Periodontology 2000, Vol. 55, 2011, 1635 Printed in Singapore.

All rights reserved

2011 John Wiley & Sons A/S

PERIODONTOLOGY 2000

Dental plaque biolms: communities, conict and control

P H I L I P D. M A R S H , A N N E T T E M O T E R & D E I R D R E A. D E V I N E

From the very beginning of the discipline of microbiology, the dogma has been to isolate bacteria in pure culture in order to be able to dene their individual properties. This process also involved the use of conventional broth (planktonic) culture to prepare biomass and to determine the phenotype of particular species. This approach provided a sound foundation for contemporary investigations of classical infectious diseases. Recently, however, there has been a renaissance in our understanding of microbial behaviour in natural habitats, and a recognition that chronic diseases can have a complex aetiology. It is now accepted that, in nature, bacteria exist for the most part attached to a surface as a biolm, often as a member of a polymicrobial community (or consortium) of interacting species. If biolms were merely planktonic-like cells that had adhered to a surface and the properties of a multi-species microbial community were just the sum of the constituent populations, then the scientic and clinical imperative for their study would be low. However, application of novel imaging (confocal or epiuorescence microscopy, uorescence in situ hybridization, live dead stains, etc.) and molecular techniques (16S rRNA gene amplication and sequence comparison, proteomics, transcriptomics, reporter gene technology, etc.) has radically altered our understanding of the biology of multi-species biolms (Table 1), and key developments that are pertinent to the control of dental plaque are highlighted in this review. Another major shift in our understanding of microbial behaviour has come from our increased knowledge of microbial ecology (3), and recognition of the intimate relationship between the resident human microora and the host. Changes in the host environment have a direct impact on gene expression, and thereby inuence the metabolic activity,

competitiveness and composition of the microora, while the action of resident microorganisms can have consequences for the host. An appreciation of this dynamic relationship is critical to fully understand the relationship between the oral microora and the host in health or disease.

The mouth as a microbial habitat

The human body is estimated to be composed of more than 1014 cells, of which only 10% are mammalian (125, 161). The majority are the microorganisms that make up the resident microora found on all environmentally exposed surfaces of the body, and this human microbiome is reported to have a metabolic capacity equivalent to that of the human liver. The microora of the skin, mouth, digestive and reproductive tracts, etc. are distinctive because of the characteristic biological and physical properties of each site (161), despite the potential movement of microorganisms between sites. This observation illustrates a key concept; namely, that the properties of the habitat are selective and dictate which organisms are able to colonize, grow and be minor or major members of the community. The mouth is similar to other habitats within the body in having a characteristic microbial community that provides benets for the host. The mouth is warm and moist, and is able to support the growth of a distinctive collection of microorganisms (viruses, mycoplasma, bacteria, Archaea, fungi and protozoa) (90). Bacteria are the most numerous group and initially were characterized using cultural approaches. Over time, it became clear that there was a discrepancy between the number of bacteria in a sample that could be grown by these conventional

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Plaque biolms and communities

Table 1. Properties of biolms and microbial communities (adapted from Ref. 90)
General property Open architecture Microbial protection Host protection Enhanced tolerance to antimicrobials* Neutralization of inhibitors Novel gene expression* Coordinated gene responses Communication with host Dental plaque example Presence of channels and voids Production of extracellular polymers to form a functional matrix; physical protection from phagocytosis Colonization; resistance Reduced sensitivity to chlorhexidine and antibiotics; gene transfer b-lactamase production by neighbouring cells to protect sensitive organisms Synthesis of novel proteins on attachment or on binding to host molecules; upregulation of gtfBC in mature biolms Production of bacterial cell-to-cell signalling molecules (e.g. CSP, AI-2) Downregulation of pro-inammatory responses by resident oral bacteria; remodelling of the cytoskeleton of epithelial cells pH and O2 gradients; co-adhesion Obligate anaerobes in an overtly aerobic environment Complete catabolism of complex host macromolecules (e.g. mucins) by microbial consortia (food chains and food webs) Pathogenic synergism in periodontal diseases

Spatial and environmental heterogeneity Broader habitat range More efcient metabolism Enhanced virulence

*One consequence of altered gene expression may be increased tolerance to antimicrobial agents.

approaches and those that were observed directly by microscopy (27, 110). It is estimated that <50% of the resident oral microora can currently be cultivated in pure culture in the laboratory (151, 152). This may reect our naivety in attempting to isolate microbes in pure culture that have evolved over millennia to grow as part of a consortium. Our knowledge of the richness and diversity of the resident oral microora has been enhanced by the recent application of culture-independent molecular approaches. Numerous studies of various surfaces and sites based on amplication, cloning and sequencing of the 16S rRNA gene have identied approximately 700 species in the mouth. Most sites (mucosal or plaque) yielded 2030 different predominant species, and the number of species per individual mouth ranged from 34 to 72 (1). However, these gures may be an underestimate, as the use of a more discriminatory pyrosequencing approach detected several thousand phylotypes in samples of saliva and supragingival plaque from healthy adults (61). This study used a next-generation high-throughput sequencing technique, which increases the number of clones that can be sequenced, thereby increasing the probability of detecting low-abundance taxa.

The fact that certain microorganisms are consistently isolated from a habitat indicates that all of their growth requirements are being met. Primary nutrients are obtained from endogenous sources, such as amino acids, proteins and glycoproteins in saliva and gingival crevicular uid; the host diet plays only a minor role in providing nutrients for the resident microora. Catabolism of the more complex host molecules requires the sequential or concerted action of consortia of bacteria (see below), in which their metabolic capabilities are combined, and is an example of microbial cooperation (10, 57, 58, 108, 113, 142). Saliva also plays a major role in maintaining the oral pH at around neutrality, which is suitable for the growth of many microorganisms. Although the mouth is aerobic, oxygen is rapidly consumed by early bacterial colonizers that are aerobic (e.g. Neisseria spp.) or facultatively anaerobic (e.g. Streptococcus and Actinomyces spp.), and other gases (CO2, H2) and reduced compounds are released that lower the redox potential, especially in dense biolms such as dental plaque, creating conditions suitable for obligate anaerobes (90). This often results in a precise spatial organization of interacting bacteria in plaque biolms, e.g. for streptococci and Fusobacterium nucleatum (Fig. 1).

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Marsh et al.

Fig. 1. Fluorescence in situ hybridization of a subgingival biolm showing the close spatial relationship between facultatively anaerobic Streptococcus spp. (orange) and obligately anaerobic Fusobacterium spp. (magenta). Subgingival biolms of periodontitis patients were obtained using a carrier system as described previously (156). Bacteria were visualized in 3 lm cross-sections of the biolms using the following probes simultaneously: probe EUB338, which detects most bacteria (green), probe Strep1 2 (49), which shows streptococci, probe FUS664, which detects most Fusobacterium spp., and non-specic nucleic acid stain DAPI (blue). Details of oligonucleotide probes are available at probeBase, an online resource for rRNA-targeted oligonucleotide probes (80) (http://www. microbial-ecology.net/probebase/).

Biolms develop on mucosal and dental surfaces within the mouth, but the composition of the oral microora varies signicantly at distinct surfaces within the mouth (1, 109, 123), again emphasizing the important link between the properties of the habitat and the organisms that are able to predominate. The remainder of this review focuses on the properties of dental plaque as a biolm and a microbial community, and on the ways in which our current knowledge of biolms can be exploited in order to improve plaque control. Due to the breadth of the topic, readers are also directed to other reviews that emphasize complementary aspects of dental plaque as a biolm (67, 81, 130, 131).

Impact of the habitat on microbial gene expression

Microorganisms are capable of adapting to changes in environmental conditions, and alter their pattern

of gene expression in order to survive (17, 46, 92). In the mouth, there are signicant changes to the habitat during disease (Fig. 2). Caries is associated with an increase in the frequency of sugar consumption and rapid conversion of these carbohydrates to acidic fermentation products. Repeated conditions of low pH in dental plaque biolms select and enrich for acidogenic and acid-tolerating species (for example, mutans streptococci, lactobacilli and other acidloving streptococci) at the expense of those bacteria with a preference for growth at neutral pH (17, 82, 138). In periodontal disease, the inammatory response to biolm accumulation results in an increase in the ow of gingival crevicular uid, sometimes with bleeding, and a local rise in temperature. The increase in ow of gingival crevicular uid not only provides components of the host defences but also introduces a range of host proteins and glycoproteins that can be exploited as substrates by, and provide essential cofactors for the growth of, many of the obligate anaerobic and proteolytic species present in subgingival biolms (142, 143). This proteolytic pattern of metabolism results in a small increase in pH. Signicantly for the ecology of the subgingival environment, the pH range for the growth of many bacteria implicated in periodontal disease, such as Porphyromonas gingivalis, Prevotella intermedia and F. nucleatum, extends above pH 7.0, and the optimum is often around pH 7.5 (92, 93, 121); thus, a rise in local pH increases the competitiveness of these putative pathogens within the subgingival community during inammation. These changes in environment associated with inammation further alter gene expression. For example, P. gingivalis becomes more proteolytic (e.g. higher gingipain activity) in response to an increase in haemin availability, and an increase in environmental pH results in further upregulation of gingipain activity (91, 93). More recent transcriptomic and proteomic studies have shown the differential expression of 70 proteins by P. gingivalis depending on haemin concentration, with upregulation of a protein associated with cell invasion during growth under haemin limitation (32). In contrast, a high temperature resulted in P. gingivalis downregulating protease activity (112). Thus, as the subgingival environment gradually changes, there is a shift in both the competitiveness and aggressiveness of previously minor components of the microora. If sustained, this can disrupt the natural balance of organisms within the biolm community, resulting in a shift in the composition of the microora of a site and increasing the risk of disease (Fig. 2) (82). As stated previously, an awareness of the

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Plaque biolms and communities

Homeostatic mechanisms

Health Caries
Acidogenic/aciduric:

- Mutans streptococci - Lactobacilli - Other acidogenic/ aciduric streptococci

Plaque community stability

Periodontal diseases Gram-negative anaerobes: - Spirochaetes (e.g. Treponema denticola) - Porphyromonas gingivalis - Tannerella forsythia - Aggregatibacter actinomycetemcomitans

Frequent sugar/ low pH challenges Low saliva flow

Inflammation/ increased gingival crevicular fluid flow Immune suppression

Ecological perturbation

Fig. 2. Ecological shifts in the dental plaque microora in health and disease (adapted from Ref. 90). Homeostatic mechanisms involving microbial interactions help maintain a stable benecial microbial community that is associated with oral health. Severe changes to the habitat (ecological perturbations) can alter this equilibrium by selecting for organisms that are more competitive in the altered environment, and this can predispose sites to disease.

dynamic balance between the environment and the microora can help explain how the normally benecial relationship between the oral microora and the host can be lost and disease can occur, providing an opportunity for novel interventions.

the microbial cell surface and that produced by the conditioning lm (8, 19). Microorganisms are usually transported passively to the surface by the ow of saliva or gingival crevicular uid; a few species (e.g. Wolinella, Selenomonas and Campylobacter spp.) found subgingivally have agella and are motile.

Dental plaque a classical multispecies biolm

Dental plaque has been dened as the microbial community that develops on the tooth surface, embedded in a matrix of polymers of bacterial and salivary origin (90). Dental plaque forms via an ordered sequence of events, resulting in a structurally and functionally organized species-rich microbial biolm (66, 67, 83, 130). The distinct stages in plaque biolm formation are described below.

Irreversible adhesion
Irreversible adhesion involves interactions between specic molecules on the microbial cell surface (adhesins) and complementary molecules (receptors) present in the acquired pellicle. These adhesin receptor interactions are strong and operate over a relatively short distance (159), and are targets for possible novel interventions to block colonization.

Co-adhesion
During co-adhesion, secondary and late colonizers adhere via cell-surface adhesins to receptors on already attached bacteria (65), leading to an increase in microbial diversity within the developing biolm (microbial succession) (Fig. 3) (67). Many of the secondary colonizers have fastidious growth requirements.

Formation of a conditioning lm
Molecules are adsorbed to the tooth surface within seconds immediately after cleaning or following initial exposure to the oral environment, and remain functional (53). These molecules are derived mainly from saliva, but, in the subgingival region, molecules originate from gingival crevicular uid. The conditioning lm alters the properties of the surface, and bacteria interact directly with the constituent molecules.

Multiplication of the attached cells

Multiplication of the attached cells leads to an increase in biomass and synthesis of exopolymers to form a biolm matrix (5, 15). A matrix is a common feature of all biolms, and is more than a chemical scaffold to maintain the shape of the biolm. It makes a signicant contribution to the structural

Reversible adhesion
Reversible adhesion involves weak, long-range, physico-chemical interactions between the charge on

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Fig. 3. Fluorescence in situ hybridization of subgingival biolm showing stratication of species. Small cocci predominate in the bottom layer of the biolm, detected by the bacterial probe (green). Fusobacterium nucleatum canifelinum (magenta) is predominantly found as a secondary colonizer, whereas the motile group II treponemes (yellow, Treponema denticola-related) are found in both layers. Details of the probes EUB338, FUNU and TREII can be obtained at http://www.microbial-ecology.net/ probebase/. At the gingival side of the biolm, autouorescent erythrocytes (red, arrowhead) and a few host cell nuclei stained by non-specic nucleic acid stain DAPI (blue, arrow) are visible.

retain water, nutrients and enzymes within the biolm. The chemistry of the matrix may also exclude or restrict the penetration of other molecules (55, 141), including some charged antimicrobial agents (e.g. chlorhexidine, quaternary ammonium compounds) (5, 15). The close proximity of cells to one another in a biolm facilitates numerous synergistic and antagonistic interactions between neighbouring species, and food chains and food webs develop (see below) (72, 90). The metabolism of the microorganisms produces gradients within the biolm; for example, in nutrients and fermentation products, and in pH and redox potential (Eh). Bacteria respond to these uctuating changes in environmental conditions by altering their patterns of gene expression (see below) (32, 46). The gradients in plaque are not necessarily linear, and the environmental heterogeneity results in a mosaic of microenvironments (150). This environmental heterogeneity over relatively short distances helps to explain how microorganisms with apparently contradictory growth requirements can co-exist in biolms such as dental plaque. These processes lead to the establishment of a mature biolm (Fig. 4) with a relatively stable composition.

integrity and general tolerance of biolms to environmental factors (e.g. desiccation) and antimicrobial agents. The matrix can be biologically active and
A B

Detachment from biolms

Bacteria are able to sense changes to their environment, for example by two-component signal

Fig. 4. Mosaic architecture of 5-day-old subgingival biolms with various oral species detected by uorescence in situ hybridization. (A) Clusters of Fusobacterium nucleatum canifelinum (magenta) and Prevotella intermedia (yellow). (B) Porphyromonas gingivalis (magenta) alternates with Tannerella forsythia (yellow). The uo-

rescence in situ hybridization probes FUNU and PRIN (A) or POGI and TAFO (B) were used in combination with EUB338 (green) and DAPI (blue). Further information regarding the uorescence in situ hybridization probes can be obtained at http://www.microbial-ecology.net/ probebase/.

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transduction systems. If conditions deteriorate, some species (e.g. Prevotella loescheii and Aggregatibacter actinomycetemcomitans) respond by upregulating enzymes that cleave their adhesins, enabling the cell to detach and colonize elsewhere (23, 60). Once the biolm has formed, the species composition at a site is characterized by a degree of stability or balance among the component species, despite regular minor environmental stresses following, for example, periodic oral hygiene, food intake or diurnal changes in saliva ow. Importantly, this stability (termed microbial homeostasis) is not due to any biological indifference among the resident organisms, but is due to a dynamic balance imposed by numerous microbial interactions, including examples of both synergism and antagonism (see below) (3, 84). Bacteria respond to environmental change (see above), and microbial homeostasis can break down if a key parameter exceeds the threshold that is compatible with community stability. A consequence of homeostasis breakdown is re-organization of the structure and composition of the microbial community, with previous species that were only minor components becoming more competitive under the new conditions, and, as a result, more dominant. Such a change in community composition and activity can predispose a site to disease. Insight into the organization and architecture of oral biolms has improved with technological developments in microscopy (129). Confocal scanning laser microscopy can visualize specimens in their natural, hydrated state. When dental plaque was allowed to develop naturally on enamel slices placed in removable devices in the mouth and imaged by confocal microscopy, the architecture of the resultant biolms was far more open than previously seen using conventional electron microscopy (164). Channels or pores, possibly lled with extracellular polymers, penetrated the biolm; the presence of this matrix could inuence the distribution and movement of molecules in plaque (Fig. 5) (55, 139, 141, 145, 155). Use of live dead stains with confocal microscopy suggests that bacterial vitality may vary throughout plaque, with most viable and active bacteria being present in the central part of the biolm and lining the channels, while a combination of uorescence in situ hybridization and confocal or uorescence microscopy enables the spatial distribution of species within oral biolms to be observed (36, 98, 145, 156), as highlighted in many of the gures in this review. In the gingival crevice, plaque biolms have a thin densely adherent layer on the root surface, with the

Fig. 5. Fluorescence in situ hybridization on a crosssection of a mature subgingival biolm showing pores or channels (arrowheads) against the background (green) of bacteria. These presumed channels are surrounded mainly by Fusobacterium nucleatum canifelinum (magenta) and Tannerella forsythia (yellow).

bulk of the biolm having a looser structure, especially where it comes into contact with the epithelial lining of the gingival crevice periodontal pocket. Thus there is opportunity for hostmicrobe crosstalk. Many bacterial associations have been observed subgingivally by electron microscopy in these outer layers, in which cocci are arranged along the length of lamentous organisms, e.g. corn cob, test tube brush or rosette formations. Fluorescence in situ

Fig. 6. High numbers of group I treponemes (orange) in a subgingival biolm, most of which are as yet uncultured. The carrier section was hybridized with probe TRE I together with FUNU for detection of Fusobacterium nucleatum canifelinum (light blue), which forms a cluster in the lower left corner, and DAPI (dark blue).

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hybridization has shown the presence of many currently unculturable bacteria, including spirochaetes (Fig. 6) and the TM7 phylum, in large numbers in samples of subgingival biolms (99, 100, 107).

Plaque as a biolm and community consequences for the microorganisms

Dental plaque was the rst biolm to be studied in terms of both its microbial composition and its sensitivity to antimicrobial agents. In the 17th century, Antonie van Leeuwenhoek pioneered the approach of studying biolms by direct microscopic observation when he reported on the diversity and high numbers of animalcules present in scrapings taken from around human teeth. He also conducted early studies on the novel properties of surface-grown cells when he failed to kill plaque bacteria on his teeth by prolonged rinsing with wine vinegar, even though the organisms were killed if they were rst removed from his molars and mixed with vinegar in vitro. It is only in recent years, with the advent and application of new molecular and imaging technologies, that a more complete understanding of the biology of dental plaque as a biolm and microbial community has been possible. Some of the implications of this surface-associated, community-driven lifestyle, and the opportunities for biolm control, are described below.

Biolm regulation of microbial gene expression

Bacteria in biolms display a phenotype that is distinct from that exhibited by the same cells growing planktonically. The initial attachment of bacteria can result in sudden changes in gene expression, especially in terms of upregulation of exopolymer synthesis. For example, adhesion of Pseudomonas aeruginosa to a surface leads to upregulation of genes involved in alginate synthesis within 15 min of the initial contact (33), while proteomic studies have demonstrated that these cells alter the expression of 4060% of their proteome during biolm formation (111, 127). Oral bacteria also modify their patterns of gene expression during biolm formation, although the effects may be less dramatic than those observed for environmental bacteria because of the obligate biolm lifestyle of the former organisms (18). For tech-

nical reasons, most studies of oral bacteria have been performed on bacteria that predominate in supragingival plaque (e.g. streptococci), but similar principles apply to subgingival organisms. During the initial stages of biolm formation (rst 2 h following attachment), 33 proteins were differentially expressed (25 proteins were upregulated; eight were downregulated) by Streptococcus mutans, and there was an increase in the relative synthesis of enzymes involved in carbohydrate catabolism (158). In contrast, some glycolytic enzymes involved in acid production were downregulated in older biolms (3-day), while proteins involved with a range of biochemical functions including protein folding and secretion, amino acid and fatty acid biosynthesis, and cell division were upregulated (135). Novel proteins of as yet unknown function were expressed by biolm but not planktonic cells. Genes associated with glucan (gtfBC) and fructan synthesis (ftf) in S. mutans (and hence matrix formation) were also differentially regulated in biolms (75). There was little inuence of surface growth on gene expression in early biolm formation (<48 h), but gtf expression was markedly upregulated in older biolms (7-day), and ftf activity was repressed. These ndings demonstrate that growth in biolms can have either a direct effect (i.e. as a result of initial attachment) or indirect effect (i.e. due to the altered environmental conditions within the biolm, e.g. substrate concentration, pH, etc.) on gene expression by plaque bacteria. Recently, transcriptomic and proteomic studies of the effect of biolm formation on gene expression by periodontal pathogens have been undertaken (70, 79). When P. gingivalis was grown as a biolm, approximately 18% of the genome was differentially expressed, with genes involved in growth and biosynthesis of cofactors being downregulated, while transport and binding proteins were upregulated (79). During plaque formation, bacteria bind to host proteins and co-aggregate with other organisms, and the potential impact of these cues on gene expression is now being explored. Exposure of Streptococcus gordonii to saliva resulted in the induction of genes (sspA B) encoding adhesins that can bind to salivary glycoproteins and engage in co-aggregation with Actinomyces spp. (40). Differential expression of proteins by P. gingivalis when part of a community with S. gordonii and F. nucleatum has also been observed, with 403 proteins downregulated and 89 proteins upregulated by P. gingivalis during community development (70).

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Microbial interactions, cellcell communication and gene transfer

In biolms, microorganisms are in close physical proximity to one another and interact as a consequence (97). Many conventional metabolic interactions (synergistic and antagonistic) have been described among oral bacteria, and the development of food chains or food webs is common, in which the metabolic product of one organism becomes the primary nutrient for a second. Bacteria collaborate in order to catabolize complex host molecules (proteins, glycoproteins) (10, 142), while obligately anaerobic bacteria such as P. gingivalis can survive in aerobic environments if they partner with and co-aggregate to oxygen-consuming species such as Neisseria (Fig. 7) (12, 13). Antagonistic interactions involve the production of inhibitory compounds (bacteriocins, acids, H2O2, etc.) to inhibit neighbouring cells, and can provide the producer cells with a competitive advantage (120). This might explain, in part, why some bacteria appear in plaque biolms as discrete clusters of cells (Fig. 8). Puried natural molecules such as bacteriocins are being evaluated as novel inhibitors of specic bacteria in plaque biolms. Bacteria from plaque can coordinate their gene expression and communicate with one another in a cell density-dependent manner via small diffusible

Fig. 8. Fluorescence in situ hybridization of subgingival biolm showing separation of species in distinct areas. In the lower part of the image, long rods detected by a bacterial probe (green) and DAPI (blue) mix with Fusobacterium (magenta, probe FUS664) and group I treponemes (yellow, probe TRE I). The latter do not move into the upper parts of the biolm, which are inhabited by cocci and a dense cluster of small fusobacteria, suggesting competition between these populations.

Fig. 7. Fluorescence in situ hybridization image of a sectioned subgingival biolm hybridized with bacterial probe EUB338 (green) and probes specic for Fusobacterium nucleatum canifelinum (magenta) or Tannerella forsythia (yellow). The species appear to co-localize in upper part of the biolm.

molecules (quorum sensing), using strategies similar to those described for other biolms (31, 64, 134). In S. mutans, quorum sensing is mediated by a competence stimulating peptide (CSP), which increases the transformation frequency of biolm-grown S. mutans 10600-fold (77). Lysed cells in biolms could act as donors of chromosomal DNA, thereby increasing the opportunity for horizontal gene transfer in dental plaque. Mutations in some of the genes involved in the CSP signalling system (comC, comD, comE and comX) result in defective biolms, indicating that CSP is directly involved in biolm formation. CSP also increases acid tolerance in recipient cells within the biolm (76). Other communication systems may function between different oral species (64). A survey of gramnegative periodontal bacteria suggests that these organisms do not posses the acyl homoserine lactone-dependent signalling circuits that are common in environmental gram-negative bacteria (48).

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Instead, LuxS genes encoding auto-inducer-2 (AI-2), have been detected in several genera of oral grampositive and gram-negative bacteria, implying that AI-2 may have a broader species range (35, 117). Several periodontal bacteria [F. nucleatum, P. intermedia, P. gingivalis and Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans] secrete a signal related to AI-2 (45, 48). In A. actinomycetemcomitans, LuxS-dependent signalling induced expression of leukotoxin and a transport protein involved in iron acquisition, whereas, in P. gingivalis, LuxSdependent quorum sensing modulated protease and haemagglutinin activities, but was not essential for virulence (16). AI-2 defects in some bacteria can be complemented by molecules produced by other (but not all) species (167). AI-2 produced by A. actinomycetemcomitans complemented a luxS mutation in P. gingivalis, and AI-2 secretion by P. gingivalis could stimulated biolm formation by F. nucleatum (45, 124), suggesting a major role for these molecules in intra- and inter-species communication and coordination of activities. Signalling events can occur between metabolically interacting organisms. Expression of a-amylase by S. gordonii increased when in co-culture with Veillonella atypica (43). A surface protein of Streptococcus cristatus can repress P. gingivalis mbrial gene expression and has an impact on biolm formation and the potential virulence of the anaerobe (165). The diversity of signalling opportunities within microbial communities, and the signicant role of these molecules in coordinating gene expression and promoting biolm formation, have provided impetus to investigate the potential of inhibitory analogues to disrupt these networks, thereby providing mechanisms to control or inuence the development of dental plaque. In addition, CSP has been fused to an antimicrobial peptide domain to generate a specically targeted antimicrobial peptide that is capable of selectively eliminating S. mutans from multi-species biolms, while leaving benecial members of the consortium unaffected (41). A similar approach has been tested in vitro for targeted killing of P. gingivalis using an antimicrobial peptide (SMAP-28) linked to IgG specic for P. gingivalis surface components (47). Cells also communicate and interact with one another in biolms via horizontal gene transfer. As discussed above, signalling molecules such as CSP markedly increase the ability of recipient cells in biolms to take up DNA by transformation (77). The transfer of conjugative transposons encoding tetracycline resistance between streptococci has been demonstrated in model biolms (119). The recovery

from the naso-pharynx of resident (Streptococcus mitis, Streptococcus oralis) and pathogenic (Streptococcus pneumoniae) streptococci with penicillin resistance genes showing a common mosaic structure, conrms that gene transfer can occur in vivo (39, 52). Similar evidence suggests the sharing of genes responsible for penicillin-binding proteins among commensal and pathogenic Neisseria (9). Gene transfer between Treponema denticola and S. gordonii has also been demonstrated in the laboratory (154). The presence of pathogenicity islands in periodontal pathogens such as P. gingivalis is also indirect evidence for horizontal gene transfer having occurred in plaque biolms at some distant time in the past, and may explain the evolution of more virulent strains (25).

Tolerance to antimicrobial agents

Bacteria growing in biolms such as dental plaque display an increased tolerance to antimicrobial agents, including those used in dentifrices and mouth rinses (63, 89, 115, 162). For example, the concentrations of chlorhexidine and amine uoride required to kill Streptococcus sobrinus growing as an established biolm were 300 and 75 times greater, respectively, than the minimum bactericidal concentration against planktonic cells (128). Similarly, it was necessary to administer 1050 times the minimum inhibitory concentration of chlorhexidine to eliminate Streptococcus sanguinis biolms (74). The age of the biolm is also a signicant factor; older biolms of S. sanguinis (95) or A. actinomycetemcomitans (137) were more tolerant of chlorhexidine or antibiotics, respectively, than younger biolms. Biolms of several species of oral bacteria have also been shown to be more tolerant of antibiotics (e.g. amoxycillin, doxycycline, minocycline and metronidazole) than planktonic cells (73, 104, 130, 137), although the lack of sensitivity varies with the organism, the model system and the inhibitor used. Confocal microscopy of in situ established natural biolms showed that chlorhexidine only affected the outer layers of cells in 24 and 48 h plaque biolms (168), suggesting either quenching of the agent at the biolm surface or a lack of penetration. Fluoride (which can inhibit bacterial enzymes in addition to its effects on enamel biochemistry) showed an uneven distribution within natural biolms that developed on an in situ device worn by volunteers (155). Using time-lapse confocal microscopy, the spatial and temporal effects of mouth rinses on model oral biolms was continuously monitored, showing

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different penetration velocities and activity patterns between chlorhexidine, ethanol and an enzyme-based antimicrobial formulation (140). Bacteria growing in the depths of biolms generally divide slowly, and slow-growing cells are always less sensitive to antimicrobial agents, while a sensitive cell can be protected by resistant neighbouring organisms that produce a neutralizing factor (see below).

conditions that are suitable even for obligate anaerobes to proliferate. This can involve close physical contact between oxygen consumers and oxygensensitive bacteria (12, 13). Similar arguments apply to organisms with a specic pH or nutritional requirement (24). More efcient metabolism Endogenous substrates are the primary source of nutrients, but pure cultures of oral bacteria are generally unable to fully catabolize complex host macromolecules, especially glycoproteins such as mucins; these can only be degraded efciently by the concerted action of consortia of oral bacteria (10, 58). Mutualistic interactions were detected when combinations of A. actinomycetemcomitans, F. nucleatum and a Veillonella sp. were grown as biolms using saliva as a substrate (113). Communities also interact to sequentially break down these substrates to the simplest products (e.g. CH4, CO2, H2, NH3, etc.) by the formation of food chains (Fig. 10) (22). Increased tolerance to inhibitory agents and host defences A drug-sensitive pathogen can be rendered apparently resistant to an antibiotic if neighbouring commensal bacteria produce a neutralizing or
Antagonism (e.g. bacteriocins)

Benets of a microbial community lifestyle

The ability of plaque bacteria to interact with neighbouring cells in biolms provides compelling support for the concept that oral bacteria do not exist as independent entities but rather function as a coordinated, spatially organized and metabolically integrated microbial community (81, 87, 88) (Fig. 9). The benets of a community lifestyle for plaque microorganisms are similar to those described for other microbial communities, and are listed below. Broader habitat range The majority of bacteria isolated from dental plaque are obligately anaerobic despite inhabiting an aerobic environment. As discussed above, early biolm colonizers consume oxygen, which eventually creates

Cell densitydependent signalling (e.g. via CSP; AI-2)

Co-adhesion; spatial organization

Food webs/concerted metabolic activity

Group protection from antimicrobials Gene transfer

R R R R R R R
Conditioning film

Adhesin receptor

Bacterialhost cross-talk

Oral surface (tooth or mucosal)

Fig. 9. Schematic representation of the types of interaction (inter-bacterial and bacterialhost) that occur in a microbial community, such as dental plaque, growing as a biolm (adapted from Refs 81 and 90). Bacteria adhere via adhesinreceptor interactions either to the conditioning lm (derived either from saliva or gingival crevicular uid) or to already attached cells (co-adhesion). Bacteria interact synergistically to metabolize complex endogenous molecules (e.g. glycoproteins), and food webs can develop. Bacteria communicate with each other in a cell densitydependent manner via diffusible signalling molecules, and with host cells. Cells are more tolerant of antimicrobial agents either because of the physical attributes of the biolm, via gene transfer of resistance genes, or through protection by neighbouring cells that produce neutralizing enzymes. Cells may also gain advantage by production of inhibitory molecules.

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Dietary Sugar

Dietary carbohydrate
Concerted action

Host glycoprotein
Concerted action Proteinase Sulfatases

Polymer-degrading bacteria

Oligosaccharides

Peptides

Sulfate

Monosaccharides
Glycolysis

Peptidase Glycosidase

Fatty & hydroxy acids Alcohols

Acetogenesis Methanogenesis

Amino acids
Deamination Decarboxylation

Sulfatereducing bacteria

H2, CO2, CH4

CO2, NH3

H2S

Fig. 10. The concerted and sequential breakdown of endogenous and exogenous substrates by communities of oral bacteria present in dental plaque biolms with complementary enzyme activities.

drug-degrading enzyme. In the mouth, gingival crevicular uid can contain sufcient b-lactamase to inactivate any penicillin delivered to the site (56, 149, 153). Pathogens can also nd a safe haven within biolms to escape surveillance by the host defences, including phagocytic cells. Enhanced virulence A wide range of virulence traits are required by an organism in order to cause disease. For the development of periodontal diseases, subgingival bacteria must adhere, gain nutrients from the host and multiply, overcome or evade the host defences, invade cells and induce tissue damage. Individually, many subgingival bacteria are unable to satisfy all of the requirements necessary to cause disease, and combine forces to do so, forming a more virulent consortium of interacting bacteria (pathogenic synergism) (Fig. 11) (148). Within such consortia, individual species could play more than one role in disease, while different species could perform identical functions in consortia of different composition at other sites. This explains why communities with varying bacterial composition have been found at sites with similar disease, and is consistent with the concept of complexes associated with health and disease (51, 132). Evidence for pathogenic synergism has come from abscess models in animals, in which different combinations of oral bacteria displayed increasing pathogenicity and tissue damage (6, 44, 133). The infective dose of P. gingivalis was reduced by 1,000-fold when cells were co-inoculated with F. nucleatum into a subcutaneous chamber in mice (94).

Plaque as a biolm and community consequences for the host

The complex biolms that develop on oral surfaces continually interact with the host, and provide

Fig. 11. Pathogenic synergy by microbial communities in the aetiology of periodontal diseases (adapted from Ref. 90). Bacteria capable of causing tissue damage directly (e.g. species X) may be dependent on the presence of other cells (e.g. organisms C and D) for essential nutrients (e.g. via a food chain) or attachment sites (co-adhesion) so that they can grow and resist the removal forces provided by the increased ow of gingival crevicular uid. Similarly, both of these groups of bacteria may be reliant for their survival on other organisms (e.g. A and C) to modulate the host defences. Individual bacteria may have more than one role (e.g. organism C), or niche, in the aetiology of disease.

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benets or can cause conict. Examples that relate to plaque biolms are described below.

Benets of the resident microora

The resident microora at any site does not have merely a passive relationship with the host. The rapid suppression of the resident human oral microora by administration of antibiotics can result in overgrowth by previously minor components of the microora (e.g. yeasts in the oral cavity), or colonization by exogenously acquired (and often pathogenic) microorganisms (86). Thus, the resident microora acts directly as an important component of the host defences by forming a signicant barrier against exogenous populations, termed colonization resistance. The mechanisms involved in colonization resistance include the enhanced competitiveness of indigenous species, the occupation of potential attachment sites by resident microbes, the production of inhibitory compounds, and the development of environments that are not conducive to the establishment of invading organisms. The resident microbiota also contributes to the normal development of the physiology, nutrition and defence systems of the organism (160). Most of our understanding of these functional relationships comes from studies of the gastrointestinal tract (recently reviewed in Refs 21, 42, 59, 102, 122). The gut of germ-free animals is poorly developed, but when these animals are colonized by members of the natural resident microora, many of these anatomical and physiological deciencies are reversed (160). Likewise, humans on long-term antibiotic treatment can suffer from nutrient deciencies due to poor absorption or metabolism of vitamins. The gut resident microora also contributes 515% of the total energy requirement of the human host through generation of short chain fatty acids (7). Short chain fatty acids are also effector molecules that inuence immune responses, cellular differentiation and growth and production of reactive oxygen species (leading to multiple homeostatic cellular responses). While the contribution of the oral microbiota to host nutrition is unlikely to be as signicant as in the gut, many oral bacteria produce short chain fatty acids as end products of metabolism, so similar effects on host cellular function may be expected within the subgingival environment. In vivo and in vitro models have shown that the resident microora of the gut is important to the normal physiology of the epithelium, enhancing epithelial barrier function, cellular metabolism and

proliferation, and wound healing responses (21, 59, 102, 105, 106). It additionally provides low-level stimulation of the innate immune system to provide a basal inammatory tone that contributes to intestinal homeostasis and health (102). The resident gut microora exerts cytoprotective effects through regulation of adaptive immune responses, and experiments with germ-free animals have indicated a role for resident microorganisms in the normal development of the mucosal immune system (26, 42, 102, 122). Studies with germ-free mice have also indicated a role for resident oral bacteria in determining normal expression of immune mediators (38). Resident bacteria in the subgingival plaque may help to maintain healthy tissue by regulating low levels of expression of intracellular adhesion molecule-1, E-selectin and interleukin-8, which in turn can regulate the establishment of a protective layer of neutrophils strategically positioned between subgingival plaque bacteria and the junctional epithelium (37). In general, the microora of a site lives in harmony with the host, and both parties benet from the association. Disruption of the hostmicrobe balance and loss of regulation of resident populations can have detrimental effects in terms of development of infections (20) or chronic inammatory disorders (102, 122, 157). Therefore, the aim of oral care programmes should be to control the levels and activity of the oral microora, rather than trying to eliminate plaque, in order to retain the benecial functions of the resident organisms and keeping the oral microora at levels compatible with health.

Microbehost signalling
The ndings described above demonstrate that communication not only occurs between bacterial cells, but also between bacteria and the host. The binding of bacteria to specic receptors on host cells can trigger substantial changes in gene expression in both bacterial and host cells, e.g. immediately following the attachment of Escherichia coli to uroepithelial cells (2). It is now clear that the natural resident microora of humans is actively engaged in cross-talk with the host. Host cell-pattern recognition receptors such as Toll-like receptors and NOD-like receptors sample the extracellular and intracellular environments and recognize microbe-associated molecular patterns (e.g. lipopolysaccharide, lipoteichoic acid, nucleic acids), activating multiple signalling pathways, many

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of which converge on nuclear factor jB. Microbeassociated molecular patterns are present on, or released from, all microbial cells. It is essential that the host is able to tolerate resident microorganisms without initiating a damaging inammatory response, while also being able to mount an efcient defence against pathogens. Pathogenic and non-pathogenic bacteria may initiate different intracellular signalling pathways and innate immune responses in epithelial cells (20, 59, 102). In the gastro-intestinal tract, commensal bacteria such as Bidobacterium infantis and Lactobacillus salivarius do not induce pro-inammatory responses, unlike an exogenous pathogen such as Salmonella typhimurium (105). Some gut commensals ensure they are tolerated by causing functional modulation of immunity through Toll-like receptor expression and signalling (116), while others are able to suppress inammatory responses in epithelial cells by inhibiting activation of nuclear factor jB (29, 62, 103, 146) or by increasing the secretion of anti-inammatory cytokines, such as interleukin-10 (50). The resident oral microora may also be engaged in similar cross-talk in the mouth, and oral microorganisms are tolerated using similar strategies to gut commensals. Oral commensals and pathogens may activate distinct response pathways in oral epithelial cells (28, 54, 69, 105, 114). Certain oral streptococci have been shown to suppress epithelial cell cytokine expression (54, 114). Streptococcus salivarius K12 not only downregulated epithelial cell inammatory responses by inhibiting the nuclear factor jB pathway, but also actively stimulated benecial pathways, including type I and II interferon responses, and exerted signicant effects on the cytoskeleton and adhesive properties of the host cell (30). Surface components of subgingival bacteria are involved in adhesion to epithelial cells at the start of colonization and biolm formation, and there is also evidence that they are involved in bacteriumhost cell cross-talk. Fimbriated P. gingivalis cells can induce formation of integrin-associated focal adhesions, with subsequent remodelling of the actin and tubulin cytoskeleton in primary gingival epithelial cells (166). It has been argued that these complex interactions reect a possible evolutionary relationship between P. gingivalis and host cells, resulting in a balanced association whereby the organism can survive within epithelial cells without causing excessive harm. P. gingivalis-mediated disease may result in part from a disruption of this balance by factors that trigger virulence or lead to host immune systemmediated tissue damage (166).

Biolms and communities in conict

It has been emphasized that the oral microora has a harmonious and positively benecial relationship with the host, and that, once established, the composition of the microora is relatively stable over time (microbial homeostasis). However, homeostasis can break down if there is a substantial change to the habitat that disrupts this normal balance and drives selection of previously minor components of the microora. The bacteria that predominate in the various types of periodontal disease are different to those that are prevalent in the healthy gingival crevice. However, numerous studies using sensitive molecular techniques have detected putative periodontal pathogens at healthy sites but only in low numbers. In general, the putative periodontal pathogens are non-competitive with other members of the resident subgingival microora at healthy sites, and remain at low levels; such levels are not clinically signicant. However, if plaque is allowed to accumulate beyond levels that are compatible with health, the host mounts an inammatory response. The ow of gingival crevicular uid is increased, and this introduces into the crevice not only components of the host defences but also complex host molecules (e.g. transferrin, haemoglobin, etc.) that can be catabolized and used as a nutrient source by the proteolytic gram-negative anaerobes that predominate in advanced periodontal lesions (142144). Organisms such as P. gingivalis have an absolute requirement for haemin for growth, and obtain this cofactor from the catabolism of host glycoproteins. This proteolytic metabolism leads to an increase in local pH and a decrease in the redox potential, which, as discussed above, promotes upregulation of some of the virulence factors associated with these putative pathogens (e.g. gingipain activity by P. gingivalis), and favours their growth at the expense of the species associated with gingival health (i.e. increases the competitiveness of the potential pathogens). If sustained, this leads to a re-arrangement in community structure and a selective increase in the proportions of the anaerobic and proteolytic components of the microora (Fig. 2). These bacteria often include members of the red complex (P. gingivalis, T. denticola and Tannerella forsythia) (132), but other bacteria with similar or relevant traits are also selected. This explains the lack of absolute specicity in the microbial aetiology of periodontal diseases, and

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re-afrms the need to understand the function or role (niche) (3) of microorganisms within plaque biolm communities rather than simply concentrating on determining their bacterial identity. Distinct bacterial species could occupy the same niche (i.e. have the same function) at different sites. This situation is analogous to the increase in mutans streptococci and lactobacilli (and also other acidogenic and acid-tolerating species) (126) as a response to conditions of low pH following repeated ingestion of fermentable dietary carbohydrates (Fig. 2) (11, 14, 82, 138). Evidence that such bacterial population shifts can be driven by environmental change has come from laboratory studies. Growth of subgingival plaque on human serum (used to mimic gingival crevicular uid) led to the selection of species associated with periodontal destruction, such as black-pigmented anaerobes, anaerobic streptococci, Fusobacterium spp. and spirochaetes; most of these species could not be detected in the original samples (142144). Likewise, in the laboratory, an increase in pH from 7.0 to 7.5 (as can occur during inammation) allowed the proportions of P. gingivalis in a microbial community of three species of black-pigmented anaerobe to increase from <1% to >99% (92). The ecological plaque hypothesis has been proposed to describe and explain this dynamic relationship between the resident microora and the host in health and disease in ecological terms (Fig. 12) (82, 85). The theory underpinning this hypothesis in the context of periodontal disease is that changes in the environment increase the competitiveness of the putative pathogens (which, if present in health, are generally only at low and clinically insignicant levels) at the expense of speGingival health
Reduced plaque Reduced inflammation

cies associated with oral health, and upregulate the expression of virulence factors. Importantly, there is acknowledgement of a clear link between local environmental conditions and the activity and composition of the biolm community. Any change to the environment induces a response in the microora, and vice versa. Implicit in this hypothesis is that, although disease can be treated by targeting the putative pathogens directly [e.g. with antimicrobial agents, or via new approaches such as photodynamic therapy (4, 68)], long-term prevention will only be achieved by interfering with the underlying changes in the environment that drive the deleterious shifts in the microora (82), e.g. by reducing the severity of the inammatory response (147) or by altering the redox potential of the pocket to restrict growth of the obligate anaerobes (163). Indeed, in this hypothesis, it is accepted that disease will inevitably recur unless the underlying predisposing factors are addressed. Manipulation of the resident subgingival microora by use of pre- or probiotics (34) or by replacement therapy with benecial bacteria (101, 136) is also under evaluation. Other relevant changes in the local environment that could perturb the hostmicrobe balance could result from trauma, an alteration in the immune status of the host (e.g. during systemic disease or after drug therapy), or tobacco smoking.

Points for discussion

Adoption of a biolm and community lifestyle has important consequences for microorganisms and the habitat in which they reside (Table 1), and there
Gingival health
Low gingival crevicular fluid flow Grampositive bacteria

Stress
Increased plaque Increased inflammation

Environmental change
High gingival crevicular fluid flow, bleeding, raised pH & temperature, low Eh

Ecological shift
Gramnegative anaerobes

Gingivitis

Periodontitis

Fig. 12. A schematic representation of the ecological plaque hypothesis in relation to periodontal disease (adapted from Refs 82, 85 and 90). Plaque biolm accumulation can produce an inammatory host response; this causes changes in the local environmental conditions and introduces novel host proteins and glycoproteins that

favour the growth of proteolytic and anaerobic gramnegative bacteria. In order to prevent or control disease, the underlying factors responsible for driving the selection of the putative pathogens must be addressed, otherwise disease will recur.

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is a direct relationship between the two. This lifestyle also has consequences for those who decide to investigate them! For example, how should we attempt to isolate and grow these bacteria? Traditionally, samples of plaque are vigorously dispersed to break up co-adherent clumps of cells, and individual phylotypes are separated from their neighbours in order to obtain pure cultures, and yet, as discussed at the very start of this review, organisms have evolved with us over millenia to grow in mutualistic combinations. This reductionist approach may be fundamental to our inability to culture more than 50% of the cells that we can observe microscopically. The fact that subgingival biolms are composed of diverse, interacting consortia of microorganisms also has implications for the development of diagnostic methods, and caution must be exercised regarding interpretation of antibiotic sensitivity testing based on planktonic cultures of isolated species. A range of sensitive molecular techniques are now available to detect putative periodontal pathogens, but the selection of discriminatory biomarker species and their diagnostic signicance is still under discussion (78, 96, 118). Rather than relying on the mere presence or absence of a species, a measure of the proportions and combinations (complexes) of subgingival bacteria (132), combined with the lack of, or a reduction in, benecial bacteria may be necessary in order to guide therapeutic decisions (71). Therefore, interpretation of microbial diagnostics in the context of periodontal diseases remains a challenge, particularly if the information is being used to determine antimicrobial therapies. Finally, how should we dene the properties of our isolated microorganisms? Conventionally, their phenotype is characterized and dened in reference texts based on studies performed in pure culture, but it is clear from the evidence provided here that the properties of an organism can be radically enhanced and extended when they are a member of a consortium or a biolm. These properties include their substrate utilization pattern, atmosphere requirement, pathogenic potential and drug sensitivity, etc. Finally, perhaps these oral biolm communities should be regarded more holistically as primitive multicellular organisms. They are spatially and functionally organized, have communication networks, and display a division of (metabolic) labour. Collectively, these features challenge some of our existing concepts and paradigms on how to investigate and interpret data from studies of plaque biolm

communities. Without re-assessment of our approaches to this topic, our ability to make advances in the control and prevention of plaque-mediated diseases will be limited.

Acknowledgments
The authors would like to thank Dr Jimmy Walker (CEPR) for assistance with this review. We further thank Annett Petrich, Stef Siemoneit and Julia Hubner (Berlin) for excellent technical assistance. The carriers for subgingival biolms were kindly provided by Anton Friedmann (Berlin).

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