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9. BIOFILM AND PERIODONTAL MICROBIOLOGY

For a comprehensive reading on this topic, please refer to CHAPTER 8 – BIOFILM AND
PERIODONTAL MICROBIOLOGY in Carranza’s Clinical Periodontology, 13th ed.,
2018.

1. The Oral Cavity from a Microbe’s Perspective

From an ecologic viewpoint, the oral cavity, which communicates with the pharynx,
should be considered as an “open growth system” with an uninterrupted ingestion and removal of
microorganisms and their nutrients. Most organisms can survive in the oropharynx only when
they adhere to either the soft tissues or the hard surfaces. Otherwise, they may be removed by:

•Swallowing, mastication, or blowing the nose

•Tongue and oral hygiene procedures
•The wash-out effect of the salivary, nasal, and crevicular fluid outflow
•The active motion of the cilia of the nasal and sinus walls

On the basis of physical and morphologic criteria, the oral cavity can be divided into six
major ecosystems (also called niches), each with the following distinct ecologic determinants:
1).The intraoral and supragingival hard surfaces (teeth, implants, restorations, and prostheses);
2).Subgingival regions adjacent to a hard surface, including the periodontal/peri-implant pocket;
3).The buccal palatal epithelium and the epithelium of the floor of the mouth; 4).The dorsum of
the tongue; 5).The tonsils; 6).The saliva.
The soft tissue surfaces are actively involved in the process of bacterial adhesion and
colonization (153). They use a variety of mechanisms to prevent the adhesion of pathogenic
organisms, with shedding being one of the most important.
Bacteria also adhere to hard tissues. From a microbiologic viewpoint, teeth and implants
are unique for two reasons: (1) they provide a hard, non-shedding surface that allows for the
development of extensive structured bacterial deposits; and (2) they form a unique ectodermal
interruption. A special seal of epithelium (junctional epithelium) and connective tissue is present
between the external environment and the internal parts of the body. The accumulation and
metabolism of bacteria on these hard surfaces are considered the primary causes of caries,
gingivitis, periodontitis, and peri-implantitis.
Oral bacteria and especially pathogenic bacteria, such as Porphyromonas gingivalis (Fig.
8.2H and J) and Aggregatibacter actinomycetemcomitans (Fig. 8.2K), have a large battery of
virulence factors, one of which is the ability to adhere to hard intraoral surfaces and/or to the
oral mucosae (Fig. 8.3) (56-58, 110-377).
In the periodontal pocket, different strategies contribute to bacterial survival, such as
adhesion to the pocket epithelium and, when dentin is encountered, the colonization of the dentinal
tubules (305).

2.Bacteria and Their Biofilm Mode of Living

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Biofilms are composed of microbial cells encased within a matrix of extracellular

polymeric substances, such as polysaccharides, proteins, and nucleic acids. Oral bacteria, like
other bacteria that grow in multispecies biofilms, interact closely with neighboring cells.
Sometimes these interactions are mutually beneficial, as is the case when one organism removes
another’s waste products and uses them as an energy source. In other instances, bacteria compete
with their neighbors by secreting antibacterial molecules such as inhibitory peptides
(bacteriocins) or hydrogen peroxide (H2O2). In addition, the biofilm mode of growth facilitates
cell-cell signaling and deoxyribonucleic acid (DNA) exchange between bacteria.
Dental plaque (biofilm) and other biofilms contain microcolonies of bacterial cells. Water
channels are commonly found in biofilms, and these can form a primitive circulatory system that
removes waste products and brings fresh nutrients to the deeper layers of the film (Fig. 8.9).
There is a clear evidence of open fluid-filled channels running through the plaque mass (64, 65-
449) (Fig. 8.6). Nutrients make contact with the sessile (attached) microcolonies by diffusion
from the water channels to the microcolony, rather than from the matrix. The bacteria exist and
proliferate within the intercellular matrix through which the channels run. The matrix confers a
specialized environment that distinguishes the bacteria that exist within the biofilm (the so-called
attached state) from those that are free floating (the so-called planktonic state in solutions such
as saliva or crevicular fluid). The biofilm matrix functions as a barrier. Substances produced by
bacteria within the biofilm are retained and concentrated, which fosters metabolic interactions
among the different bacteria.
The intercellular matrix consists of organic and inorganic materials derived from saliva,
gingival crevicular fluid, and bacterial products. Organic constituents of the matrix include
polysaccharides, proteins, glycoproteins, lipid material, and DNA (226). Glycoproteins from the
saliva are important components of the pellicle that initially coats a clean tooth surface, but they
also become incorporated into the developing plaque biofilm. Polysaccharides produced by
bacteria also contribute to the organic portion of the matrix. They play a major role in
maintaining the integrity of the biofilm. The inorganic components of plaque are predominantly
calcium and phosphorus, with trace amounts of other minerals such as sodium, potassium, and
fluoride. The source of inorganic constituents of supragingival plaque is primarily saliva.

3.Structure of a Mature Dental Plaque Biofilm

Dental plaque (Fig. 8.9) is defined clinically as a structured, resilient, yellow-grayish

substance that adheres tenaciously to the intraoral hard surfaces, including removable and fixed
restorations (30). The tough extracellular matrix makes it impossible to remove plaque by rinsing
or with the use of sprays. Plaque can thus be differentiated from other deposits that may be found
on the tooth surface, such as materia alba and calculus. Materia alba refers to soft accumulations
of bacteria, food matter, and tissue cells that lack the organized structure of dental plaque and
that are easily displaced with a water spray. Calculus is a hard deposit that forms via the
mineralization of dental plaque and that is generally covered by a layer of unmineralized plaque
(Table 8.2).
Dental plaque is composed primarily of microorganisms. One gram of plaque (wet
weight) contains approximately 1011 bacteria (358, 380). In a periodontal pocket, counts can
range from 103 bacteria in a healthy crevice to more than 10 8 bacteria in a deep pocket. It has
been estimated that more than 750 distinct microbial phylotypes can be present as natural inhabitants
of dental plaque (1).
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Any individual may harbor hundreds of different species. Next to bacteria, nonbacterial
organisms can also be found in the dental plaque biofilm, including archaea, yeasts, protozoa,
and viruses (62, 217).
Dental plaque is broadly classified as supragingival or subgingival on the basis of its
position on the tooth surface toward the gingival margin.
•Supragingival plaque is found at or above the gingival margin; when in direct contact with the
gingival margin, it is referred to as marginal plaque.
•Subgingival plaque is found below the gingival margin, between the tooth and the gingival
pocket epithelium.

Supragingival plaque typically demonstrates the stratified organization of a multilayered

accumulation of bacterial morphotypes (Fig. 8.12) (462). Gram-positive cocci and short rods
predominate at the tooth surface, whereas Gram-negative rods, filaments, and spirochetes
predominate in the outer surface of the mature plaque mass.
In general, the subgingival microbiota differs in composition from the supragingival
plaque, primarily because the environmental parameters of the subgingival region differ from
those of the supragingival region. The gingival crevice or pocket is bathed by the flow of
crevicular fluid, which contains many substances that bacteria may use as nutrients. Host
inflammatory cells and mediators are likely to have considerable influence on the establishment
and growth of bacteria in the subgingival region.
There are distinctions between the tooth-associated regions and the soft tissue-associated
regions of subgingival plaque (Fig. 8.7A to C) (222-268). The tooth-associated cervical plaque
that adheres to the root cementum does not markedly differ from that observed in gingivitis. At
this location, filamentous microorganisms dominate, but cocci and rods also occur. This plaque
is dominated by Gram-positive rods and cocci, including S. mitis, S. sanguinis, Actinomyces oris,
Actinomyces naeslundii, and Eubacterium spp. (Fig. 8.2N). The apical border of the plaque mass
is separated from the junctional epithelium by a layer of host leukocytes, and the bacterial
population of this apical-tooth-associated region shows an increased concentration of Gram-
negative rods (Fig. 8.7D). Bacteria are also found within the host tissues, such as in the soft
tissues (Fig. 8.4), and within epithelial cells (Fig. 8.13), as well as in the dentinal tubules (Fig.
8.14) (344, 345).
The composition of the subgingival plaque depends on the pocket depth. The apical part
is more dominated by spirochetes, cocci, and rods, whereas in the coronal part more filaments are
observed.

4.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
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on tooth surfaces consists of more than 180 peptides, proteins, and glycoproteins, including
keratins, mucins, proline-rich proteins, phosphoproteins, histidine-rich proteins, and other
molecules that can function as adhesion sites (receptors) for bacteria (366, 367-453). 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.
Initial Adhesion/Attachment of Bacteria. Tooth-brushing removes most but not all
bacteria from the exposed surfaces of teeth. 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 (82, 273, 427).
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.
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. Fusobacteria coaggregate with all other human oral bacteria, whereas
Veillonella spp., Capnocytophaga spp. (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.
Secondary colonizers (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 gingival margin involves a shift
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in the microbial population from primarily Gram-positive organisms to high numbers of Gram-
negative bacteria. Therefore, during the later stages of plaque formation, coaggregation among
different Gram-negative species is likely to predominate. Examples are the coaggregation of
F.nucleatum with P.gingivalis or Treponema denticola (Fig. 8.2L) (173, 180, 186).
The changes within the subgingival microbiota during the first week after mechanical
debridement showed an only partial reduction of around 3 logs (from 10 8 bacterial cells to 105
cells) followed by rapid regrowth toward nearly pretreatment levels (−0.5 log) within 7 days
(118, 145, 242). The fast recolonization was explained by followings: the remaining bacteria
after mechanical debridement, the pathogens penetrating the soft tissues or the dentinal tubules,
and subgingival irregularities.

Bacterial “complexes” in subgingival plaque

Bacterial adhesive preferences as well as other interaction affinities could explain the
presence of color-coded “complexes” in the subgingival plaque. The color-coded complexes of
periodontal microorganisms tend to be found together in health or disease. The composition of
the different complexes was based on the frequency with which different clusters of
microorganisms were recovered, and the complexes were color coded for easy conceptualization
(Fig. 8.8) (384).
Interestingly, the early colonizers are either independent of defined complexes (A.
naeslundii, A. oris) or members of the yellow (Streptococcus spp.) or purple complexes (A.
odontolyticus) (see Fig. 8.2D).
The microorganisms primarily considered secondary colonizers fell into the green,
orange, and red complexes.
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The green complex includes Eikenella corrodens, A. actinomycetemcomitans serotype a,
and Capnocytophaga spp.
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The orange complex includes Fusobacterium, Prevotella, and Campylobacter spp. (Fig.
8.2R). The green and orange complexes include species recognized as pathogens in
periodontal and nonperiodontal infections.
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The red complex consists of P.gingivalis, T. forsythia, and T. denticola. This complex is
of particular interest because it is associated with bleeding on probing (384).

The existence of complexes of species in plaque is another reflection of bacterial

interdependency in the biofilm environment.

5. Characteristics of Biofilm Bacteria

Metabolism of Dental Plaque Bacteria

Most nutrients for dental plaque bacteria originate from saliva or gingival crevicular
fluid, although the host diet provides an occasional but nevertheless important food supply. The
transition from Gram-positive to Gram-negative microorganisms observed in the structural
development of dental plaque is paralleled by a physiologic transition in the developing plaque.
The early colonizers (e.g., Streptococcus and Actinomyces spp.) use oxygen and lower the
redox potential of the environment, which then favors the growth of anaerobic species (80, 428).
Many of the Gram-positive early colonizers use sugars as an energy source. The bacteria that

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predominate in mature plaque are anaerobic and asaccharolytic (i.e., they do not break down
sugars), and they use amino acids and small peptides as energy sources (231).
There are many metabolic interactions among the different bacteria found in dental
plaque (Fig. 8.23).

Communication Between Biofilm Bacteria

Bacterial cells do not exist in isolation. In a biofilm, bacteria have the capacity to
communicate with each other. One example of this is quorum sensing, in which bacteria secrete
a signaling molecule that accumulates in the local environment and triggers a response such as a
change in the expression of specific genes once they reach a critical threshold concentration. The
threshold concentration is reached only at a high cell density, and therefore bacteria sense that
the population has reached a critical mass or “quorum”. Quorum sensing appears to play diverse
roles in, for example, modulating the expression of genes for antibiotic resistance, encouraging
the growth of beneficial species in the biofilm, and discouraging the growth of competitors.

Interactions Between Dental Plaque Bacteria

Nonpathogenic organisms in subgingival dental plaque can modify the behavior of
periodontal pathogens. In multispecies biofilms in which many bacteria are juxtaposed to cells
of different species, interactions among genetically distinct microorganisms can be mutually
beneficial (Fig. 8.5A). However, many examples of competitive interactions between different
bacteria exist (Fig. 8.24). For example, S. mutans produces antimicrobial peptides that have
broad activity against bacteria in vitro (195).
Another example of competitive interaction exists between streptococci and
periodontopathogens. S.sanguinis, S.salivarius, and S.mitis have been shown to inhibit hard and
soft tissue colonization of A. actinomycetemcomitans, P.gingivalis, and P.intermedia in vitro
(370, 405-422).

Biofilms and Antimicrobial Resistance

Bacteria growing in microbial communities adherent to a surface do not “behave” the
same way as bacteria growing suspended in a liquid environment (i.e., in a planktonic or
unattached state). For example, the resistance of bacteria to antimicrobial agents is dramatically
increased in the biofilm (9, 65, 147, 302). Almost without exception, organisms in a biofilm are
1000 to 1500 times more resistant as compared with antibiotics in their planktonic state. The
mechanisms of this increased resistance differ from species to species, from antibiotic to
antibiotic, and for biofilms growing in different habitats.
In addition, extracellular enzymes such as P-lactamases, formaldehyde lyase, and
formaldehyde dehydrogenase may become trapped and concentrated in the extracellular matrix,
and as such inactivate some antibiotics. Some antibiotics, such as the macrolides, are unaffected
by this process.
“Superresistant” bacteria were identified within a biofilm. These cells have multidrug
resistance pumps that can extrude antimicrobial agents from the cell (38). Because these pumps
place the antibiotics outside of the outer membrane, the process offers protection against
antibiotics that target, for example, cell wall synthesis. The penetration and efficacy of
antimicrobials against biofilm bacteria are critical issues for the treatment of periodontal
infections.

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Antibiotic resistance may be spread through a biofilm via the intercellular exchange of
DNA. The high density of bacterial cells in a biofilm facilitates the exchange of genetic
information among cells of the same species and across species and even across genera.

6.Microbiologic Specificity of Periodontal Diseases

Ecological Plaque Hypothesis.

According to the ecologic plaque hypothesis, both the total amount of dental plaque and
the specific microbial composition of plaque may contribute to the transition from health to
disease.
The health-associated dental plaque microbiota is considered to be relatively stable over
time and in a state of dynamic equilibrium or “microbial homeostasis”. The host controls
subgingival plaque to some extent by a tempered immune response and low levels of gingival
crevicular fluid flow. Perturbations to the host response may be brought about by an excessive
accumulation of nonspecific dental plaque, by plaque-independent host factors (e.g., the onset of
an immune disorder, changes in hormonal balance [e.g., during pregnancy]), or by environmental
factors (e.g., smoking, diet).
Changes in the host status, such as inflammation, tissue degradation, and/or high gingival
crevicular fluid flow, may lead to a shift in the microbial population in plaque. As a result of
microenvironmental changes, the number of beneficial species may decrease, whereas the
number of potentially pathogenic species increases. This gradual shift in the entire microbial
community, known as dysbiosis, may result in a chronic disease state such as periodontitis (23).
The ecologic plaque hypothesis is entirely consistent with observations that disease-associated
organisms are minor components of the oral microbiota in health; these organisms are kept in
check by interspecies competition during microbial homeostasis. Disease is associated with the
overgrowth of specific members of the dental plaque biofilm when the local microenvironment
changes, but it is not necessarily the same species in each case. An important consideration of the
ecologic plaque hypothesis is that therapeutic intervention can be useful on a number of different
levels. Eliminating the etiologic stimulus—whether it is microbial, host, or environmental—will
help to restore microbial homeostasis. Targeting specific microorganisms may be less effective
because the conditions for disease will remain.

Keystone Pathogen Hypothesis and Polymicrobial Synergy and Dysbiosis Model

The keystone pathogen hypothesis indicates that certain low-abundance microbial
pathogens can orchestrate inflammatory disease by remodeling a normally benign microbiota
into a dysbiotic one. It may provide a novel conceptual basis for the development of targeted
diagnostic and treatment modalities for complex dysbiotic diseases (131, 133).
Some evidence indicates that certain pathogens may trigger the disruption of microbial
homeostasis, thereby leading to the development of periodontal disease, even when they are
present only in low numbers. For example, P.gingivalis subverts the host immune system and
changes the microbial composition of dental plaque, ultimately leading to periodontal bone loss.
On this basis, P.gingivalis was labeled a “keystone” pathogen; this means that it is an organism
that is central to the disease process, even when it is at a relatively low abundance.
The keystone pathogen hypothesis has been extended to include the concept of a
disrupted homeostasis in addition to the important roles of keystone pathogens in the
polymicrobial synergy and dysbiosis model of disease (132). In this model, interspecies
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communication between keystone pathogens and other members of the community (known as
accessory pathogens) is considered one important factor that leads to overgrowth of the more
pathogenic microbiota and to a dysbiotic microbial community.

7. The Transition from Health to Disease

The oral microbial ecology is dynamic. The presence and numbers of particular
microorganisms, even as a part of a community colonizing a niche in a human being, are
controlled by the type and quantity of nutrients present (nutritional determinant), their ability to
tolerate the specific physicochemical factors (physicochemical determinants), and their ability to
cope with antimicrobial compounds (biologic determinants) or mechanical removal forces
(mechanical determinants) (445). Although these determinants are well defined from a theoreti-
cal point of view, in practice they overlap each other, especially in more complex environments
(i.e., multispecies environments). Inherent to their biologic nature, bacteria interact with each
other, with their environment, and vice versa.
Therefore, the microbial ecology will change its composition or the composition of the
microbial ecology will be changed when transitioning from a healthy status to a diseased status
or vice versa. A change in the composition of a bacterial community as the result of external,
nonmicrobial factors is termed allogenic succession. Smoking is a good example of such
interaction. In autogenic succession (i.e., a change in the composition of a microbial community
that arises from microbial activities), interbacterial and viral-bacterial interactions are involved.
The number and proportions of different subgingival bacterial morphotypes differ between
healthy and diseased sites (Figs. 8.31 and 8.32) (223, 373, 380). The total number of bacteria,
which was determined by microscopic counts per gram of plaque, was twice as high in peri-
odontally diseased sites as compared with healthy sites (380). Because considerably more plaque
is found at diseased sites, this suggests that, in general, the total bacterial load in diseased sites is
greater than that of healthy sites. The microbiota in periodontally healthy sites consists
predominantly of Gram-positive facultative rods and cocci (approximately 75%) (373). The
recovery of this group of microorganisms is decreased proportionally in gingivitis (44%) and
periodontitis (10% to 13%). These decreases are accompanied by increases in the proportions of
Gram-negative rods, from 13% in health to 40% in gingivitis and 74% in advanced periodontitis.
The current concept regarding the etiology of periodontal diseases considers three
groups of factors that determine whether active periodontal destruction will occur in a subject: a
susceptible host, the presence of pathogenic species, and the absence or small proportion of so-
called beneficial bacteria (389, 448). The clinical manifestations of periodontal destruction will
result from a complex interplay among these etiologic agents. In general, small amounts of
bacterial plaque can be controlled by the body’s defense mechanisms without destruction;
however, when dysbiosis happens (e.g., as a result of increased susceptibility, high bacterial
load, or pathogenic infections), periodontal destruction could occur (23).

8. Pathogenic Bacteria

The second essential factor for disease initiation and progression is the presence of one
or more pathogens of the appropriate clonal type and in sufficient numbers. Despite the
difficulties inherent in characterizing the microbiology of periodontal diseases, a small group of
pathogens is recognized because of their close association with disease. Obvious data support
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the designation of A. actinomycetemcomitans, T. forsythia, T.denticola, and P.gingivalis as

key pathogens because they are strongly associated with periodontal disease status, disease
progression, and unsuccessful therapy. For the following list of bacteria, moderate evidence
for etiology has been reported - at least if their concentration passes a certain threshold level:
P.intermedia, Prevotella nigrescens (Fig. 8.2I)- C.rectus, P.micra (Fig. 8.2J), F.nucleatum,
Eubacterium nodatum (Fig. 8.2N)- and various spirochetes (12, 379, 382, 389, 448). The
significance of the role of these key pathogens is largely based on epidemiologic data - the
ability of these microorganisms to produce disease when inoculated in animals- and their
capacity to produce virulence factors. However, the mere presence of putative (suspected)
periodontopathogens in the gingival crevice is not in itself sufficient to initiate or cause
periodontal inflammation.
An elevation in the relative proportion or number of these pathogens to reach a critical
mass seems more crucial to mount an effective tissue-damaging process. Indeed, even in health,
periodontopathogens are or can be present in the gingival crevice - albeit in low numbers, as
members of the normal resident flora (236).