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86 BOWDEN& Ll ADV DENT RE S APRIL1997salivary planktonic phase influence adherence in vivo, theirsignificance in the process is not clear. There is no doubt thatthe mechanisms of adherence involving specific receptors oncell surfaces, predicted on the basis of in vitro tests, aregenerally borne out by examinationsin vivo.

E V E N T S I N B A C T E R I A L B I O F I L M SEarly accumulation, growth,and shedding of adherent bacteriaBacterial biofilms develop in a series of stages that arecommon to a diverse range of habitats (Gilbert et al., 1993).The events in the planktonic phase described above govern,in part, the adherence of cells. Once cells are adherent to thesurface, usually as a dispersed monolayer on impervioussubstrata, they are primarily dependent on nutrients from theplanktonic environment for growth, and they develop to apoint where thei r extent and mass are l imi ted by thecharacteristics of the environment. In vitro, the stages ofadherence, lag, and rapid growth of biofilms of oral bacteriaoccur during the first 12 to 20 hrs, while in vivo,the period isfrom 18 to 48 hrs. Subsequently, the plaque num bers becom erelatively stable.

Bright and Fletcher (1983) suggested that the localenvironment is probably a more significant influence on thephysiological act iv i ty of adherent cel l s than i s thei ra s s o c i a t i o n w i t h s u r f a c e s . H o w e v e r , s o m e d e t a i l e dphysiology and specific examples where surfaces affect cellsare described by Fletcher (1991) and Costerton et al.(1995).It has been reported that the growth rates of adherent cellscan exceed those of cells in the associated planktonic phases(El lwood et al, 1982; F le tcher , 1991; Li and Bow den,1994a), although cell number doubling time (Beckers andvan der Hoeven, 1982) may be a better term, given thedif ferent processes which can be involved in cel laccumulation (Li and Bowden, 1994a). Van Loosdrechtet al.(1990) discussed the complexity of identifying true surfaceeffects on utilization of substrates and growth rate andconcluded that the effects may be negative, positive, ornonexistent, a conclusion also reached by Fletcher (1991).Development n v troThe control of growth rates of biofilm cells on membranesthrough perfused nutrient limitation has been described byAllison and Gilbert and their co-workers (Gilbertet al., 1989;Allison et al., 1990a,b), where steady-state conditions wereachieved by balance between the growth and shedding ofc e l l s . A l l i s o n et al. (1990a) found that the sur faceh y d r o p h o b i c i t i e s o f c h e m o s t a t a n d b i o f i l m c e l l s o fEscherichia coli were equivalent and decreased wi thincreased growth rates. However, daughter cells shed frombiofilms were more hydrophilic than either the biofilm orplanktonic cells, suggesting that decreased hydrophobicityduring biofilm cell division facilitated the liberation ofd a u g h t e r c e l l s . D i f f e r e n t r e s u l t s w e r e o b t a i n e d w i t hPseudomonas aeruginosa, where, in keeping with others,Allison et al. (1990b) found increased hydrophobicity ofbiofilm and chemostat cells with increased growth rate.

However, as with E. coli, liberated daughter cells were morehydrophilic. The authors suggest that changes in biofilm cellsurfaces during growth, possibly the reduction of surfaceproteins during division, promote the liberation of daughtercells. These observations aggree with the suggestion madeabove for variations in the surfaces of planktonic cells andadherence.Most recently, James et al. (1995) have proposed that theavailability of nutrients influences both cell adherence andrelea se of cel l s from a b iof i lm . U sing a s t ra in ofAcinetobacter a s a m o d e l , t h e s e a u t h o r s s h o w e dmorphological transitions from small coccoid to rod forms,associated with adherence and the release of biofilm cells.Reduced nutrient promoted the development of small cocci,which formed the biofilm. Subsequent increases in nutrientlevels resulted in division and the production of rod forms,which spread over the surface and were also released into theplanktonic phase.Examination of the effects of excess glucose (glucosepulse) on the initial stages ofin vitro development of biofilms

of S. mutans, Streptococcus sanguis, Streptococcus mitis,Actinomyces naeslundii geno mic specie s 1 and 2, andLactobacillus casei (Li and Bowden, 1994a) showed thatcells accumulated rapidly, and the early lag stages of biofilmdevelopment (0-6 hrs), seen in this model under glucoselimitation, did not occur. After 6 hrs, the total biofilm cellnumbers were signif icantly increased in glucose excess,relative to limitation. Moreover, the cell number doublingtimes of the biofilm cells were changed. W ith the exceptionof S. mitis, the cell number doubling times (0-2.0 hrs) werereduced, although by 4.0-6.0 hrs, with the exception of S.sanguis, cell number doubling times were similar to thoseunder glucose limitation. This 'immediate' response to excessglucose was common to all strains tested and suggests thatpioneer cells adherent to a surface can respond and growrapidly (Jameset al., 1995), under the appropriate co nditions.Such responses to excess carbohydrate by pioneer biofilmcells in vivocould result in localized increases in numbers ofselected species during early biofilm accumulation. Beightonand Hay day (1986 ) showed some influence of carboh ydrateon the bacterial composition of six-hour dental plaque inmonkeys, when S. mutans was favored by sucrose diets.However, they did not see significant changes in bacterialcel l numb er do ubl in g t im es for the f i r s t 6 hrs ofaccumulation.

An indi rect ef fect of carbohydrate on the p laqueenvironment is the lowering of the local pH. Recently, Leee tal . (1996) found that low environmental pH promoted theshedding of cells from monolayer biofilms of S. mutans.Previously (Lee, 1995), i t had been shown that surfaceproteins were released from a strain of S. mutans by enzymeactivity that was optimal at pH 5.0. Examination of thedetachment of adherent cells from hydroxyapatite indicatedthat a similar enzymic release of bound cells occurred frombiofilms during the early stages of development (Lee et al.,1996) . Studies of the retention of biofilm cells (Li andBowden, 1995; and unpubl ished) suggest that enzymicdetachment of biofilm cells is more significant during early



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VOL.U l) NUTRITION AND BIOFILM DEVELOPM ENT 87

Bacterial SpeciesNumber

Mono-associatedS. mutans

A. viscosus N yl

Di-associatedS. mutansT2

development and may beresponsible for the loss ofs m a l l e r p r o p o r t i o n s o fcells from older (five-day)biof i lms and those wi ths i g n i f i c a n t a m o u n t s o fextracellular matrix.The influence of acidenvironments and excesscarbohydrate on the actionof fluoride on growth ofbiofilm cells has also beent e s t e d i n a c h e m o s t a tbiof i lm model (Li andBowden, 1994b). Fluoridefrom the substratum signi-f icantly reduced the cellnumber doubling times ofS. mutansandA . naeslundii(0-6 hrs of accumulation)

under conditions of lowp H a n d c a r b o h y d r a t eexcess. Biofilm cells of L.casei were not affected. Int h e s e e x p e r i m e n t s , t h eeffect of f luoride onbiofilm cells reflected itsactivity on cells in chemo-s t a t c u l t u r e ( B o w d e n ,1990), suggesting that cellsin early biofilm accumu-lations retain some of thec h a r a c t e r i s t i c s o f t h eassociated planktonic cells.

Col lect ively , these invitro s t u d i e s o f m o n o -cul ture b iof i lms suggestthat d i rect and indi recteffects of nutrient levels inplanktonic environmentscan inf luence the de-v e l o p m e n t o f b i o f i l m s ,although the effects mayvary amo ng di f ferentbacteria. The situation inthe oral cavity is somewhatsimilar, although saliva isconstantly present. Saliva, whichindependent of dietprovides selective conditioning films (Scannapieco, 1994)and suitable substrates for oral bacteria, supports biofilmaccumulation in the absence of diet. As w e shall see later, thedirect and indirect influences of nutrition on the planktonicenvironment m ay be more obvious in early biofilm formationthan for the more complex 'older' biofilms, which seem tomaintain a 'biofilm environment' (Fletcher, 1991).Development nvivoThe impact of available carbohydrate on the composition and

TABLE 2E F F E C TS O F D I F FE R E N T D I E T R E G IM E N S O N C E LL N U M B E R D O U B LI N G TI M E SOF BACTERIA IN ORAL BIOFILMS IN GNO TOB IOTIC RATS(Beckers and van der Hoeven, 1982, 1984)

Dietary Regimen

A. viscosus N y l

Sucrose

Fasted

Sucrose

FastedSucrose

Glucose

Fasted

Sucrose

Glucose

Fasted

A c c u m u l a t i o n T i m e / C e l lD oubling Time, (td)/hoursAccumulation t,a

2-1212-4848-726-1212-246-24

24-4848-722-242-1212-2448-722-1212-2448-722-1212-242-3030-482-3030-486-2424-36

1.17.538.01.34.42.93.924.02.81.52.225.01.42.223.01.62.42.75.32.522.02.310.0

a Standard errors omitted.accumulation of biofilms in vivo (Carlsson and Egelberg,1965; de Stoppelaar et ai, 1970; van Houte et a/., 1976a,b;Beckers and van der Hoeven, 1982, 1984; van der Hoevenetal , 1985b; B eighton and Hayday 1986; de Jong et a/., 1986;Tanzer, 1986) is well-known. The early stages of biofilmformation in vivo are not restricted by carbohydrate limitation(Beckers and van der Hoeven, 1982, 1984; Beighton andHayday, 1986; Beightonet al, 1986), which is thought to bea feature of mature biofilms.Study of biofilms in experimental animals has providedsome of the most valuable data on the effects of nutrients onbiofilm development. Various animals, including gnotobiotic



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BOWDEN & Li ADV DENT RE S APRIL1997

rats and conventional rats and monkeys, have been used. Insome cases, the studies have been directed specifically atquestions of biofilm biology, while others have concentratedon the relationships between diet and caries. Although theformer studies are most valuable, studies of the caries processhave included the influence of diet on colonization ofexper imental animals . Thus, s tudies by van Houte andUpeslacis (1976) and van Houteet al (1976a,b) showed that,although sucrose was not essential to the establishment of S.mutans in conventional rats, extracellular polysaccharideproduction by this organism on sucrose diets favored itsretention on tooth sufaces.

Studies by van der Hoeven and his co-workers haveexamined the development of biofilms of Streptococcus andActinomyces mono-associated in rats (Beckers and van derHoeven, 1982; de Jong et al, 1985). B efore the results areexam ined, a point should be made wi th regard to thedefinition of A. naeslundii an d A. viscosus. Up to 1990(Johnson et al, 1990), A. viscosus and A. naeslundii wereseparated by relatively few phenotypic tests and the catalasereaction. A. naeslundii was negative and A. viscosus waspositive, and this test was significant in differentiation.However , Johnson et al. (1990) , using DNA homology,proposed that the human strains of A. naeslundii should bedesignated A. naeslundii genom ic species 1, and humanstrains of A. viscosus, A. naeslundii genomic species 2. A.viscosus was retained for strains of animal origin. Therefore,it is possible that some human strains designated A. viscosusin the literature are closely related to A. naeslundii. The strainoften used by van der Hoeven,i.e.,Ny 1, isA. viscosus,and itis also likely thatA. viscosus colonizes monkeys. Care shouldbe taken in the assessment of different results obtained withorganisms designated A. viscosus and A. naeslundii on thebasis of the catalase test.

Early experiments (van der Hoeven, 1974) described theenrichment of an extracellular polysaccharide-producingstrain of A.viscosus (Ny 1) in the plaque of conventional ratson a glucose diet . Beckers and van der Hoeven (1982)showed that development of biofilms of S. mutans and A.viscosus in gnotobiotic rats followed the series of stagesdescribed for biofilms in nature, i.e., a lag stage, a period ofe x p o n e n t i a l g r o w t h , an d a p l a t e a u . Tw o s i g n i f i c a n tobservations were made with respect to carbohydrate. Theinclusion of sucrose in the diet did not influence the cellnumber doubling times of biofilm cells of A. viscosus or S.mutans, and the same cell number doubling times were alsoseen in plaque of animals starved for 24 hrs (Table 2). Theresults showed that diet did not influence the early stages ofbiofilm accumulation of these organisms, and that substratelimitation did not seem to be a feature in early biofilmdevelopment. However, the authors suggested that substratel imi ta t ion would occur wi th th icker b iof i lms la ter inaccumulation. De Jong et al. (1985) confirmed that, in fact,dietary carbohydrates influenced the numbers of S. mutansand A. viscosus in plaque accumulated for 28 days in rats.They concluded, in agreement with others (van Houte et al.,1976b), that production of ex tracellular polysaccharides by S.mutans on sucrose diets and A. viscosus on sucrose and

glucose diets favored the retention of cells and enhancedplaque formation. Similarly, in humans, sucrose favorsplaque accumulation (Carlsson and Egelberg, 1965) and oralcolonization byS. mutans(Krasseet al., 1967).NUTRIENT AND COMPETITIONAMONG BIOFILM CELLS

Bacterial competitionThe nature, theory, and predicted outcomes of microbialcompetit ion for nutr ients have been well-described forlaboratory systems (Fredrickson and Stephanopoulos, 1981;Gottschal, 1992), and such competition is reflected in nature(Smith , 1993b) . The s igni f icance of resources in theenvironment, relative to competit ion and succession inecology, have been recognized in the resource-ratio theory(Tillman, 1990), which can be used to predict the outcome ofcompetit ion among species or strains. Recently (Smith,1993a,b), this theory has been reviewed with respect tomicrobial ecology, and Smith (1993b) gives examples whereresource-ratio theory could be applied to infectious disease.To date, resource-ratio theory has not been used to describethe outcome of competition among oral bacteria.Spatially dispersed chemostat culture lends i tself todemonstrating competition between bacteria in mixed culture,and this technique has been used extensively to examine theoutcome of interaction between oral bacteria in controlledenvironments (van der Hoeven and de Jong, 1984; Marsh,1995). In general, chemostats have been used to mimic andexplain mechanisms which have the potential to influencebiofilm formation in vivo,or to predict thein vivooutcome ofintervention strategies. In particular, studies have described

roles for l imiting substrate in growth, competit ion, andcoexistence of Streptococcus andActinomyces (van derHoeven and de Jong, 1984; van der Hoeven et al., 1985a;Rogers et al., 1986; van der Hoeven and Gottschall, 1989;Bowden and Hamilton, 1989; Bradshaw et al, 1989, 1990;Marsh, 1995). These studies carried out in suspended cultureare used to predict events in biofilm communities, and, ingeneral, the predictions are borne out by in vivo studies inexperimental animals.The most diverse of the chemostat communities of oralbacteria studied is that of McKee et al. (1985), which iscomprised of 9 organisms. Using this model, Bradshaw et al.

(1989) showed that pulses of carbohydrate in environmentsof pH 7.0 caused little change in the proportions of thepopulations in the community. However, pulses of glucosewithout pH control resulted in periodic lowering of thee n v i r o n m e n t a l p H a n d a n i n c r e a s e i n S. mutans an dLactobacillus.In contrast to acid production, it has been recognized formany years that n i t rogen metabol ism by oral bacter iap r o d u c e s b a s e , w h i c h c a n p r o v i d e a b a l a n c e t o a c i dproduction (Kleinberg et al, 1979; Curtis and Kemp, 1984;Sissons et al, 1985). Mo reover, base is produced fromsalivary urea, peptides, and am ino acids, which, unlike excesscarbohydrate, are present consistently. Therefore, nutrientswhich promote base production in the environment may



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VOLll(l) NUTRITION AND BIOFILM DEVELOPMEN T 89

0.2 mil glucot*0.0125 mucln

O

CBi-blof i lm:Sm 6.2An 7.8Mono-blofilm:Sm5.4An 7.5

S. mutans BM71A. naeslundii WVU627

10 20Time (h)

30

S. mutans BM71A. naeslundii WVU627

Fig. 1Bi-culturebiofilms ofS.mutansBM71 andA. naeslundiiWVU627growing on epon-hydroxyapatiterods in mucin-supplementedmedium, pH 7.0,D =O.lh'1, withlimiting glucose(A) and withoutglucose (B).

inf luence bac ter ia l compet i t ion di rec t ly or modi fy pHchanges introduced through carbohydrate metabolism. Anarginine-supplemented die t enhanced the numbers of 5 .sanguis and S. milleri relative to S. mutans in the plaque ofgnotobiotic rats (van der Hoeven et al, 1985b). Selectiveut i l i za t ion of a rginine by S. sanguis an d S. milleri inc h e m o s t a t c u l t u r e p r o v i d e d a n e x p l a n a t i o n f o r t h i senhancement (Rogersetal, 1987).Competition in biofilms nvitroAfter the early stages of accumulation, biofilms reach a'steady state' or plateau of accumulation. In some cases,biofilms reach a balance with their environment to form aclimax community. The final bacterial composition of thebiof i lm i s the resul t of ecologica l success ion, and theextracellular matrix of the biofilm results from inclusion ofmolecules from the environment and also bacterial products.Both bacterial succession and extracellular matrix can beinfluenced by available nutrient.

R e c e n t l y , b i o f i l m m o d e l s h a v e b e e n u s e d t o s t u d ys u c c e s s i o n a n d t h e e s t a b l i s h m e n t of m i x e d - b i o f i l mcommunit ies of oral bacteria (Marsh et ah, 1994, 1995;B r a d s h a w et ai, 1 9 9 5 ; G a n d e r t o n et al., 199 5; Li andBowden, 1996).Marsh et ai (1994) studied the developm ent of a 10-species biofilm in a chemostat model for periods of up to 4days. A series of daily glucose pulses was given to providealternating glucose excess. pH control was removed for 6 hrsafter each pulse, when the pH was returned to pH 7.0 for 18hrs. The lowest pH achieved in the planktonic phase was pH4.3. The direct and indirect influences of alternating carbonexcess and low pH produced a reduct ion in Neisseria,Fusobacterium, Prevotella, an d Porphyromonas, an d

increases in S. mutans an d Lactoba cillus casei. Similarpopulation shifts, but to a greater extent, were seen in theassociated planktonic phase.Substrate effects on the development of two-speciesbiofi lms have also been made by Li and Bowden (1996;unpublished). Strains of A. naeslundii W VU 627 and 5.mutans BM 71 were grown together in a semi-defined mucin-

supplemented medium, pH 7.0, with and without limitingg l u c o s e . T h e d e v e l o p m e n t o f t h e b i o f i l m s o n e p o n -hydroxyapati te rods was monitored for 24 hrs by viablecounts . In medium wi th l imi t ing glucose , both bac ter iasurvived in a mixed steady-state in the planktonic culture;however, in the absence of glucose, S. mutans washed outfrom the chemostat. The results of accumulation of 24-hourbiofilms under both conditions are shown in Fig. 1. Fig. 1Ashows that, in medium with limiting glucose and mucin, bothorganisms accumulated in the biofilm. It is significant thatthe sequence of stages and the final numbers of biofilm cellsin the mixed cul ture were s imi la r to those when theorganisms were grown in monoculture (Li and Bowden,1994a). That is, the numbers of cells (xlO 6 cfu/cm2) after 24hrs were, S.mutans monoculture 5.4, bi-culture 6.2, and A.naeslundii mo nocul ture 7.5, bi -cul ture 7.8. These da tasuggest that , under the environmental condit ions of themodel, the organisms adhered to and accumulated on thesurface for 24 hrs, without any interact ion. Absence ofinteraction between species is not unknown in ecology, and alack of interaction between S. mutans an d A. viscosus wasalso noted by B eckers and van der Hoeven (1984) during thefirst 24 hrs of accumulation of these species in biofilms onteeth of germ -free rats.The nutri t ional influence of glucose on S. mutans is

emphasized by reference to Fig. IB . Here, in medium withoutby guest on August 11, 2011 For personal use only. No other uses without permission.adr.sagepub.comDownloaded from


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90 BdWDEN & Ll ADV DENT RES APRILI997

Iu.O

CMO

U .oc


12/20

NUTRITION AND BIOFILM D EVELOPMENT

S. mutans, it facilitates retention and gives a

in vitro,increaseS. mutans.In contrast to conventional animals, gnotobiotic animals

in vivo. Di-associated gnotobiotic rats fed glucose orHoeven 1984) to study competition between S. mutans A. viscosus in dental plaque. There was no evidence of

the organisms Table 2); however, the rate of24 hrs) in the starved group than inVariations in the biofilm populations ofA. viscosuswere

S. mutans in the biofilm were 4 times those ofA. In con trast, on the sucro se diet, A. viscosus S. mutans by a factor of two. The authors suggestS. mutans to compete on the A. viscosus from glucose. The A.polymer would enhance the retention ofS. mutans in

A role for carbohydrate de Jong et al.,1986) and arginineet al.,1985b) in nutrient competition amongS. sanguisandS. milleriwere S. mutans in gnotobiotic rats on glucose,

S. mutans by sucrose but also (S. sanguis)with the highest metabolic

S. mutans and 5. sanguis or S. milleri. S. sanguis an d S. milleri, which utilize S. mutans, which cannot

Studies by Beighton 198 5), Beighton et al. 1986) ,Hayday 198 6), and Smith and Beighton

10 8 .

3u.o 10 7 .

106

Planktonlc populationsS.mutansBM71(95.7 )A.naeslundii WVU627 (4.3 )

pre-pulse 1

CMO

oca

8 .1

1 7

1

iofilm populationsE3 S.mutansBM71(81.5 )E3 A.naeslundii WVU627 (18.5 )

pre-pulse 1Day

Fig. 3 Shifts in the bacterial popu lations o fS. mutansBM71 andA. naeslundiiWVU 627 in mixedplanktonic (A)and the associated biofilm culture (B) during daily glucosepulses for 5 days in m ucin-supplemented medium atD =0 1h 1without pH control (terminal pH = 4.8).

support a resident flora. Therefore, the results of studies onthe bacterial composition of plaque in monkeys represent theoutcome of compet i t ion among the res ident p laquecommunity.Beighton 1985) determ ined the colon ization of thetongue, cheek, and incisor teeth erupted by 4 weeks) omonkeys by A. viscosus and A. naeslundii from 3 days aftebirth until 12 weeks of age. Five weeks after eruption of theteeth, the two species were isolated from 26/27 of themonkeys. Subsequently, 6 weaned juvenile monkeys, whichhad been fed a maintenance diet for 32 weeks, were given ahigh-sucrose diet for 26 weeks. Samples taken during theperiod on the sucrose diet showed that the frequency ofby guest on August 11, 2011 For personal use only. No other uses without permission.adr.sagepub.comDownloaded from


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92

Diet

BOWDEN & Ll

TABLE 3ADV DENT RE S APRIL1997

NUM BE RS (mean log10 ) A N D M E A N C E L L N U M B E R D O U B L I N G T IM E S ( td)O F TH E P R E D O M I N A N T S T RE P T O C O C C I IN P L A Q U E F RO M M O L A R T E E T HO F MON KEYS O N DIFFEREN T D IET REGIMENS (Beighton and Hayday, 1986)Predominant Streptococcus Mean tda Numbers of Cells (mean Iog 10 )aand Plaque Age (hrs)

0 6 24 96Commercial S. sanguisMaintenance S. sanguisS. 'mitior'S. mutansHigh sucrose S. sanguisS. mutans

5.154.75.74.062.964.93

2.59 3.01 4.48 3.723.32.783.254.582.43

4.082.584.044.115.16

5.154.375.325.526.31

5.464.125.204.526.19

a Standard deviations omitted.

isolation of A. viscosus at all sites was significantly reduced.Isolation of A.naeslundii from the soft-tissue surfaces did notchange on sucrose diets; however, the isolation frequencyfrom incisor teeth was significantly increased.Beighton and Hayday (1986) demonst ra ted tha t thestreptococcal populations recolonizing cleaned tooth surfaceson monkey molars were also influenced by diet. Three dietswere used, a maintenance diet, a commercial diet, and a high-sucrose diet.S. sanguis was isolated from samples taken fromall three diets and was the predominant streptococcus on thecommercial diet. Streptococcus 'mitior', S. sanguis, an d S.

mutans were isolated on the maintenance diet. All monkeyson the high-sucrose diet harbored S. mutans and S. sanguis;isolat ion of 5. 'mit ior ' was variable (Table 3). Monkeysconsuming the maintenance or commercial diet were alsofasted for 18 hrs. Interest ingly , the numbers of bacteriacolonizing the tooth surfaces of monkeys were equivalent tothose when the monkeys were fed. Also, the population of S.'mitior' in the plaque of fasted monkeys was significantlyhigher than when they were given food. In these monkeys,the doubling times of S. mutans an d S. 'mitior' during thefirst 24 hrs of plaque re-growth were not influenced by diet.However, sucrose significantly increased the doubling timesof 5.sanguis (Table 3).In a similar study, Beighton et al. (1986) extended theirexamination of plaque regrowth to include data on morespecies and noted a significant increase in the numbers ofNeisseria mucosa after the monkeys had been fasted andgiven distilled water. However, glucose supplement in thedrinking water of fasted monkeys modified this result, andalthough the numbers ofN. m ucosa increased, the differencewas not significant. Based on their results, Beighton and hisco-workers concluded that carbon was not a growth-limitingfactor during plaque formation, but that the nature of dietcould influence the numbers and proportions of bacterialpopulations in the comm unity.T o e x p l o r e f u r t h e r t h e p o s s i b i l i t y t h a t s a l i v a r y

glycoproteins were responsible for the support of bacterialbiofilm development in monkeys, Beightonet al. (1986) andS m i t h a n d B e i g h t o n ( 1 9 8 6 ) e x a m i n e d p l a q u e f o rexoglycosidase enzymes. Smith and Beighton (1986) assayed13 exoglycosidases in plaque from monkeys fed either amaintenance or a sucrose diet, and in plaque formed in theanimals after fasting. The results showed significant increasesin a-L-fucosidase, 6-N-acetyl-D-glucosaminidase, 6-N-acetylgalactoaminidase, and neuraminidase in plaque from fastedmonkeys previously eat ing the starch-based maintenanced i e t ; t h e l e v e l s o f B - L - f u c o s i d a s e , a - N - a c e t y l - D -galactosaminidase, and B-N-acetyl-D-glucoaminidase werereduced. In contrast, plaque from fasted animals previouslyfed sucrose did not show increases in any of the enzymeactivities. The authors suggest that removal of the starch dietinduced increases in the enzymes, to allow for continuedg r o w t h o f t h e o r g a n i s m s t h r o u g h m e t a b o l i s m o f t h ecarbohydrate residues of salivary glycoproteins. However,the presence of excess carbohydra te a l so necess i ta tedincreases in these enzymes, not to provide carbohydrate, butto supply the nitrogen from the exposed protein component ofthe glycoprotein necessary to maintain growth with excesscarbohydrate.

Studies in monkeys and rats suggest that diet does not playa significant role during the early development of plaque. Thestudies of Beighton and his co-workers have added furtheremphasis to the role of salivary glycoprotein as a significantnutrient in the oral cavity. In the absence of diet, organismsable to metabolize sal ivary glycoproteins, amino sugars(Beighton and Smith, 1986), and amino acids will have acompetitive advantage over those more dependent on refinedcarbohydrates. The nature of the diet can influence bacterialsuccession later in the accumulation of plaque.Study of competition among the populations of biofilmcommunities in humans in relation to diet is difficult. Veryoften, s tudies on plaque development in humans haveinvolved in situ models (Bowden, 1995) . Inte rbac ter ia l



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NUTRITION ANDBIOFILM DEVE LOPMENT

10

10

10

9 .

10 8 .

10 7

1 0 '

S. sanguis SK78E3 A naeslundii WVU627

25.2 74.8

22.2% 77.8

DaysPriming

10

10

1 0 ,

9

1 0 '

10 7

1 0

S. sanguis SK78A. naeslundiiWVU627

B82.2 17.8

42.5% 57.5

DaysGlucose Pulses

ProportionsofcellsofS. sanguisSK78andA.naeslundiiWV U 627 accumulatedinbiofilmsinm ucin-supplementedat D =0.1h1, pH7.0,for 1and5days A) andafter exposureofthe b iofilmsto anenvironmat D =0.1h1 withoutpHcontrol.

is oneaspectof thesuccession of bacterialandtherefore these models cou ld be usedtotheeffects of diet,but such studiesmay be deemedthe of diet on bacterial succession by exposingatosucrose outsideof themouth. Minahet. (1981), usinga removable device, madea detailed study the microbiological effects of sucrose compared withof experimental plaque from caries-freeand In caries-susceptible persons, inV eillonella, Lactoba cillus,and, to a lesser extent,S.mutans. ofN eisseria and 5.sanguis declined. Increasesin were associated with reduction inS. mutans.ofandN eisseriaand lower levelsofLactobacillus.a similar study, Macpherson et al. (1990), using enamelin aremovable device, examinedtheinfluenceofextra- on colonization of the slabs after

of themouthsof the subjects with theirownofS.mutans.Under these conditions, colonizationbywasenhanced and thenumbersof Lactobacillusin the plaque increased, sugges t ing a low pH The p r o p o r t i o n s of S. sanguis and declined.Theresults from thelimited of studiesof theeffects of sucrose using in situinplaque invivocan by s u c r o s e . In addi t ion, s tudies of the of caries have shown that, in certain casesarehighincarbohydrate and/orof low pH,such

in biofilmscan berelatedtodental disease (Bo wden, 1991)E S T A B L I S H M E N T OF A B I O F I L ME N V I R O N M E N T

Early accumulationsofbiofilms often consistofsingle layeof cellsorsmall aggregatesonsurfaces. Later stages resulta multi-cell layer with intercellular matrices, which veoften consist of carbohydra te (Coster ton et al.,19ChristensenandChara cklis, 1990; Gilbertetal., 1993). Thec o m p l e x - s t r u c t u r e d b i o f i l m s p r o v i d e a s e q u e s t e r eenvironment (Costertonet al, 1981,1987, 1994;BrownaGilb ert , 19 93), different from that in the a s s o c i a t eplanktonic phase. Examination of the fluid phaseof dentplaque, i.e.,plaqu e fluid, con firms thattheenvironmentbiofilm cellsin themouth is quite distinct from thatofassociated saliva (Edgar andHigham, 1990;T atevossia1990;Vogelet al.,1990; MargolisandMo reno, 1994). Alscellsin mature biofilms can be more resistant than thosefluid cultureto harmful agencies, suchas some antibioti(Costertonet al.,1987; Brownand Gilbert, 1993).Theearstagesof biofilm formation involve cells with relatively littextracellular matrix, whichcan be dividing rapidly, quitecontrastto mature biofilms. Therefore, theinfluence ofplanktonic environment, including the nutrients availablmaybedifferent betweentheinitialandsteady-state 'ma turaccumulationsof biofilm cells. Multi-species mature biofilmrepresent consortia where the interactive metabolismnutrients which cannotbe easily predicted or studiedcoccur. However, elegant microscopic techniquesforanalysof mature biofilms arebeing developed (Costerton et a



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94 BOWDEN & Li ADV DENT RE S APRIL1997TABLE 4

N U M B E R S O F C E LLS O FS. sanguis AN DA. naeslundiiI N B I O F ILM S G R O W NUND ER GLUCO SE LIMITATION AT pH 7.0B E F O R E A N D A F TE R E X P O S U RETO A N E N V IR O N M E N T W I TH F LU C TU A TIN GG LU C O S E E X C E S S A N D LO W p H

Priming Biofilms

One DayS. sanguis SK 78A. naeslundiiW VUFive DavsS. sanguis SK 78A. naeslundii W VU

62 7

627

CF U

311

2674

x 10 6/cm 2

.2.2

.3

.8

CFU x 106/cm2after 5 D ays'Glucose Pulse

5.88.0

59 5129

1994; James et ai, 1995). The ability to survive and grow inacidic environments i s , as has been shown above, animportant competit ive advantage to bacterial populationsmetabolizing carbohydrate in dental plaque. I t has beensuggested that early biofilm accumulations may be moreaffected by the local environment than the later , morecomplex biofilms (Fletcher, 1991).Initial experiments on bi-cultures in a chemostat modelprovide some support for this concept (Li and Bowden,

unpubl ished) . B i -cul ture b iof i lms of S. sanguis and A.naeslundii were produce d as primary biofilms. Primarybiofilms were grown on rods in mucin-supplemented basalmedium with limiting glucose (0.2 mmol/L) at pH 7.0, D =0.1 h 1 in a chemostat for periods of 5 days (multilayer ofcel ls) and 24 hrs (cel l monolayer ) . Subsequent ly , thechemostat culture was pulsed daily with glucose (7.5%, 10mL) without pH control for 5 days. The results are shown inFig. 4 and Table 4. The primary 24-hour and f ive-daybiofilms contained similar proportions of both species, but, asexpected, the five-day biofilms had higher numbers of cells.Twenty-four-hour primary biofilms in the stressing glucosepulse environment (Fig. 4B) for 5 days did not increase theircell numbers, which were sl ightly reduced, al though theproportions of the cells changed. In contrast , f ive-dayprimary biofilms increased their cell numbers in the glucosepulse environment (Fig . 4B) . Also , S. sanguis b e c a m edominant under conditions of fluctuating pH compared withA. naeslundii, which was dominant in five-day biofilms inglucose-limiting culture at pH 7.0 (Fig. 4A). These datasupport the concept that cells in mature biofilms resist harshenvironments better than earlier accumulations and alsosuggest that mature biofilms develop their own 'biofilmenvironment'. The change in proportions of the two speciesseen in Fig. 4B shows that populations in mature biofilms canrespond to changes in the availability of nutrient, which

influences bacterial succession.C o n s i d e r a t i o n o f t h e in vitro studie s of b iof i lmdevelopment and nutrition indicates that the predictions oncompetition and survival of mixed cultures from suspendedchemostat culture are often borne out by study of biofilms.Some significant features have to be considered, however, inrelation to the nutritional influences on biofilms. The first isthat, by their very nature, biofilms ensure the retention ofcells within a habitat, independent of relatively harsh director indirect nutritional influences. This further emphasizes thesignificance of adherence and the early establishment of cellson a surface. D orman t adherent cells probably retain thec a p a c i t y f o r g r o w t h w h e n e n v i r o n m e n t a l c o n d i t i o n s ,including nutrient, are suitable and therefore provide areservoir of biofilm populations. In addition, mature biofilmsmay develop their own localized environments that dictatethe metabolic activities of cells and protect them to someextent against changes in the environment of the associatedplanktonic phase. I t must be recognized, however, thatnutrient can produce changes within the environment ofmature b iof i lms, such as var ia t ions in pH (Edgar andHigham, 1990), so that the ability to survive or adapt tonutritional and other changes in environments within maturebiofilms remains an important aspect of the ecology ofbiofilm cells.

NUTRITION ND THE PRODUCTIONOF INTERCELLUL R M TRIX

Cells in mature biofilms are generally supported in anextracellular matrix (Guggenheim, 1970; Costerton et al.,1981; Christensen and Characklis, 1990; Gilbertet ai, 1993).This matrix serves to stabilize the biofilm on the surface andprovides support for the individual biofilm cells in thecommunity. Although extracellular carbohydrate polymersassociated with a wide range of medical and other bacteriahave been described, with some exceptions, relatively little isknown of the composition of biofilm matrices (Costerton etal , 1981; Christensen and Characklis, 1990; Fletcher, 1991).

The association between refined carbohydrate diets andcaries, together with the demonstration that strains of S.mutans produced extracellular polysaccharides from sucrose,led, early on (Guggenheim, 1970), to the recognition of apotential role for the plaque matrix in dental disease. Thiswas reinforced by the f inding of glucans in the water-insoluble matrix of plaque (Hotzet al.9 1972). Consequently,since the 1960s, the study of the production, nature, andenzymes involved in the synthesis of various extracellularpolysaccharides by oral bacteria, in particular streptococci(Doyle and Ciardi, 1983), has been a significant area forresearch. Although emphasis has been placed on streptococciand sucrose, i t has also been recognized that other oralbacteria will produce extracellular polysaccharides fromsucrose, glucose, or other carbohydrates. Strains resemblingNeisseria mucosa an d N eisseria subflava synthesize twotypes of polysaccharides from sucrose (Birkhed et al.,1979a); Actinomyces viscosus and A. naeslundii can producecomplex heteropolymers and levans (van der Hoeven, 1974;



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voL.ua NUTRITION AND BIOFILM DEVE LOPMENT 95Birkhed et ai, 1979b; O oshima amd Kuramitsu, 1981), andStomatococcus mucilagenosus produces a complexheteropolysaccharide surface slime on standard carbohydrate-containing media (Bowden, 1969).

Given th is potent ia l for the product ion of var iouscarbohydrate polymers by oral bacteria, it is not surprisingthat the intercellular matrix of oral biofilms can include thesematerials. Often, sucrose is the substrate for these polymers,and consequently, diets containing sucrose can increaseplaque mass (Carlsson and Egelberg, 1965) and facilitate theretention of streptococci and their colonization of the oralcavity in experimental animals (see above) and humans(Krasseet al, 1967). Glucose favored the establishment of A.viscosus in experimental animals (van der Hoeven, 1974).Thus,carbohydrates in the diet would be expected to promotean increase in bacterial extracellular polymers, and changesin the proportions of extracellular polymers to cells may berelated to the cariogenicity of plaque (Dibdin and Shellis,1988; Van Houte ^ a/., 1989).

CONCLUSIONSHuman diets are often comprised of a high proportion off e r m e n t a b l e c a r b o h y d r a t e s , a n d c o n s u m p t i o n o f h i g h -carbohydrate diets is related to the development of dentalcaries. The bacteria involved in demineralization of enamelare localized to the tooth surface as a biofilm community,supported by an extracellular matrix. Methods to prevent andcontrol dental caries are directed, either indirectly or directly,at modification of the activities of biofilms, to a state that iscompatible with health. Therefore, modification of thebiology of biofilms, be it in their formation, final bacterialcomposition, metabolism, or chemical structure, is one key tocontrol caries. Since a similar but more complex relationshipe x i s t s b e t w e e n b i o f i l m s a n d p e r i o d o n t a l d i s e a s e ,understanding biofilm biology is a signif icant area ofresearch in oral microbiology. W e already have a firm basisof understanding of aspects of the biology of oral biofilms. Inthe future, a more complete understanding of the formation ofbiofilms and, most particularly, of the activities of biofilmcells in consortia may lead to recognition of new approachesto control dental disease. Certainly, improved understandingwill allow us to modify our current approaches to make themmore effective.

ACKNOWLEDGMENTG.H.W .B . is supported by grant MT 7611 from the MedicalResearch Council of Canada.

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Implantation of caries-inducing streptococci in the humanoral cavity.Arch Oral Biol 12:231-236.Leach SA, Saxton CA (1966). An electron microscopic studyof the acquired pellicle and plaque formed on the enamelof human incisors.Arch Oral Biol 11:1081-1094.Lee SF (1995) . Act

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