Accumulation of a Dental Plaque Biofilm

The process of plaque formation can be divided into several phases: (1) the formation of the
pellicle on the tooth surface, (2) the initial adhesion/attachment of bacteria, and (3)
colonization/plaque maturation.
Formation of the Pellicle
All surfaces in the oral cavity, including the hard and soft tissues, are coated with a layer of
organic material known as the acquired pellicle. The pellicle on tooth surfaces consists of more
than 180 peptides, proteins, and glycoproteins, including keratins, mucins, proline-rich proteins,
phosphoproteins (e.g., statherin), histidine-rich proteins, and other molecules that can function as
adhesion sites (receptors) for bacteria. The salivary pellicle can be detected on clean enamel
surfaces within 1 minute after their introduction into the mouths of volunteers.143 By 2 hours,
the pellicle is essentially in equilibrium between adsorption and detachment, although further
pellicle maturation can be observed for several hours. Transmission electron microscopy shows
the pellicle to be composed of two layers: a thin basal layer that is very difficult to remove, even
with harsh chemical and mechanical treatments, and a thicker globular layer, up to 1 µm or more,
that is easier to detach. From these observations, it can be concluded that dental enamel is
permanently covered with an acquired pellicle from the moment that teeth erupt. Consequently,
bacteria that adhere to tooth surfaces do not contact the enamel directly but interact with the
acquired enamel pellicle. However, the pellicle is not merely a passive adhesion matrix. Many
proteins retain enzymatic activity when they are incorporated into the pellicle, and some of these,
such as peroxidases, lysozyme, and α-amylase, may affect the physiology and metabolism of
adhering bacterial cells.
In addition, a close ecologic relationship between the pellicle and its associated microbiology
seems to exist. Walker and coworkers reported that dental plaque samples produced in vitro
biofilms only if the surface on which they were grown contained a salivary pellicle belonging to
the patient who donated the plaque sample. No biofilm could be grown on a pellicle that came
from a different subject.
Initial Adhesion/Attachment of Bacteria
Colonization of a surface begins immediately after its introduction in the oral cavity. Colonizing
bacteria can be detected within 3 minutes after the introduction of sterile enamel into the
mouth.139 The initial steps of transport and interaction with the surface are essentially
nonspecific (i.e., they are the same for all bacteria). The proteins and carbohydrates that are
exposed on the bacterial cell surface become important when the bacteria are in loose contact
with the acquired enamel pellicle. The specific interactions between microbial cell surface
“adhesin” molecules and receptors in the salivary pellicle determine whether a bacterial cell will
remain associated with the surface. Only a relatively small proportion of oral bacteria possess
adhesins that interact with receptors in the host pellicle, and these organisms are generally the
most abundant bacteria in biofilms on tooth enamel shortly after cleaning. Over the first 4 to 8
hours, the genus Streptococcus tends to dominate, usually accounting for >20% of bacteria
present. Other bacteria that commonly present at this time include species that cannot survive
without oxygen (obligate aerobes), such as Haemophilus spp. and Neisseria spp., as well as
organisms that can grow in the presence or absence of oxygen (facultative anaerobes), including
Actinomyces spp. and Veillonella spp.1,79 These species are considered the “primary
colonizers” of tooth surfaces. The primary colonizers provide new binding sites for adhesion by
other oral bacteria. The metabolic activity of the primary colonizers modifies the local
microenvironment in ways that can influence the ability of other bacteria to survive in the dental
plaque biofilm. For example, by removing oxygen, the primary colonizers provide conditions of
low oxygen tension that permit the survival and growth of obligate anaerobes. The initial steps in
colonization of teeth by bacteria occur in three phases. Phase 1 is transport to the surface, phase
2 is initial reversible adhesion, and phase 3 is strong attachment.
Colonization and Plaque Maturation
The primary colonizing bacteria (Table 8.3) adhered to the tooth surface provide new receptors
for attachment by other bacteria as part of a process known as coadhesion.185 Together with the
growth of adherent microorganisms, coadhesion leads to the development of microcolonies (Fig.
8.15) and eventually to a mature biofilm. Cell–cell adhesion between genetically distinct oral
bacteria also occurs in the fluid phase (i.e., in saliva). In the laboratory, interactions between
genetically distinct cells in suspension result in clumps or coaggregates that are macroscopically
visible (Fig. 8.16). Different species—or even different strains of a single species— have distinct
sets of coaggregation partners (see eFig. 8.6 online). Fusobacteria coaggregate with all other
human oral bacteria, whereas Veillonella spp., Capnocytophaga spp. (see Fig. 8.2Q), and
Prevotella spp. bind with streptococci and/or actinomyces.184,186,442 Each newly accreted cell
becomes itself a new surface and therefore may act as a coaggregation bridge to the next
potentially accreting cell type that passes by. Many coaggregations among strains of different
genera are mediated by lectin-like adhesins (proteins that recognize carbohydrates) and can be
inhibited by lactose and other galactosides or by amino acids such as L-arginine. The
significance of coaggregation in oral colonization has been documented in studies of biofilm
formation in vitro as well as in animal model studies. Well-characterized interactions of
secondary colonizers (see Table 8.3) with early colonizers include the coaggregation of F.
nucleatum with S. sanguinis, Prevotella loescheii with A. oris, and Capnocytophaga ochracea
with A. oris.170,172,437–439 Streptococci show intrageneric coaggregation, which allows them
to bind to the nascent monolayer of already bound streptococci.155,181,274,368 Secondary
colonizers (see Table 8.3) such as P. intermedia, P. loescheii, Capnocytophaga spp., F.
nucleatum, and P. gingivalis do not initially colonize clean tooth surfaces but rather adhere to
bacteria that are already in the plaque mass.184 The transition from early supragingival dental
plaque to mature plaque growing below the
Phase 1: Transport to the Surface
The first stage involves the initial transport of the bacterium to the tooth surface. Random
contacts may occur, for example, through Brownian motion (average displacement, 40 µm/hour),
through sedimentation of microorganisms, through liquid flow (several orders of magnitude
faster than diffusion), or through active bacterial movement (chemotactic activity). However,
relatively few oral bacteria are motile, and forces such as saliva flow or mechanical contact
between oral soft tissues and teeth are almost certainly more important than swimming for
bringing the primary colonizing bacteria into contact with teeth.
Phase 2: Initial Adhesion
The second stage results in an initial reversible adhesion of the bacterium. This is initiated when
the bacterial cell comes into close proximity to the surface (separation distance, ≈50 nm). Long-
and short-range forces, including van der Waals attractive forces and electrostatic repulsive
forces, operate at this distance. The behavior of bacterial cells can be reasonably described by the
Derjaguin– Landau–Verwey–Overbeek theory of colloid stability.148 According to this theory,
the total interaction energy (also called the total Gibbs energy [GTOT]), is the sum of the
attractive forces (GA) and the electrostatic repulsion (GR). At the physiologic ionic strength of
saliva, the van der Waals forces result in a net attraction of bacterial cells at distances of tens of
nanometers from the surface. Approximately 10 nm from the surface is a secondary net energy
minimum. Electrostatic repulsion prevents bacterial cells from being even closer to the surface.
At distances of approximately 10 nm from the surface, bacterial cells are reversibly bound. It is
thought that stronger binding at this point is the consequence of interactions between bacterial
adhesins and receptors in the salivary pellicle. It has been estimated that 10 to 50 ligand-receptor
interactions are required to attain essentially irreversible binding of a bacterial cell to the
pellicle.44 Although the Derjaguin–Landau–Verwey–Overbeek theory presents a neat picture of
the initial stages of bacterial adhesion, in reality even these early steps in adhesion are extremely
complex. Bacteria are not perfect spheres, and many cells possess structures, such as fimbriae,
that protrude from the cell surface. Lewis acid–base interactions (hydrophobicity) also influence
cell surface interactions.148 In addition, microbial cell surfaces are not uniformly coated with a
negative charge. Some regions of the cell surface may be positively charged, and, for these areas,
electrostatic interactions with a negatively charged surface will tend to be attractive. Finally,
observations of early dental plaque often show bacteria associated with shed epithelial cells, thus
indicating that host cells may act as transporters to bring bacteria into close proximity with the
teeth. Therefore, it is difficult to predict how initial attachment will be affected by modifying the
prevailing environment.
Phase 3: Strong Attachment
After initial adhesion, a firm anchorage between the bacterium and the surface is established. On
a rough surface, bacteria are more irreversible binding may occur more easily and more
frequently. The substratum surface free energy becomes important because the water film
between the interacting surfaces has to be removed before short-range forces can be involved.
The binding between the bacteria and the pellicle is mediated by specific adhesins on the
bacterial cell surface (usually proteins) and complementary receptors (proteins, glycoproteins, or
polysaccharides) in the acquired pellicle. Many proteins in the acquired pellicle can act as
receptors for streptococci, including α-amylase, acid proline-rich proteins, statherin, and salivary
agglutinin glycoprotein gp340.353 The specific adhesins of primary colonizing bacteria have
been the subject of many investigations because these represent potential targets for interference
with the formation of dental plaque. One of the best-characterized interactions is the binding
between the antigen I/II family adhesins of oral streptococci and gp340.166,167,301 Antigen I/II
family adhesins are 160- to 180-kDa proteins expressed on the surfaces of many oral
streptococci, including S. mutans, S. sobrinus (see Fig. 8.2P), S. gordonii, and S. intermedius.
Gp340 is present in fluid-phase saliva, and it is a component of the salivary pellicle. In the fluid
phase, interactions between antigen I/II proteins and gp340 result in the aggregation of bacteria.
Large clumps of bacterial cells do not stick well to surfaces, and they are probably removed by
swallowing.220 By contrast, bacterial binding to immobilized gp340 results in the retention of
cells within the biofilm. Interestingly, the recognition of fluid-phase and immobilized gp340 by
streptococci appears to involve different mechanisms.235 It is likely that gp340 changes its
conformation after adhesion to a surface and thus exposes different receptors for bacterial
binding. Similar conformational changes have been noted for a number of host proteins,
including proline-rich proteins and statherin.93,117 The selective binding of fluid-phase or
surface-bound proteins may modulate the ability of bacterial cells to colonize salivary pellicle-
coated surfaces.
Coaggregation is a direct interaction; it is distinct from agglutination, which occurs when cells
are stuck together by molecules in solution. At least 18 genera from the oral cavity have shown
some form of coaggregation.183 All oral bacteria possess surface molecules that foster some sort
of cell–cell interaction (eFig. 8.6).184 The initial stages of coaggregation or coadhesion are
essentially the same as the first steps involved in bacterial binding to surfaces: bacterial cells
come into contact through passive or active transport and bind weakly through nonspecific
hydrophobic, electrostatic, and van der Waals forces.84,102,179,184 These steps can be
dramatically accelerated in vitro by vigorously mixing dense suspensions of bacterial cells.185
Strong cell–cell binding is then determined by the presence of adhesin proteins or carbohydrates
on one partner and complementary receptor proteins or carbohydrates on the other. Note that
adhesin–receptor interactions are mediated by the fundamental physicochemical forces (i.e.,
hydrophobic, electrostatic, and van der Waals), but they are highly specific.