Periodontal Microbiology

1. PERIODONTAL
MICROBIOLOGY
DR. IBRAR HUMAYUN
ASSISTANT PROFESSOR
PERIODONTOLOGY

2. INTRODUCTION
• The colonization of the oral cavity also starts close to the time of birth.
• Streptococcus salivarius and Streptococcus mitis have been identified as the first and
most dominant oral microbes to colonize the oral cavity of newborn infants.
• The species that colonize the teeth after eruption include Streptococcus sanguinis,
Lactobacillus spp., and Streptococcus oralis.
• Oral streptococci, including S. oralis, Streptococcus anginosus, mutans streptococci,
and Streptococcus gordonii are commonly reported to be present after the first year
of life.
• In addition, anaerobes, including Fusobacterium spp. and Prevotella spp. are
reported in young children.

3. ORAL CAVITY FROM A MICROBE’S
PERSPECTIVE
• The oral cavity can be divided into six major ecosystems:
• The intraoral and supragingival hard surfaces (teeth, implants, restorations, and
prostheses)
• Subgingival regions adjacent to a hard surface, including the periodontal/peri-
implant pocket
• The buccal palatal epithelium and the epithelium of the floor of the mouth
• The dorsum of the tongue
• The tonsils
• The saliva

4. FROM A MICROBE’S PERSPECTIVE
• In the human body, teeth and nails are the only naturally occurring nonshedding surfaces.
• Artificial nonshedding surfaces of medical importance include prosthetic devices such as
catheters, artificial joints, dental implants, and heart valves.
• From a microbiologic viewpoint, teeth and implants are unique for two reasons: (1) they
provide a hard, nonshedding 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, peri-implantitis, and, sometimes, bad
breath.

5. PLAQUE BIOFILM
• Organic constituents of the matrix include polysaccharides, proteins, glycoproteins,
lipid material, and DNA.
• Albumin, which probably originates from crevicular fluid, lipid material from
disrupted cells and Glycoproteins from the saliva are important components of the
pellicle.
• The inorganic components of plaque are predominantly calcium and phosphorus,
with trace amounts of other minerals such as sodium, potassium, and fluoride.
• Supragingival calculus is frequently found adjacent to salivary ducts and this reflects
the high concentration of minerals available from saliva.
• Minerals required for subgingival plaque are derived from crevicular fluid and are
therefore seen to be dark green or brown in colour.

6. DIFFERENT TOOTH DEPOSITS

7. MATERIA ALBA

8. PLAQUE

9. CALCULUS

10. TYPES OF PLAQUE
• Supragingival plaque is found at or above the gingival margin.
• Subgingival plaque is found below the gingival margin, between the tooth and the
gingival pocket epithelium.
• Gram-positive cocci and short rods in supragingival plaque predominate at the tooth
surface, whereas gram-negative rods, filaments, and spirochetes predominate in the
outer surface of the mature plaque mass.
• However, in the deeper parts of the pocket, the filamentous organisms become fewer
in subgingival plaque and shows an increased concentration of gram-negative rods,
cocci and spirochetes.

11. ACCUMULATION OF 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

12. FORMATION OF THE PELLICLE
• All hard and soft tissues are coated with a layer of organic material known as the
acquired pellicle consisting of more than 180 peptides, proteins, and glycoproteins.
• The salivary pellicle can be detected on clean enamel surfaces within 1 minute after
their introduction.
• It is composed of two layers: a thin basal layer that is very difficult to remove, and a
thicker globular layer, up to 1 μm or more, that is easier to detach.
• Consequently, bacteria that adhere to tooth surfaces do not contact the enamel
directly but interact with the acquired enamel pellicle.

13. INITIAL ADHESION
• Colonizing bacteria can be detected within 3 minutes after the introduction of sterile enamel
into the mouth.
• 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.
• Over the first 4 to 8 hours, the genus Streptococcus tends to dominate, usually accounting
for >20% of bacteria present, along with Actinomyces and Veillonella spp termed together as
primary colonisers.
• The primary colonizers provide new binding sites and microenvironment for adhesion and
survival of other oral bacteria.
• 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.

14. 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).

15. PHASE 2: INITIAL ADHESION
• Long- and short-range forces, including van der Waals attractive forces and
electrostatic repulsive forces operate at this distance.
• According to Derjaguin–Landau–Verwey–Overbeek 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 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.

16. STRONG ATTACHMENT
• On a rough surface, bacteria are more protected against shear forces so that a change
from reversible to irreversible binding may occur more easily and more frequently.
• 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.
• In the salivary fluid phase, interactions between antigen I/II proteins of Streptococci
spp. and gp340 protein result in the aggregation of bacteria and conformational
changes.
• Coaggregation or coadhesion involves bacterial cells to contact eachother through
passive or active transport and bind weakly through nonspecific hydrophobic,
electrostatic, and van der Waals forces followed by strong binding using adhesion-
receptor interaction.

18. COLONIZATION AND PLAQUE
MATURATION
• The primary colonizing bacteria adhered to the tooth surface provide new receptors for
attachment by other bacteria as part of a process known as coadhesion.
• Cell–cell adhesion between genetically distinct oral bacteria also occurs in the fluid phase
(i.e., in saliva).
• 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., and Prevotella spp. bind with streptococci and/or actinomyces.
• 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.

19. PLAQUE MATURATION
• Well-characterized interactions of secondary colonizers with early colonizers include
the coaggregation of F. nucleatum with S. sanguinis, Prevotella loescheii with A.
oris, and Capnocytophaga ochracea with A. oris.
• Secondary colonizer 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.
• The transition from early supragingival dental plaque to mature plaque growing
below the gingival margin involves a shift in the microbial population from primarily
gram-positive organisms to high numbers of gram-negative bacteria.

20. FACTORS AFFECTING SUPRAGINGIVAL
PLAQUE FORMATION
• During the first 24 hours when starting with a clean tooth surface, plaque growth is
negligible from a clinical viewpoint (i.e., <3% coverage of the vestibular tooth
surface).
• During the following 3 days, coverage progresses rapidly to the point at which, after
4 days, an average of 30% of the total coronal tooth area will be covered with
plaque.
• The microbial composition of the dental plaque will change, with a shift toward a
more anaerobic and a more gram-negative flora, including an influx of fusobacteria,
filaments, spiral forms, and spirochetes.
• With this ecologic shift within the biofilm, a transition occurs from the early aerobic
environment with gram-positive facultative species, to a highly oxygen-deprived
environment, in which gram-negative anaerobic microorganisms predominate.

21. TOPOGRAPHY OF THE
SUPRAGINGIVAL PLAQUE
• Early plaque formation on teeth follows a typical topographic pattern, with initial
growth along the gingival margin and from the interdental spaces.
• This pattern may change severely when the tooth surface contains irregularities that
offer a favorable growth path.
• Plaque formation may also originate from grooves, cracks, perikymata, or pits.
• Early colonization of the enamel surface starts from surface irregularities in which
bacteria shelter from shear forces, thereby permitting them the time needed to change
from reversible to irreversible binding.

22. BACTERIAL COMPLEXES

23. SURFACE ROUGHNESS
• Rough intraoral surfaces (e.g., crown margins, implant abutments, denture bases)
accumulate and retain more plaque and calculus in terms of thickness, area, and
colony-forming units.
• Ample plaque also reveals an increased maturity or pathogenicity of its bacterial
components, which is characterized by an increased proportion of motile organisms
and spirochetes and/or a denser packing of them.
• Smoothing an intraoral surface decreases the rate of plaque formation.
• There seems to be a threshold level for surface roughness (Ra ≈ 0.2 μm) above which
bacterial adhesion will be facilitated.

24. INDIVIDUAL VARIABLES THAT
FAVOUR PLAQUE FORMATION
• The rate of plaque formation differs significantly among subjects, and these
differences may overrule surface characteristics.
• The difference in rate of plaque formation can also be explained by factors such as
diet, chewing fibrous food, smoking, and the presence of copper or amalgam.
• Moreover, tongue and palate brushing, the colloid stability of bacteria in the saliva,
antimicrobial factors present in the saliva, the chemical composition of the pellicle,
and the retention depth of the dentogingival area affect plaque formation.

25. VARIATION WITHIN THE DENTITION
Within a dental arch, large differences in plaque growth rate can be detected:
• In general, early plaque formation occurs faster: in the lower jaw (as compared with
the upper jaw);
• in molar areas;
• on the buccal tooth surfaces (as compared with palatal sites, especially in the upper
jaw); and
• in the interdental regions (as compared with the buccal or lingual surfaces).

26. IMPACT OF GINGIVAL
INFLAMMATION AND SALIVA
• Several studies clearly indicate that early in vivo plaque formation is more rapid on
tooth surfaces facing inflamed gingival margins than on those adjacent to healthy
gingivae.
• Studies suggest that the increase in crevicular fluid production containing minerals,
proteins, carbohydrates favor both the initial adhesion and/or the growth of the early
colonizing bacteria.
• In addition, it is known that, during the night, the plaque growth rate is reduced by
some 50%.

27. EFFECT OF AGE
• Although older studies were contradictory, more recent reports clearly indicate that a
subject’s age does not influence de novo plaque formation.
• In a study by Fransson and colleagues,no differences could be detected in de novo
plaque formation between a group of young patients and a group of older subjects
who abolished mechanical tooth cleaning measures for 21 days.
• However, the developed plaque in the older patient group resulted in more severe
gingival inflammation, which seems to indicate an increased susceptibility to
gingivitis with aging.

28. DE NOVO SUBGINGIVAL PLAQUE
FORMATION
• A critical review of the effectiveness of subgingival debridement, for instance,
revealed that a high proportion of treated tooth surfaces (5% to 80%) still harbored
plaque and/or calculus after scaling.
• These remaining bacteria were considered the primary source for the subgingival
recolonization.
• Some pathogens penetrate the soft tissues or the dentinal tubules and eventually
escape instrumentation.
• These investigators also observed that smooth surfaces harbored significantly less
plaque and concluded that subgingival irregularities shelter submerged
microorganisms.

29. METABOLISM OF 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 early colonizers (e.g., Streptococcus and Actinomyces spp.) use sugars as an
energy source.
• Lactate and formate are byproducts of the metabolism of streptococci and
Actinomyces spp., which are used in the metabolism of Veillonella spp. and A.
actinomycetemcomitans.
• The growth of P. gingivalis is also enhanced by metabolic byproducts such as
succinate produced by C. ochracea or T. denticola and protoheme from
Campylobacter rectus.

30. METABOLISM OF PLAQUE BACTERIA
• Metabolic interactions also occur between the host and the plaque microorganisms.
• The bacterial enzymes that degrade host proteins mediate the release of ammonia,
which may be used by bacteria as a nitrogen source.
• Hemin iron from the breakdown of host hemoglobin may be important in the
metabolism of P. gingivalis.
• Increases in steroid hormones are associated with significant increases in the
proportions of P. intermedia found in subgingival plaque.

31. COMMUNICATION BETWEEN
BACTERIA
• Quorum sensing, involves a signaling molecule that accumulates in the local environment
and triggers a response such as a change in the expression of specific genes, encouraging the
growth of beneficial species in the biofilm, and discouraging the growth of competitors.
• Two types of signaling molecules have been detected from dental plaque bacteria: peptides
released by gram-positive organisms during growth and a “universal” signal molecule called
autoinducer 2 (AI-2).
• The streptococcal peptides are known as competence-stimulating peptides because the major
response to these signals is the induction of competence (uptake of DNA), while S. mutans,
respond to competence-stimulating peptides by lysing (dissemination of DNA following
lysing).
• AI-2 is produced and detected by many different bacteria such as S. oralis and A. oris
causing wide-ranging changes in gene expression, in some cases affecting up to one-third of
the entire genome.

32. INTERACTIONS AMONG PLAQUE
BACTERIA
• The expression of long fimbriae of P. gingivalis is down-regulated in the presence of
Streptococcus cristatus, and short fimbriae are down-regulated by S. gordonii, S.
mitis, or S. sanguinis.
• However, competitive interactions exist between different bacteria such as S. mutans
producing antimicrobial peptides that have broad activity against bacteria.
• 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.
• Studies have shown that these interactions can also influence the host such as S.
cristatus attenuates the ability of F. nucleatum to stimulate interleukin-8 production
by host oral epithelial cell and S. gordonii–induced increased expression of a
complement resistance protein, ApiA, in A. actinomycetemcomitans.

33. BIOFILM AND ANTIMICROBIAL
RESISTANCE
• Organisms in a biofilm are 1000 to 1500 times more resistant as compared with antibiotics
in their planktonic state.
• It is generally accepted that the resistance of bacteria to antibiotics is affected by their
nutritional status, growth rate, temperature, pH, and prior exposure to subeffective
concentrations of antimicrobial agents.
• Extracellular enzymes such as β-lactamases, formaldehyde lyase, and formaldehyde
dehydrogenase may become trapped and concentrated in the extracellular matrix, and as
such inactivate some antibiotics.
• “Superresistant” bacteria have multidrug resistance pumps that can extrude antimicrobial
agents from the cell.
• Antibiotic resistance may be spread through a biofilm via the intercellular exchange of DNA
via mechanism of conjugation, transformation, plasmid and transposon transfer.

34. NON-BACTERIAL INHABITANTS OF
THE ORAL CAVITY
• In addition to bacteria, viruses, fungi and protozoa can be present in the mouth.
• Herpesviruses, human papillomaviruses, picornaviruses, and retroviruses can all
contribute to the development of oral ulcers, tumors, mononucleosis, Sjögren
syndrome, osteomyelitis, osteonecrosis, oral leukoplakia, and oral lichen planus.
• Together with C. albicans, some of the most common opportunistic fungal candidal
pathogens in humans are C. tropicalis, C. glabrata, C. krusei, C parapsilosis, C.
guilliermondii, and C. dubliniensis causing various fungal diseases.

35. NON-SPECIFIC PLAQUE HYPOTHESIS
• The nonspecific plaque hypothesis, was supported by epidemiologic studies that
correlated both age and the amount of plaque with evidence of periodontitis.
• Several observations contradicted these conclusions.
• First, some individuals with considerable amounts of plaque and calculus, as well as
gingivitis, never developed destructive periodontitis.
• Furthermore, individuals who did present with periodontitis demonstrated
considerable site specificity with regard to the pattern of disease.
• In the presence of a uniform host response, these findings were inconsistent with the
concept that all plaque was equally pathogenic.

36. SPECIFIC PLAQUE HYPOTHESIS
• The specific plaque hypothesis underlines the importance of the qualitative
composition of the resident microbiota.
• This concept encapsulates that plaque that harbors specific bacterial pathogens may
provoke periodontal disease because key organisms produce substances that mediate
the destruction of host tissues.
• Acceptance of the specific plaque hypothesis was spurred by the recognition of A.
actinomycetemcomitans as a pathogen in localized aggressive periodontitis.
• In addition, these studies have shown that periodontal disease can occur even in the
absence of defined “pathogens,” such as red complex bacteria, and conversely that
“pathogens” may be present in the absence of disease.

37. ECOLOGIC 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.
• Perturbations to the host response may be brought about by an excessive
accumulation of nonspecific dental plaque, plaque-independent host factors (e.g.,
• the onset of an immune disorder, changes in hormonal balance [e.g., during
pregnancy], environmental factors (e.g., smoking, diet), changes in the host status,
such as inflammation, 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 leading to
a concept called dysbiosis.

38. KEYSTONE PATHOGEN HYPOTHESIS
• 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.
• Studies have shown specific pathogen-free mice exposed to P. gingivalis developed
periodontal bone loss even when the pathogen was present in less than 0.1% of the total
microbiota.
• P. gingivalis subverts the host immune system and changes the microbial composition of
dental plaque, ultimately leading to periodontal bone loss that resulted it to be labeled as a
“keystone” pathogen.
• In this model, interspecies communication between keystone pathogens and other members
of the community is considered one important factor that leads to overgrowth of the more
pathogenic microbiota and to a dysbiotic microbial community.

39. PATHOGENIC BACTERIA

40. BENEFICIAL SPECIES
These bacteria can affect the pathogenic species in different ways and thus modify the
disease process as follows:
• by passively occupying a niche that may otherwise be colonized by pathogens,
• by actively limiting a pathogen’s ability to adhere to appropriate tissue surfaces,
• by adversely affecting the vitality or growth of a pathogen,
• by affecting the ability of a pathogen to produce virulence factors, or
• by degrading the virulence factors produced by the pathogen.

41. PERIODONTAL HEALTH
• The bacteria associated with periodontal health are primarily gram-positive
facultative species and members of the genera Streptococcus and Actinomyces.
• Small proportions of gram-negative species are also found, most frequently P.
intermedia, F. nucleatum spp., F. periodonticum, Capnocytophaga spp, Neisseria
spp., and Veillonella spp.
• Certain bacterial species have been proposed to be protective or beneficial to the
host, including S. sanguinis, Veillonella parvula and C.ochracea.

42. VIRULENCE FACTORS
Virulence factors of periodontal microorganisms can be subdivided as follows:
• factors that promote colonization (adhesins),
• toxins and enzymes that degrade host tissues, and
• mechanisms that protect pathogenic bacteria from the host.

43. ADHESIVE SURFACE PROTEINS
• To colonize the periodontal pocket, bacteria must adhere to cells or tissues in the region,
such as teeth, the existing microbial biofilm, or the pocket epithelium.
• Fimbriae or pili are polymeric fibrils that are composed of repeating subunits that can extend
several micrometers from the cell membrane.
• Strains of P. gingivalis produce two types of fimbriae, which are known as the major
fimbriae (Fim A) and the minor fimbriae (Mfa 1) interact with oral streptococci such as S.
gordonii. P. gingivalis.
• Fim A binds to glyceraldehyde-3-phosphate dehydrogenase, whereas Mfa1 interacts with the
S. gordonii cell surface adhesin B.
• Major fimbriae have also been shown to bind host extracellular matrix proteins fibronectin
and type I collagen, salivary proline-rich proteins and statherin, and epithelial cells.

44. TISSUE DESTRUCTION PROMOTING
FACTORS
• Many bacterial proteins that interact with host cells are recognized by the immune
system, and they may trigger immune responses.
• Most of the tissue destruction in periodontal pockets is actually caused by host
matrix metalloproteinases (MMPs), but bacterial proteases play important roles in
activating the host enzymes.
• In the case of P. gingivalis, three enzymes known as gingipains are responsible for at
least 85% of the total proteolytic activity of the bacterium.
• Leukotoxin (LtxA) produced by A. actinomycetemcomitans act by delivering an
adenylate cyclase domain into cells, which catalyzes the uncontrolled conversion of
adenosine triphosphate to cyclic adenosine monophosphate.

45. STRATEGIES FOR EVADING HOST
DEFENSES
Subverting the host immune system, includes the following microbial strategies:
• the production of an extracellular capsule,
• the proteolytic degradation of host innate or acquired immunity components,
• the modulation of host responses by binding serum components on the bacterial cell
surface, and
• the invasion of gingival epithelial cells.