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com

DENTAL PLAQUE..........UNVEILING THE BIOFILM INSIDE

Shantipriya Reddy Professor and Head, Sanjay Kaul Professor, Prasad MGS Reader, Hrishikesh Asutkar
Postgraduate student, Nirjhar Bhowmik Postgraduate student, Jaya Senior Lecturer, Department of Periodontics,
Dr. Syamala Reddy Dental College, Hospital and Research Center, Bengaluru, Karnataka, India.
Correspondence: Nirjhar Bhowmik, Postgraduate student, Department of Periodontics, Dr. Syamala Reddy Dental College, Hospital
and Research Center, Bengaluru, Karnataka, India. E-mail: nirbhowmik@yahoo.co.in

Received Feb 08, 2012; Revised Mar 13, 2012; Accepted Mar 24, 2012

ABSTRACT
Dental plaque is the diverse microbial community, embedded in a matrix of host and bacterial polymers, growing on teeth
as a biofilm. Dental plaque develops naturally, and contributes to the host defences by preventing colonization by
exogenous species. The composition of dental plaque varies at distinct surfaces as a result of the inherent biological
and physical properties at these sites; the balance of the predominant bacterial populations shifts in disease. Bacteria
growing on a surface display a novel phenotype; one consequence of which is an increased resistance to antimicrobial
agents. Such biofilm-associated traits, affect the mode of action and efficacy of antimicrobials. Agents with a broad
spectrum of activity in laboratory studies may display a far narrower inhibitory profile in the mouth. This may result in
a selective inhibition of species implicated in disease, or reduced production of virulence factors, while preserving the
benefits associated with a resident oral microflora.
Key Words: Dental plaque, open architecture, diffusion reaction theory, selective inhibition.

Changing Views of Dental Plaque
Over the past 50 years, the understanding and
characterization of dental plaque have undergone
significant evolution. Loesche 6 proposed both a
nonspecific and a specific plaque hypothesis for periodontal
disease initiation and progression. The nonspecific plaque
hypothesis proposed that the entire microbial community
of plaque that accumulated on tooth surfaces and in the
gingival crevice contributed to the development of
periodontal disease. Plaque bacteria produced virulence
factors and noxious products that initiated inflammation,
challenged the host defence system, and resulted in the
destruction of periodontal tissues. Under this hypothesis,
the quantity of plaque was considered to be the critical
factor in the development of periodontal disease. Thus,
increases in the amount of plaque (quantity), as opposed
to specific pathogenic microorganisms (quality) found in
the plaque, were viewed as being primarily responsible for
inducing disease and disease progression 7,8. Studies on
the microbial aetiology of various forms of periodontitis
support the specific plaque hypothesis, which proposes
that only certain microorganisms within the plaque complex
are pathogenic. Despite the presence of hundreds of
species of microorganisms in periodontal pockets, fewer
than 20 are routinely found in increased proportions at
periodontally diseased sites. These specific virulent
bacterial species activate the hosts immune and
inflammatory responses that then cause bone and soft
tissue destruction 6,8,9. Socransky and colleagues 4,10
recognized that early plaque consists predominantly of
gram-positive organisms and that if the plaque is left
undisturbed it undergoes a process of maturation resulting
in a more complex and predominantly gram-negative flora.
These investigators assigned the organisms of the
subgingival microbiota into groups, or complexes, based
on their association with health and various disease
severities4,10. Colour designations were used to denote the
association of particular bacterial complexes with
periodontal infections. The blue, yellow, green, and purple
complexes designate early colonizers of the subgingival
flora. Orange and red complexes reflect late colonizers
associated with mature subgingival plaque. Certain bacterial
complexes are associated with health or disease10,11. For
example, the bacteria in the red complex are more likely to
be associated with clinical indicators of periodontal disease
such as periodontal pocketing and clinical attachment loss.
Plaque Recognized as a Biofilm (Table: 1)
Research over the past decade has led to the recognition
of dental plaque as a biofilm - a highly organized
accumulation of microbial communities attached to an
environmental surface. Biofilms are organized to maximize

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energy, spatial arrangements, communication, and
continuity of the community of microorganisms. Biofilms
protect bacteria living within their structures and thereby
provide an advantage over free-floating (planktonic)
bacteria. The slimy extracellular matrix produced by biofilm
bacteria encloses the microbial community and protects it
from the surrounding environment, including attacks from
chemotherapeutic agents. Chemotherapeutic agents have
difficulty penetrating the polysaccharide matrix to reach
and affect the microorganisms1, 11-13. Thus, the matrix helps
to protect bacteria deep within the biofilm from antibiotics
and antiseptics, increasing the likelihood of the colonies
survival. Furthermore, the extracellular matrix keeps the
bacteria banded together, so they are not flushed away by
the action of saliva and gingival crevicular fluid. Mechanical
methods, including toothbrushing, interdental cleaning, and
professional scaling procedures, are required to regularly
and effectively disrupt and remove the plaque biofilm.
Antiseptics, such as mouthrinses, can help to control the
biofilm but must be formulated so as to be able to penetrate
the plaque matrix and gain access to the pathogenic bacteria.
Biofilms have a definite architectural structure. The bacteria
are not uniformly distributed throughout the biofilm; rather,
there are aggregates of microcolonies that vary in shape
and size. Channels between the colonies allow for
circulation of nutrients and by-products and provide a
system to eliminate wastes14, 15. Microorganisms on the
outer surface of biofilms are not as strongly attached within
the matrix and tend to grow faster than those bacteria deeper
within the biofilm. Surface microorganisms are more
susceptible to detachment, a characteristic that facilitates
travel to form new biofilm colonies on nearby oral structures
and tissues. Bacteria in biofilm communicate with each other
by a process called quorum sensing. This dynamic,
sophisticated communication system enables bacteria to
monitor each others presence and to modulate their gene
expression in response to the number of bacteria in a given
area of the biofilm8. In addition, as a result of quorum
sensing, portions of the biofilm can become detached in
order to maintain a cell density compatible with continued
survival.
Stages of Biofilm Formation
The growth and development of biofilm are characterized
by 4 stages: initial adherence, lag phase, rapid growth, and
steady state. Biofilm formation begins with the adherence
of bacteria to a tooth surface, followed by a lag phase in
which changes in genetic expression (phenotypic shifts)
occur. A period of rapid growth then occurs, and an
exopolysaccharide matrix is produced. During the steady
state, the biofilm reaches growth equilibrium. Surface
detachment and sloughing occur, and new bacteria are
acquired.
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Initial Adherence and Lag Phase
The first phase of supragingival biofilm formation is the
deposition of salivary components, known as acquired
pellicle, on tooth surfaces. This pellicle makes the surface
receptive to colonization by specific bacteria. Salivary
glands produce a variety of proteins and peptides that
further contribute to biofilm formation. For example, salivary
mucins, such as MUC5B and MUC7, contribute to the
formation of acquired pellicle16, 17, and statherin, a salivary
acidic phosphoprotein, and proline-rich proteins promote
bacterial adhesion to tooth surfaces18. Acquired pellicle
formation begins within minutes of a professional
prophylaxis; within 1 hour, microorganisms attach to the
pellicle. Usually, gram-positive cocci are the first
microorganisms to colonize the teeth. As bacteria shift from
planktonic to sessile life, a phenotypic change in the bacteria
occurs requiring significant genetic up-regulation (gene
signaling that promotes this shift). As genetic expression
shifts, there is a lag in bacterial growth.
Rapid Growth
During the rapid growth stage, adherent bacteria secrete
large amounts of water-insoluble extracellular
polysaccharides to form the biofilm matrix. The growth of
microcolonies within the matrix occurs. With time, additional
varieties of bacteria adhere to the early colonizers - a
process known as coaggregation and the bacterial
complexity of the biofilm increases. These processes involve
unique, selective molecular interactions leading to structural
stratification within the biofilm. Coaggregation and
subsequent cell division also increase the thickness of
biofilm19-21.
Steady State/Detachment
During the steady state phase, bacteria in the interior of
biofilms slow their growth or become static. Bacteria deep
within the biofilm show signs of death with disrupted
bacterial cells and other cells devoid of cytoplasm; bacteria
near the surface remain intact. During this phase, crystals
can be observed in the interbacterial matrix that may
represent initial calculus mineralization 22. As noted above,
during the steady state stage, surface detachment and
sloughing also occur, with some bacteria travelling to form
new biofilm colonies.
Biofilm Structure
Extracellular Polymeric Substances
Biofilms are composed primarily of microbial cells and EPS.
EPS may account for 50% to 90% of the total organic carbon
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of biofilms23 and can be considered the primary matrix
material of the biofilm. EPS may vary in chemical and
physical properties, but it is primarily composed of
polysaccharides. Some of these polysaccharides are neutral
or polyanionic, as is the case for the EPS of gram-negative
bacteria. The presence of uronic acids (such as D-
glucuronic, D-galacturonic, and mannuronic acids) or ketal-
linked pryruvates confers the anionic property24. This
property is important because it allows association of
divalent cations such as calcium and magnesium, which
have been shown to cross-link with the polymer strands
and provide greater binding force in a developed biofilm23.
In the case of some gram-positive bacteria, such as the
staphylococci, the chemical composition of EPS may be
quite different and may be primarily cationic. Hussain et
al25, found that the slime of coagulase-negative bacteria
consists of a teichoic acid mixed with small quantities of
proteins. EPS is also highly hydrated because it can
incorporate large amounts of water into its structure by
hydrogen bonding. EPS may be hydrophobic, although
most types of EPS are both hydrophilic and hydrophobic24.
EPS may also vary in its solubility. Sutherland24 noted two
important properties of EPS that may have a marked effect
on the biofilm. First, the composition and structure of the
polysaccharides determine their primary conformation. For
example, many bacterial EPS possess backbone structures
that contain 1,3- or 1,4--linked hexose residues and tend
to be more rigid, less deformable, and in certain cases poorly
soluble or insoluble. Other EPS molecules may be readily
soluble in water. Second, the EPS of biofilms is not generally
uniform but may vary spatially and temporally. Leriche26 et
al. used the binding specificity of lectins to simple sugars
to evaluate bacterial biofilm development by different
organisms. These researchers results showed that different
organisms produce differing amounts of EPS and that the
amount of EPS increases with age of the biofilm.EPS may
associate with metal ions, divalent cations, other
macromolecules (such as proteins, DNA, lipids, and even
humic substances) 23. EPS production is known to be
affected by nutrient status of the growth medium; excess
available carbon and limitation of nitrogen, potassium, or
phosphate promote EPS synthesis24. Slow bacterial growth
will also enhance EPS production24 because EPS is highly
hydrated, it prevents desiccation in some natural biofilms.
EPS may also contribute to the antimicrobial resistance
properties of biofilms by impeding the mass transport of
antibiotics through the biofilm, probably by binding directly
to these agents27.
Biofilm Architecture
Tolker-Nielsen and Molin noted that every microbial biofilm
community is unique28 although some structural attributes
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can generally be considered universal. The term biofilm is
in some ways a misnomer, since biofilms are not a
continuous monolayer surface deposit. Rather, biofilms are
very heterogeneous, containing microcolonies of bacterial
cells encased in an EPS matrix and separated from other
microcolonies by interstitial voids (water channels). Liquid
flow occurs in these water channels, allowing diffusion of
nutrients, oxygen, and even antimicrobial agents. This
concept of heterogeneity is descriptive not only for mixed
culture biofilms (such as might be found in environmental
biofilms) but also for pure culture biofilms common on
medical devices and those associated with infectious
diseases. Stoodleyet al. defined certain criteria or
characteristics that could be considered descriptive of
biofilms in general, including a thin base film, ranging from
a patchy monolayer of cells to a film several layers thick
containing water channels29. The organisms composing the
biofilm may also have a marked effect on the biofilm
structure. For example, James et al showed that biofilm
thickness could be affected by the number of component
organisms. Pure cultures of either K. pneumoniae or P.
aeruginosa biofilms in a laboratory reactor were thinner (15
and 30 respectively), whereas a biofilm containing both
species was thicker (40 ). Jones et al. noted that this could
be because one species enhanced the stability of the other.
Biofilm architecture is heterogeneous both in space and
time, constantly changing because of external and internal
processes. Tolker-Nielsen et al.32 investigated the role of
cell motility in biofilm architecture in flow cells by examining
the interactions of P. aeruginosa and P. putida by confocal
laser scanning microscopy. When these two organisms were
added to the flow cell system, each organism initially formed
small microcolonies. With time, the colonies intermixed,
showing the migration of cells from one microcolony to the
other. The microcolony structure changed from a compact
structure to a looser structure over time, and when this
occurred the cells inside the microcolonies were observed
to be motile. Motile cells ultimately dispersed from the
biofilm, resulting in dissolution of the microcolony.
Biofilm and Oral Disease
Biofilms can cover surfaces throughout the oral cavity.
Microcolonies exist on oral mucosa, the tongue,
biomaterials used for restorations and dental appliances,
and tooth surfaces above and below the gingival margin. It
is important for oral health professionals to communicate
to their patients that both dental caries and periodontal
disease are infectious diseases resulting from dental plaque
biofilm accumulation. Each of these diseases requires
specific strategies for prevention and treatment. With
respect to periodontal disease, dental plaque biofilm
demonstrates a succession of microbial colonization with
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Nirjhar Bhowmik et al
changes in bacterial flora observed from health to disease.
Bacterial species contained in the yellow, green, and purple
complexes appear to colonize the subgingival sulcus first
and predominate in gingival health. In contrast, orange
complex bacteria are associated with gingivitis and gingival
bleeding. Interestingly, bacteria of the orange complex may
also be associated with red complex microorganisms
including Porphyromonas gingivalis, Tannerella
forsythensis, and Treponema denticola, organisms found
in greater numbers in diseased sites and in more advanced
periodontal disease. As the biofilm matures and proliferates,
soluble compounds produced by pathogenic bacteria
penetrate the sulcular epithelium. These compounds
stimulate host cells to produce chemical mediators
associated with the inflammatory process; Interleukin-1 beta
(IL-1 ), prostaglandins, tumor necrosis factor alpha (TNF-
), and matrix metalloproteinases are mediators that recruit
neutrophils to the area via chemotaxis and cause increased
permeability of gingival blood vessels, permitting plasma
proteins to migrate from within the blood vessels into the
tissue. As the gingival inflammatory process continues,
additional mediators are produced, and more inflammatory
cell types such as neutrophils, T cells, and monocytes are
recruited to the area. Proinflammatory cytokines are
produced in the tissues as a response to the chronic
inflammatory process, and these proteins may further
escalate the local inflammatory response and affect the
initiation and progression of systemic inflammation and
disease. The result of this chronic inflammation is a
breakdown of gingival collagen and accumulation of an
inflammatory infiltrate, leading to the clinical signs of
gingivitis. In some individuals, the inflammatory process
will also lead to the breakdown of collagen in the periodontal
ligament and resorption of the supporting alveolar bone. It
is at this point that the lesion progresses from gingivitis to
periodontitis, continuing the same challenge from
proinflammatory mediators as with chronic gingivitis. Thus,
controlling dental plaque biofilm is essential to preventing
and reversing gingivitis as well as preventing and managing
periodontitis.
The Established Community: Biofilm Ecology
The basic structural unit of the biofilm is the microcolony.
Proximity of cells within the microcolony (or between
microcolonies) provides an ideal environment for creation
of nutrient gradients, exchange of genes, and quorum
sensing. Since microcolonies may be composed of multiple
species, the cycling of various nutrients (e.g., nitrogen,
sulfur, and carbon) through redox reactions can readily
occur in aquatic and soil biofilms. The primary colonizers
which are gram positive organisms are facultative
anaerobes in nature and they make the environment
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conducive for the late colonizers. It has been proposed
that as a microbial biofilm develops the community will
ultimately form a more stable climax community. As the
community is able to adapt appropriately to outside
perturbations the term microbial homeostasis has been
suggested to reflect stability within a climax community.
Gene Transfer
Biofilms also provide an ideal niche for the exchange of
extra-chromosomal DNA (plasmids). Conjugation (the
mechanism of plasmid transfer) occurs at a greater rate
between cells in biofilms than between planktonic cells35-
37
. Ghigo38 has suggested that medically relevant strains of
bacteria that contain conjugative plasmids more readily
develop biofilms. He showed that the F conjugative pilus
(encoded by the tra operon of the F plasmid) acts as an
adhesion factor for both cell-surface and cell-cell
interactions, resulting in a three dimensional biofilm of
Escherichia coli. Plasmid-carrying strains have also been
shown to transfer plasmids to recipient organisms, resulting
in biofilm formation; without plasmids these same organisms
produce only microcolonies without any further
development. The probable reason for enhanced
conjugation is that the biofilm environment provides
minimal shear and closer cell-to-cell contact. Since plasmids
may encode for resistance to multiple antimicrobial agents,
biofilm association also provides a mechanism for selecting
for, and promoting the spread of, bacterial resistance to
antimicrobial agents.
Quorum Sensing
Cell-to-cell signalling has recently been demonstrated to
play a role in cell attachment and detachment from biofilms.
Xie et al.39showed that certain dental plaque bacteria can
modulate expression of the genes encoding fimbrial
expression (fimA) in Porphyromonas gingivalis. P. gingivalis
would not attach to Streptococcus cristatis biofilms grown
on glass slides. P. gingivalis, on the other hand, readily
attached to S. gordonii. S. cristatus cell-free extract
substantially affected expression of fimA in P. gingivalis,
as determined by using a reporter system. S. cristatus is
able to modulate P. Gingivalis fimA expression and prevent
its attachment to the biofilm. Davies et al.40 showed that
two different cell-to-cell signalling systems in P. aeruginosa,
lasR-lasI and rhlR-rhlI, were involved in biofilm formation.
At sufficient population densities, these signals reach
concentrations required for activation of genes involved
in biofilm differentiation. Mutants unable to produce both
signals (double mutant) were able to produce a biofilm, but
unlike the wild type, their biofilms were much thinner, cells
were more densely packed, and the typical biofilm
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Dental Plaque..........unveiling the biofilm inside
architecture was lacking. In addition, these mutant biofilms
were much more easily removed from surfaces by a
surfactant treatment. Addition of homoserine lactone to
the medium containing the mutant biofilms resulted in
biofilms similar to the wild type with respect to structure
and thickness.
Stickler et al.41 also detected acylated homoserine lactone
signals homoserine lactone signals in biofilms of gram-
negative bacteria on urethral catheters. Yung-Hua et al.42
showed that induction of genetic competence (enabling
the uptake and incorporation of exogenous DNA by
transformation) is also mediated by quorum sensing in S.
mutans. Transformational frequencies were 10600 times
higher in biofilms than planktonic cells.
Predation and Competition
Bacteria within biofilms may be subject to predation by
free-living protozoa, Bdellovibrio spp., bacteriophage, and
polymorphonuclear leukocytes (PMNs) as a result of
localized cell concentration. Murga et al.43 demonstrated
the colonization and subsequent predation of heterotrophic
biofilms by Hartmannella vermiformis, a free-living
protozoon. Predation has also been demonstrated with
Acanthamoeba spp. in contact lens storage case biofilms44.
James et al.31 noted that competition also occurs within
biofilms and demonstrated that invasion of a
Hyphomicrobium sp. biofilm by P. putida resulted in
dominance by the P. putida, even though the biofilm-
associated Hyphomicrobium numbers remained relatively
constant. Stewart et al.45 investigated biofilms containing
K. pneumoniae and P. aeruginosa and found that both
species are able to coexist in a stable community even
though P. aeruginosa growth rates are much slower in the
mixed culture biofilm than when grown as a pure culture
biofilm. P. aeruginosa grow primarily as a base biofilm,
whereas K. pneumoniae form localized microcolonies
(covering only about 10% of the area) that may have greater
access to nutrients and oxygen. Apparently P. aeruginosa
can compete because it colonizes the surface rapidly and
establishes a long-term competitive advantage. K.
pneumoniae apparently survives because of its ability to
attach to the P. Aeruginosa biofilm, grow more rapidly, and
out-compete the P. Aeruginosa in the surface layers of the
biofilm.
Dispersal
Biofilm cells may be dispersed either by shedding of
daughter cells from actively growing cells, detachment as a
result of nutrient levels or quorum sensing, or shearing of
biofilm aggregates (continuous removal of small portions
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of the biofilm) because of flow effects. The mechanisms
underlying the process of shedding by actively growing
cells in a biofilm are not well understood. Gilbert et al.53
showed that surface hydrophobicity characteristics of
newly divided daughter cells spontaneously dispersed from
either E. coli or P. aeruginosa biofilms differ substantially
from those of either chemostat-intact biofilms or
resuspended biofilm cells. These researchers suggested
that these differences might explain newly divided daughter
cells detachment. Hydrophobicity was lowest for the newly
dispersed cells and steadily increases upon continued
incubation and growth. Alginate is the major component of
the EPS of P. aeruginosa. Inducing alginate lyase expression
substantially decreased the amount of alginate produced,
which corresponded with a significant increase in the
number of detached cells. The authors54 suggested that
the role of algL (the gene cassette for alginate lyase
production) in wild type P. aeruginosa may be to cause a
release of cells from solid surfaces or biofilms, aiding in the
dispersal of these organisms. Polysaccharidase enzymes
specific for the EPS of different organisms may possibly be
produced during different phases of biofilm growth of these
organisms. Detachment caused by physical forces has been
studied in greater detail. Brading et al.55 have emphasized
the importance of physical forces in detachment, stating
that the three main processes for detachment are erosion
or shearing (continuous removal of small portions of the
biofilm), sloughing (rapid and massive removal), and
abrasion (detachment due to collision of particles from the
bulk fluid with the biofilm). Characklis 56 noted that the rate
of erosion from the biofilm increases with increase in biofilm
thickness and fluid shear at the biofilm-bulk liquid interface.
Sloughing is more random than erosion and is thought to
result from nutrient or oxygen depletion within the biofilm
structure 55. Sloughing is more commonly observed with
thicker biofilms that have developed in nutrient-rich
environments 56.
Possible Strategies to Control Oral Biofilm.
Disruption of biofilm matrix
The mechanical disruption of the biofilm matrix has been
one of the oldest and most successful means to control
oral biofilms, the main advantage of the technique is it is
simple to perform, gives consistent results, helps to reduce
the bacterial load, no development of resistance so may be
performed regularly as home care procedure or as
professional oral prophylaxis.
Control of nutrients
Addition of base-generating nutrients (arginine)
Reduction of GCF flow through anti-inflammatory agents
Inhibition of key microbial enzymes
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Nirjhar Bhowmik et al
Control of biofilm pH
Sugar substitutes
Antimicrobial agents
Fluoride
Stimulate base production
Control of redox potential
Redox agents
Oxygenating agents
Conclusion
The key to a more complete understanding of the role of
microorganisms in dental diseases such as periodontal
diseases and caries may depend on a paradigm shift away
from concepts that have evolved from studies of classical
medical infections with a simple and specific (e.g. single
species) aetiology to an appreciation of ecological
principles. The development of plaque-mediated disease
at a site may be viewed as a breakdown of the homeostatic
mechanisms that normally maintain a beneficial relationship
between the resident oral microflora and the host. When
assessing treatment options, an appreciation of the ecology
of the oral cavity will enable the enlightened clinician to
take a more holistic approach and consider the nutrition,
physiology, host defences, and general well-being of the
patient, as these will affect the balance and activity of the
resident oral microflora. Future episodes of disease will
occur unless the cause of any breakdown in homeostasis
is recognized and remedied.
Table 1: Basic Biofilm Properties.
Cooperating community of various types of
microorganisms
Microorganisms are arranged in microcolonies
Microcolonies are surrounded by protective matrix
Within the microcolonies are differing environments
Microorganisms have primitive communication system
Microorganisms in biofilm are resistant to antibiotics,
antimicrobials, and host response.
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