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Dental plaque biofilms:communities, conflict and

controlP H I L I P D. M ARSH, AN N E T T E M O T E R & DE I R D R E A. DE V I N E

From the very beginning of the discipline of micro-

biology, the dogma has been to isolate bacteria in

pure culture in order to be able to define their indi-

vidual properties. This process also involved the use

of conventional broth (planktonic) culture to prepare

biomass and to determine the phenotype of partic-

ular species. This approach provided a sound foun-

dation 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 biofilm, often as a

member of a polymicrobial community (or consor-

tium) of interacting species. If biofilms were merelyplanktonic-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 scientific and clinical impera-

tive for their study would be low. However, applica-

tion of novel imaging (confocal or epifluorescence

microscopy, fluorescence in situ hybridization,

live dead stains, etc.) and molecular techniques (16S

rRNA gene amplification and sequence comparison,

proteomics, transcriptomics, reporter gene technol-

ogy, etc.) has radically altered our understanding of

the biology of multi-species biofilms (Table 1), andkey 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 microflora and the host. Changes in the host

environment have a direct impact on gene expres-

sion, and thereby influence the metabolic activity,

competitiveness and composition of the microflora,

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 microflora 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 mam-

malian (125, 161). The majority are the microorgan-

isms that make up the resident microflora found on

all environmentally exposed surfaces of the body, and

this

human microbiome

is reported to have a met-abolic capacity equivalent to that of the human liver.

The microflora 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 organ-

isms 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 communitythat provides benefits 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 app-

roaches. 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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Periodontology 2000, Vol. 55, 2011, 1635

Printed in Singapore. All rights reserved

2011 John Wiley & Sons A/S

PERIODONTOLOGY 2000

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approaches and those that were observed directly by

microscopy (27, 110). It is estimated that

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Biofilms develop on mucosal and dental surfaces

within the mouth, but the composition of the oral

microflora varies significantly 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 biofilm and a microbial commu-

nity, and on the ways in which our current knowledge

of biofilms can be exploited in order to improve

plaque control. Due to the breadth of the topic,

readers are also directed to other reviews thatemphasize complementary aspects of dental plaque

as a biofilm (67, 81, 130, 131).

Impact of the habitat on microbialgene 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 significant changes to the hab-

itat 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 biofilms select and enrich for

acidogenic and acid-tolerating species (for example,

mutans streptococci, lactobacilli and other acid-loving streptococci) at the expense of those bacteria

with a preference for growth at neutral pH (17, 82,

138). In periodontal disease, the inflammatory

response to biofilm accumulation results in an in-

crease in the flow of gingival crevicular fluid, some-

times with bleeding, and a local rise in temperature.

The increase in flow of gingival crevicular fluid not

only provides components of the host defences but

also introduces a range of host proteins and glyco-

proteins that can be exploited as substrates by, and

provide essential cofactors for the growth of, many of

the obligate anaerobic and proteolytic species pres-

ent in subgingival biofilms (142, 143). This proteolytic

pattern of metabolism results in a small increase in

pH. Significantly 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 opti-

mum 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 commu-

nity during inflammation. These changes in envi-ronment associated with inflammation 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. gingi-valisdownregulating protease activity (112). Thus, as

the subgingival environment gradually changes, there

is a shift in both the competitiveness and aggres-

siveness of previously minor components of the

microflora. If sustained, this can disrupt the natural

balance of organisms within the biofilm community,

resulting in a shift in the composition of the micro-

flora of a site and increasing the risk of disease

(Fig. 2) (82). As stated previously, an awareness of the

Fig. 1. Fluorescence in situhybridization of a subgingival

biofilm showing the close spatial relationship betweenfacultatively anaerobic Streptococcus spp. (orange) and

obligately anaerobic Fusobacterium spp. (magenta). Sub-

gingival biofilms of periodontitis patients were obtained

using a carrier system as described previously (156).

Bacteria were visualized in 3 lm cross-sections of the

biofilms 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-specific

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/).

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dynamic balance between the environment and the

microflora can help explain how the normally bene-ficial relationship between the oral microflora and

the host can be lost and disease can occur, providing

an opportunity for novel interventions.

Dental plaque a classical multi-species biofilm

Dental plaque has been defined as the microbial

community that develops on the tooth surface,

embedded in a matrix of polymers of bacterial andsalivary origin (90). Dental plaque forms via an or-

dered sequence of events, resulting in a structurally

and functionally organized species-rich microbial

biofilm (66, 67, 83, 130). The distinct stages in plaque

biofilm formation are described below.

Formation of a conditioning film

Molecules are adsorbed to the tooth surface within

seconds immediately after cleaning or following initial

exposure to the oral environment, and remain func-

tional (53). These molecules are derived mainly fromsaliva, but, in the subgingival region, molecules orig-

inate from gingival crevicular fluid. The conditioning

film alters the properties of the surface, and bacteria

interact directly with the constituent molecules.

Reversible adhesion

Reversible adhesion involves weak, long-range,

physico-chemical interactions between the charge on

the microbial cell surface and that produced by the

conditioning film (8, 19). Microorganisms are usuallytransported passively to the surface by the flow of

saliva or gingival crevicular fluid; a few species (e.g.

Wolinella, Selenomonas and Campylobacter spp.)

found subgingivally have flagella and are motile.

Irreversible adhesion

Irreversible adhesion involves interactions between

specific molecules on the microbial cell surface (ad-

hesins) 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 ad-

here via cell-surface adhesins to receptors on already

attached bacteria (65), leading to an increase in micro-

bial diversity within the developing biofilm (microbial

succession) (Fig. 3) (67). Many of the secondary

colonizers have fastidious growth requirements.

Multiplication of the attached cells

Multiplication of the attached cells leads to an in-

crease in biomass and synthesis of exopolymers to

form a biofilm matrix (5, 15). A matrix is a common

feature of all biofilms, and is more than a chemical

scaffold to maintain the shape of the biofilm. It

makes a significant contribution to the structural

Homeostatic mechanisms

Ecological perturbation

Frequent sugar/

low pH challenges

Low saliva flow

Inflammation/

increased gingival crevicular fluid flow

Immune suppression

CariesAcidogenic/aciduric:

- Mutans streptococci- Lactobacilli

- Other acidogenic/aciduric streptococci

Plaquecommunity

stability

Periodontal diseases

Gram-negative anaerobes:- Spirochaetes(e.g. Treponema denticola)

- Porphyromonas gingivalis- Tannerella forsythia- Aggregatibacteractinomycetemcomitans

Health

Fig. 2. Ecological shifts in the dental

plaque microflora in health and

disease (adapted from Ref. 90).

Homeostatic mechanisms involving

microbial interactions help main-

tain a stable beneficial microbial

community that is associated with

oral health. Severe changes to the

habitat (ecological perturbations)

can alter this equilibrium by select-

ing for organisms that are more

competitive in the altered environ-

ment, and this can predispose sites

to disease.

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integrity and general tolerance of biofilms to envi-

ronmental factors (e.g. desiccation) and antimicro-

bial agents. The matrix can be biologically active and

retain water, nutrients and enzymes within the bio-

film. 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 biofilm facilitates numerous synergistic and

antagonistic interactions between neighbouring spe-

cies, and food chains and food webs develop (seebelow) (72, 90). The metabolism of the microorgan-

isms produces gradients within the biofilm; for

example, in nutrients and fermentation products,

and in pH and redox potential (Eh). Bacteria respond

to these fluctuating changes in environmental con-

ditions by altering their patterns of gene expression

(see below) (32, 46). The gradients in plaque are not

necessarily linear, and the environmental heteroge-

neity 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 biofilms such as dental plaque. These

processes lead to the establishment of a mature

biofilm (Fig. 4) with a relatively stable composition.

Detachment from biofilms

Bacteria are able to sense changes to their environ-

ment, for example by two-component signal

Fig. 3. Fluorescence in situ hybridization of subgingival

biofilm showing stratification of species. Small cocci pre-

dominate in the bottom layer of the biofilm, detected by

the bacterial probe (green). Fusobacterium nucleatum

canifelinum (magenta) is predominantly found as a sec-

ondary colonizer, whereas the motile group II treponemes

(yellow, Treponema denticola-related) are found in both

layers. Details of the probes EUB338, FUNU and TREIIcan be obtained at http://www.microbial-ecology.net/

probebase/. At the gingival side of the biofilm, autofluo-

rescent erythrocytes (red, arrowhead) and a few host cell

nuclei stained by non-specific nucleic acid stain DAPI

(blue, arrow) are visible.

A B

Fig. 4. Mosaic architecture of 5-day-old subgingival bio-

films with various oral species detected by fluorescence

in situ hybridization. (A) Clusters of Fusobacterium

nucleatum canifelinum (magenta) and Prevotella inter-

media (yellow). (B) Porphyromonas gingivalis (magenta)

alternates with Tannerella forsythia (yellow). The fluo-

rescence in situhybridization probes FUNU and PRIN (A)

or POGI and TAFO (B) were used in combination with

EUB338 (green) and DAPI (blue). Further information

regarding the fluorescence 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 biofilm has formed, the species compo-

sition at a site is characterized by a degree of stability

or balance among the component species, despite

regular minor environmental stresses following, forexample, periodic oral hygiene, food intake or diurnal

changes in saliva flow. Importantly, this stability

(termed microbial homeostasis) is not due to any

biological indifference among the resident organ-

isms, 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 com-

patible with community stability. A consequence of

homeostasis breakdown is re-organization of the

structure and composition of the microbial commu-

nity, 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 biofilms has improved with technological

developments in microscopy (129). Confocal scan-

ning laser microscopy can visualize specimens in

their natural, hydrated state. When dental plaque wasallowed to develop naturally on enamel slices placed

in removable devices in the mouth and imaged by

confocal microscopy, the architecture of the resultant

biofilms was far more open than previously seen

using conventional electron microscopy (164).

Channels or pores, possibly filled with extracellular

polymers, penetrated the biofilm; the presence of this

matrix could influence the distribution and move-

ment 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 bac-teria being present in the central part of the biofilm

and lining the channels, while a combination of

fluorescence in situ hybridization and confocal or

fluorescence microscopy enables the spatial distri-

bution of species within oral biofilms to be observed

(36, 98, 145, 156), as highlighted in many of the fig-

ures in this review.

In the gingival crevice, plaque biofilms have a thin

densely adherent layer on the root surface, with the

bulk of the biofilm having a looser structure, espe-

cially where it comes into contact with the epithelial

lining of the gingival crevice periodontal pocket.

Thus there is opportunity for hostmicrobe cross-

talk. Many bacterial associations have been observed

subgingivally by electron microscopy in these outer

layers, in which cocci are arranged along the length of

filamentous organisms, e.g. corn cob, test tube

brush or rosette formations. Fluorescence in situ

Fig. 5. Fluorescence in situ hybridization on a cross-

section of a mature subgingival biofilm showing pores or

channels (arrowheads) against the background (green) of

bacteria. These presumed channels are surrounded

mainly by Fusobacterium nucleatum canifelinum (mag-

enta) and Tannerella forsythia (yellow).

Fig. 6. High numbers of group I treponemes (orange) in a

subgingival biofilm, most of which are as yet uncultured.

The carrier section was hybridized with probe TRE I to-

gether with FUNU for detection of Fusobacterium nucle-

atum 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 cur-

rently unculturable bacteria, including spirochaetes

(Fig. 6) and the TM7 phylum, in large numbers in

samples of subgingival biofilms (99, 100, 107).

Plaque as a biofilm and community consequences for the

microorganisms

Dental plaque was the first biofilm to be studied in

terms of both its microbial composition and its sen-

sitivity to antimicrobial agents. In the 17th century,

Antonie van Leeuwenhoek pioneered the approach of

studying biofilms 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 pro-longed rinsing with wine vinegar, even though the

organisms were killed if they were first 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 biofilm and microbial community

has been possible. Some of the implications of this

surface-associated, community-driven lifestyle, and

the opportunities for biofilm control, are described

below.

Biofilm regulation of microbial geneexpression

Bacteria in biofilms display a phenotype that is dis-

tinct 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 biofilm formation

(111, 127).

Oral bacteria also modify their patterns of gene

expression during biofilm formation, although the

effects may be less dramatic than those observed for

environmental bacteria because of the obligate bio-

film lifestyle of the former organisms (18). For tech-

nical reasons, most studies of oral bacteria have been

performed on bacteria that predominate in supra-

gingival plaque (e.g. streptococci), but similar prin-

ciples apply to subgingival organisms. During the

initial stages of biofilm formation (first 2 h following

attachment), 33 proteins were differentially expressed

(25 proteins were upregulated; eight were downreg-

ulated) by Streptococcus mutans, and there was an

increase in the relative synthesis of enzymes involvedin carbohydrate catabolism (158). In contrast, some

glycolytic enzymes involved in acid production were

downregulated in older biofilms (3-day), while pro-

teins 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 biofilm but not plank-

tonic cells. Genes associated with glucan (gtfBC) and

fructan synthesis (ftf) in S. mutans(and hence matrix

formation) were also differentially regulated in bio-

films (75). There was little influence of surface growth

on gene expression in early biofilm formation

(

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Microbial interactions, cellcellcommunication and gene transfer

In biofilms, 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, whysome bacteria appear in plaque biofilms as discrete

clusters of cells (Fig. 8). Purified natural molecules

such as bacteriocins are being evaluated as novel

inhibitors of specific bacteria in plaque biofilms.

Bacteria from plaque can coordinate their gene

expression and communicate with one another in a

cell density-dependent manner via small diffusible

molecules (quorum sensing), using strategies similar

to those described for other biofilms (31, 64, 134). In

S. mutans, quorum sensing is mediated by a com-

petence stimulating peptide (CSP), which increases

the transformation frequency of biofilm-grown

S. mutans 10600-fold (77). Lysed cells in biofilms

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 biofilms,indicating that CSP is directly involved in biofilm

formation. CSP also increases acid tolerance in

recipient cells within the biofilm (76).

Other communication systems may function be-

tween different oral species (64). A survey of gram-

negative periodontal bacteria suggests that these

organisms do not posses the acyl homoserine lac-

tone-dependent signalling circuits that are common

in environmental gram-negative bacteria (48).

Fig. 7. Fluorescence in situ hybridization image of a sec-

tioned subgingival biofilm hybridized with bacterial probe

EUB338 (green) and probes specific for Fusobacterium

nucleatum canifelinum (magenta) or Tannerella forsy-

thia (yellow). The species appear to co-localize in upper

part of the biofilm.

Fig. 8. Fluorescence in situ hybridization of subgingival

biofilm showing separation of species in distinct areas. In

the lower part of the image, long rods detected by a bac-

terial probe (green) and DAPI (blue) mix with Fusobacte-

rium (magenta, probe FUS664) and group I treponemes

(yellow, probe TRE I). The latter do not move into the

upper parts of the biofilm, which are inhabited by cocci

and a dense cluster of small fusobacteria, suggesting

competition between these populations.

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Instead, LuxS genes encoding auto-inducer-2 (AI-2),

have been detected in several genera of oral gram-

positive and gram-negative bacteria, implying that

AI-2 may have a broader species range (35, 117).

Several periodontal bacteria [F. nucleatum, P. inter-

media, P. gingivalis and Aggregatibacter (formerly

Actinobacillus) actinomycetemcomitans] secrete a

signal related to AI-2 (45, 48). In A. actinomycetem-

comitans, LuxS-dependent signalling induced exp-ression of leukotoxin and a transport protein involved

in iron acquisition, whereas, in P. gingivalis, LuxS-

dependent 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. actino-

mycetemcomitans complemented a luxS mutation in

P. gingivalis, and AI-2 secretion byP. gingivaliscould

stimulated biofilm formation by F. nucleatum (45,

124), suggesting a major role for these molecules in

intra- and inter-species communication and coordi-

nation 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 Strepto-

coccus cristatuscan repress P. gingivalisfimbrial gene

expression and has an impact on biofilm formation

and the potential virulence of the anaerobe (165).

The diversity of signalling opportunities within

microbial communities, and the significant role of

these molecules in coordinating gene expression andpromoting biofilm formation, have provided impetus

to investigate the potential of inhibitory analogues to

disrupt these networks, thereby providing mecha-

nisms to control or influence the development of

dental plaque. In addition, CSP has been fused to an

antimicrobial peptide domain to generate a specifi-

cally targeted antimicrobial peptide that is capable of

selectively eliminating S. mutans from multi-species

biofilms, while leaving beneficial members of the

consortium unaffected (41). A similar approach has

been tested in vitro for targeted killing ofP. gingivalis

using an antimicrobial peptide (SMAP-28) linked toIgG specific for P. gingivalissurface components (47).

Cells also communicate and interact with one

another in biofilms via horizontal gene transfer. As

discussed above, signalling molecules such as CSP

markedly increase the ability of recipient cells in

biofilms to take up DNA by transformation (77). The

transfer of conjugative transposons encoding tetra-

cycline resistance between streptococci has been

demonstrated in model biofilms (119). The recovery

from the naso-pharynx of resident (Streptococcus

mitis, Streptococcus oralis) and pathogenic (Strepto-

coccus pneumoniae) streptococci with penicillin

resistance genes showing a common mosaic struc-

ture, confirms 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 andS. gordonii has also been demonstrated in the labo-

ratory (154). The presence of pathogenicity islands

in periodontal pathogens such as P. gingivalisis also

indirect evidence for horizontal gene transfer having

occurred in plaque biofilms at some distant time in

the past, and may explain the evolution of more

virulent strains (25).

Tolerance to antimicrobial agents

Bacteria growing in biofilms 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 concen-

trations of chlorhexidine and amine fluoride required

to kill Streptococcus sobrinus growing as an estab-

lished biofilm were 300 and 75 times greater,

respectively, than the minimum bactericidal con-

centration against planktonic cells (128). Similarly, it

was necessary to administer 1050 times the mini-

mum inhibitory concentration of chlorhexidine to

eliminate Streptococcus sanguinis biofilms (74). The

age of the biofilm is also a significant factor; olderbiofilms of S. sanguinis (95) or A. actinomycetem-

comitans (137) were more tolerant of chlorhexidine

or antibiotics, respectively, than younger biofilms.

Biofilms of several species of oral bacteria have also

been shown to be more tolerant of antibiotics (e.g.

amoxycillin, doxycycline, minocycline and metroni-

dazole) 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

biofilms showed that chlorhexidine only affected the

outer layers of cells in 24 and 48 h plaque biofilms(168), suggesting either quenching of the agent at the

biofilm surface or a lack of penetration. Fluoride

(which can inhibit bacterial enzymes in addition to

its effects on enamel biochemistry) showed an un-

even distribution within natural biofilms that devel-

oped 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

biofilms 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 biofilms generally divide slowly, and

slow-growing cells are always less sensitive to anti-

microbial agents, while a sensitive cell can be

protected by resistant neighbouring organisms that

produce a neutralizing factor (see below).

Benefits of a microbial communitylifestyle

The ability of plaque bacteria to interact with

neighbouring cells in biofilms 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 benefits 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 biofilm col-

onizers consume oxygen, which eventually creates

conditions that are suitable even for obligate anaer-

obes to proliferate. This can involve close physical

contact between oxygen consumers and oxygen-

sensitive bacteria (12, 13). Similar arguments apply to

organisms with a specific pH or nutritional require-

ment (24).

More efficient metabolism

Endogenous substrates are the primary source ofnutrients, 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 efficiently by the

concerted action of consortia of oral bacteria (10, 58).

Mutualistic interactions were detected when combi-

nations of A. actinomycetemcomitans, F. nucleatum

and a Veillonella sp. were grown as biofilms 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 appar-

ently resistant to an antibiotic if neighbouring

commensal bacteria produce a neutralizing or

Oral surface (tooth or mucosal)

Conditioning film

Food webs/concertedmetabolic activity

R

R

R

R

Group protection from

antimicrobials

Co-adhesion;spatial

organization

Gene transfer

Cell density-

dependent

signalling

(e.g. via CSP; AI-2)

Adhesinreceptor

Bacterialhost

cross-talk

Antagonism

(e.g. bacteriocins)

R

R

R

R

Fig. 9. Schematic representation of the types of interac-

tion (inter-bacterial and bacterialhost) that occur in a

microbial community, such as dental plaque, growing as a

biofilm (adapted from Refs 81 and 90). Bacteria adhere via

adhesinreceptor interactions either to the conditioning

film (derived either from saliva or gingival crevicular fluid)

or to already attached cells (co-adhesion). Bacteria inter-

act synergistically to metabolize complex endogenous

molecules (e.g. glycoproteins), and food webs can develop.

Bacteria communicate with each other in a cell density-

dependent manner via diffusible signalling molecules,

and with host cells. Cells are more tolerant of antimicro-

bial agents either because of the physical attributes of the

biofilm, via gene transfer of resistance genes, or through

protection by neighbouring cells that produce neutraliz-

ing enzymes. Cells may also gain advantage by production

of inhibitory molecules.

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drug-degrading enzyme. In the mouth, gingival cre-

vicular fluid can contain sufficient b-lactamase to

inactivate any penicillin delivered to the site (56, 149,

153). Pathogens can also find a safe haven within

biofilms 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 develop-

ment of periodontal diseases, subgingival bacteria

must adhere, gain nutrients from the host and multi-

ply, overcome or evade the host defences, invade cells

and induce tissue damage. Individually, many sub-

gingival bacteria are unable to satisfy all of the

requirements necessary to cause disease, and com-

bine forces to do so, forming a more virulent consor-

tium of interacting bacteria (pathogenic synergism)

(Fig. 11) (148). Within such consortia, individual spe-

cies 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 similardisease, and is consistent with the concept of com-

plexes associated with health and disease (51, 132).

Evidence for pathogenic synergism has come from

abscess models in animals, in which different combi-

nations of oral bacteria displayed increasing patho-

genicity and tissue damage (6, 44, 133). The infective

dose ofP. gingivaliswas reduced by 1,000-fold when

cells were co-inoculated with F. nucleatum into a

subcutaneous chamber in mice (94).

Plaque as a biofilm and community consequences for the host

The complex biofilms that develop on oral surfaces

continually interact with the host, and provide

Dietary

Sugar

Concertedaction

H2S

Sulfatases

Concertedaction

Host

glycoproteinDietary

carbohydrate

Polymer-degrading bacteria

Oligosaccharides Peptides Sulfate

Monosaccharides

Fatty & hydroxy acids

Alcohols

Amino acids

CO2, NH3H2, CO2, CH4

AcetogenesisMethanogenesis

Proteinase

Peptidase

DeaminationDecarboxylation

Glycosidase

Sulfate-reducing

bacteria

Glycolysis

Fig. 10. The concerted and sequen-

tial breakdown of endogenous and

exogenous substrates by communi-

ties of oral bacteria present in dental

plaque biofilms with complemen-

tary enzyme activities.

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 flow of gingival crevicular fluid. 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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benefits or can cause conflict. Examples that relate to

plaque biofilms are described below.

Benefits of the resident microflora

The resident microflora at any site does not have

merely a passive relationship with the host. The rapid

suppression of the resident human oral microflora by

administration of antibiotics can result in overgrowthby previously minor components of the microflora

(e.g. yeasts in the oral cavity), or colonization by

exogenously acquired (and often pathogenic) micro-

organisms (86). Thus, the resident microflora acts

directly as an important component of the host de-

fences by forming a significant barrier against exog-

enous 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 (re-

cently 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 nat-

ural resident microflora, many of these anatomicaland physiological deficiencies are reversed (160).

Likewise, humans on long-term antibiotic treatment

can suffer from nutrient deficiencies due to poor

absorption or metabolism of vitamins. The gut resi-

dent microflora 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 influence

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 hostnutrition is unlikely to be as significant 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 microflora 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 inflammatory tone that contributes to

intestinal homeostasis and health (102). The resident

gut microflora exerts cytoprotective effects through

regulation of adaptive immune responses, and

experiments with germ-free animals have indicated a

role for resident microorganisms in the normaldevelopment of the mucosal immune system (26, 42,

102, 122). Studies with germ-free mice have also

indicated a role for resident oral bacteria in deter-

mining 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 mole-

cule-1, E-selectin and interleukin-8, which in turn

can regulate the establishment of a protective layer of

neutrophils strategically positioned between sub-

gingival plaque bacteria and the junctional epi-

thelium (37).

In general, the microflora of a site lives in harmony

with the host, and both parties benefit 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 inflammatory disorders

(102, 122, 157). Therefore, the aim of oral care pro-

grammes should be to control the levels and activity

of the oral microflora, rather than trying to eliminate

plaque, in order to retain the beneficial functions of

the resident organisms and keeping the oral micro-flora at levels compatible with health.

Microbehost signalling

The findings described above demonstrate that

communication not only occurs between bacterial

cells, but also between bacteria and the host. The

binding of bacteria to specific receptors on host cells

can trigger substantial changes in gene expression in

both bacterial and host cells, e.g. immediately fol-

lowing the attachment of Escherichia coli to uro-epithelial cells (2). It is now clear that the natural

resident microflora 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. Microbe-

associated 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 inflammatory response,

while also being able to mount an efficient defence

against pathogens. Pathogenic and non-pathogenic

bacteria may initiate different intracellular signalling

pathways and innate immune responses in epithelialcells (20, 59, 102). In the gastro-intestinal tract, com-

mensal bacteria such as Bifidobacterium infantisand

Lactobacillus salivariusdo not induce pro-inflamma-

tory responses, unlike an exogenous pathogen such as

Salmonella typhimurium (105). Some gut commen-

sals ensure they are tolerated by causing functional

modulation of immunity through Toll-like receptor

expression and signalling (116), while others are

able to suppress inflammatory responses in epithe-

lial cells by inhibiting activation of nuclear factor jB

(29, 62, 103, 146) or by increasing the secretion of

anti-inflammatory cytokines, such as interleukin-10

(50).

The resident oral microflora may also be engaged

in similar cross-talk in the mouth, and oral micro-

organisms 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 salivariusK12 not

only downregulated epithelial cell inflammatory re-

sponses by inhibiting the nuclear factor jB pathway,but also actively stimulated beneficial pathways,

including type I and II interferon responses, and ex-

erted significant 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 biofilm formation, and there is also

evidence that they are involved in bacteriumhost

cell cross-talk. Fimbriated P. gingivalis cells can in-

duce formation of integrin-associated focal adhe-

sions, with subsequent remodelling of the actin and

tubulin cytoskeleton in primary gingival epithelialcells (166). It has been argued that these complex

interactions reflect a possible evolutionary relation-

ship between P. gingivalisand host cells, resulting in

a balanced association whereby the organism can

survive within epithelial cells without causing exces-

sive 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 system-

mediated tissue damage (166).

Biofilms and communities inconflict

It has been emphasized that the oral microflora has a

harmonious and positively beneficial relationship

with the host, and that, once established, the com-

position of the microflora is relatively stable over

time (microbial homeostasis). However, homeostasiscan 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

microflora.

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 tech-

niques have detected putative periodontal pathogens

at healthy sites but only in low numbers. In general,

the putative periodontal pathogens are non-com-

petitive with other members of the resident subgin-gival microflora at healthy sites, and remain at low

levels; such levels are not clinically significant.

However, if plaque is allowed to accumulate beyond

levels that are compatible with health, the host

mounts an inflammatory response. The flow of gin-

gival crevicular fluid 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 catabo-

lized 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 viru-

lence factors associated with these putative patho-

gens (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 sus-tained, this leads to a re-arrangement in community

structure and a selective increase in the proportions

of the anaerobic and proteolytic components of the

microflora (Fig. 2). These bacteria often include

members of the red complex (P. gingivalis, T. den-

ticola and Tannerella forsythia) (132), but other bac-

teria with similar or relevant traits are also selected.

This explains the lack of absolute specificity in the

microbial aetiology of periodontal diseases, and

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re-affirms the need to understand the function or role

(niche) (3) of microorganisms within plaque biofilm

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-tol-

erating species) (126) as a response to conditions oflow 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

fluid) 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 inflammation) allowed

the proportions of P. gingivalis in a microbial com-

munity of three species of black-pigmented anaerobe

to increase from 99% (92).

The ecological plaque hypothesis has been pro-

posed to describe and explain this dynamic rela-

tionship between the resident microflora 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 com-petitiveness of the putative pathogens (which, if

present in health, are generally only at low and

clinically insignificant levels) at the expense of spe-

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 biofilm community. Any change

to the environment induces a response in the mi-

croflora, and vice versa. Implicit in this hypothesis is

that, although disease can be treated by targeting

the putative pathogens directly [e.g. with antimi-crobial agents, or via new approaches such as pho-

todynamic 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 microflora (82), e.g. by

reducing the severity of the inflammatory 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 predis-

posing factors are addressed. Manipulation of the

resident subgingival microflora by use of pre- or

probiotics (34) or by replacement therapy with

beneficial bacteria (101, 136) is also under evalua-

tion. Other relevant changes in the local environ-

ment that could perturb the hostmicrobe balance

could result from trauma, an alteration in the im-

mune status of the host (e.g. during systemic disease

or after drug therapy), or tobacco smoking.

Points for discussion

Adoption of a biofilm and community lifestyle has

important consequences for microorganisms and

the habitat in which they reside (Table 1), and there

Gingival

health

Gingivitis

Reducedplaque

Reducedinflammation

Increasedplaque

Increasedinflammation

Stress

Gram-positivebacteria

Low gingivalcrevicularfluid flow

High gingivalcrevicular fluidflow, bleeding,

raised pH &temperature,

low Eh

Gram-negative

anaerobes

Environmentalchange

Ecologicalshift

Periodontitis

Gingivalhealth

Fig. 12. A schematic representation of the ecological

plaque hypothesis in relation to periodontal disease

(adapted from Refs 82, 85 and 90). Plaque biofilm accu-

mulation can produce an inflammatory 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 gram-

negative 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 life-

style 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 dis-

persed 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, organ-isms 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 biofilms are composed

of diverse, interacting consortia of microorganisms

also has implications for the development of diag-

nostic 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 bi-

omarker species and their diagnostic significance is

still under discussion (78, 96, 118). Rather than

relying on the mere presence or absence of a spe-

cies, a measure of the proportions and combina-

tions (complexes) of subgingival bacteria (132),

combined with the lack of, or a reduction in,

beneficial bacteria may be necessary in order to

guide therapeutic decisions (71). Therefore, inter-

pretation of microbial diagnostics in the context ofperiodontal diseases remains a challenge, particu-

larly if the information is being used to determine

antimicrobial therapies.

Finally, how should we define the properties of our

isolated microorganisms? Conventionally, their phe-

notype is characterized and defined 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 consor-

tium or a biofilm. These properties include their

substrate utilization pattern, atmosphere require-ment, pathogenic potential and drug sensitivity, etc.

Finally, perhaps these oral biofilm communities

should be regarded more holistically as primitive

multicellular organisms. They are spatially and

functionally organized, have communication net-

works, and display a division of (metabolic) labour.

Collectively, these features challenge some of our

existing concepts and paradigms on how to investi-

gate and interpret data from studies of plaque biofilm

communities. Without re-assessment of our ap-

proaches 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, Steffi Siemoneit and Julia

Hubner (Berlin) for excellent technical assistance.

The carriers for subgingival biofilms were kindly

provided by Anton Friedmann (Berlin).

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