Dental Plaque Biofilms-P
Transcript of Dental Plaque Biofilms-P
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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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Printed in Singapore. All rights reserved
2011 John Wiley & Sons A/S
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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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