1) Carranza’s Clinical Periodontology; 10th ed. Elsevier publication.
2) Textbook of Clinical Periodontology, Glickman; 6th ed. 3) Oral microbiology – Philip Marsh, Michael Martin – 4th ed4) Oral microbiology & immunology – Nisengard & Newman -2nd
ed5) Immunology of Oral Diseases – Thomas Lehner – 3rd ed6) Essential microbiology for dental students – Lakshman
Samaranayake – 3rd ed7) Anne. D. Haffajee & sigmund.S. Socransky. Microbial
etiological agents of destructive periodontal diseases. Periodontology 2000, Vol. 5, 1994, 78-1
8) Tatsuj Nishihara & Takeyoshi koseki. Microbial etiology of Periodontitis. Periodontology 2000, Vol. 36, 2004, 14–26.
Aerobes Anaerobes Facultative anaerobes Obligatory Capnophilic Microaerophilic
MICROBIOLOGY OF ORAL CAVITY
The colonization of the oral cavity starts about the time of birth.
Within hours - colonized by low numbers of mainly
facultative and aerobic bacteria.
Second day - anaerobic bacteria can be detected in the infant’s
Within 2 weeks - a nearly mature microbiota is established
in the gut of the newborn.
After weaning (>2 years), approximately 1014 microorganisms
consisting of more than 400 different types of bacteria.
Body contains 10 times more bacteria than human cells.
51000 bacteria were isolated
509 distinct taxa were recognized
141 were detected only once.
Teeth are the primary habitat for periopathogens. Therefore, teeth can even be considered as a port of entry for periodontopathogens.
Mucosal surfaces – dorsum of tongue & tonsils.
Dental plaque is defined as a specific but highly
variable structural entity, resulting from colonization ofmicroorganisms on tooth surface, restorations and Other parts of the oral cavity which consists of salivarycomponents like mucin, desquamated epithelial cells, debris and microorganisms all embedded in a gelatinousextracellular matrix.
STRUCTURE AND COMPOSISTION OF DENTAL PLAQUE
Dental plaque is composed primarily of
microorganisms. One gram of plaque (wet weight) contains
approximately 1011 bacteria.
In a periodontal pocket,
Healthy crevice - 103 bacteria.
Deep pocket - 108 bacteria.
Nonbacterial microorganisms that are found in
plaque include Mycoplasma species, yeasts, protozoa, and
Dental plaque is broadly classified based on its position on the
tooth surface toward the gingival margin:
* Supragingival plaque:
* Marginal plaque:
* Subgingival plaque:
Subgingival microbiota differs in composition from the
supragingival plaque, primarily
Availability of blood products and
Low oxidation reduction (redox) potential,
GCF substances which acts as nutrients characterizes the
The site specificity of plaque is significantly associated with
diseases of the periodontium.
Marginal plaque – prime importance in the initiation and
development of gingivitis.
Supragingival plaque and tooth associated subgingival
plaque – critical in calculus formation and root caries.
Tissue associated subgingival plaque – important in the
tissue destruction that characterizes different forms of
BASIC BIOFILM STRUCTURE
They are grouped in microcolonies surrounded by an
enveloping intermicrobial matrix.
The matrix is penetrated by fluid channels that conduct the
flow of nutrients, waste products, enzymes, metabolites, and
These microcolonies have micro environments with differing
pH’s, nutrient availability, and oxygen concentrations.
The bacteria in a biofilm communicate with each other by
sending out chemical signals.
These chemical signals trigger the bacteria to produce
potentially harmful proteins and enzymes.
Lower plaque layers: these are dense, microbes are bound
together in a polysaccharide matrix with other organic and
Loose layer: on top of the lower layer. Appears that is often
irregular in appearance; it can extend into the surrounding
Fluid layer: bordering the biofilm has a rather stationary
sublayer and a fluid layer in motion.
Nutrient components penetrate this fluid medium by
The intercellular matrix consists or organic and
inorganic materials derived from saliva, gingival
crevicular fluid, and bacterial products.
ORGANIC CONSTITUENTS INORGANIC
COMPONENTS - Albumin - Predominantly
- Lipid material calcium and - - Glycoproteins
phosphorous - Polysaccharides-dextran. - Trace amt, of sodium, potassium, and fluoride.
The source of inorganic constituents:Supragingival plaque - primarily salivaSubgingival plaque – Crevicular fluid (a
The fluoride component of plaque is largely derived from external sources such as fluoridated toothpastes, rinses and fluoridated drinking water.
DENTAL BIOFILM FORMATION AT THE ULTRASTRUCTURAL LEVEL
The process of plaque formation can be divided
into three major phases:
The formation of the pellicle on the tooth
Initial adhesion and attachment of bacteria.
Colonization and plaque maturation.
FORMATION OF THE PELLICLE
Within nanoseconds after a vigorously polishing the teeth, a thin,
saliva-derived layer called the acquired pellicle, covers the tooth
This pellicle consists of;
Enzymes (alpha amylase)
Other molecules which acts as adhesion sites.
INITIAL ADHESION AND ATTACHMENT OF BACTERIA
Phase 1: Transport to the surface;
Random contacts may occur. (through Brownian
motion, liquid flow or active bacterial movement).
Phase 2: Initial adhesion;
Initiated by the interaction between the bacterium and
the surface, from a certain distance (50 nm), through long
range and short range forces.
Phase 3: Attachment; A firm anchorage between bacterium and surface
will be established by specific interactions (covalent, ionic or
Eg: A. viscosus possesses fimbriae that contain adhesions that
specifically bind to proline rich proteins of the dental pellicle.
Phase 4: Colonization of the surface and biofilm formation.
COLONIZATION AND PLAQUE MATURATION
COMPLEXES OF PERIODONTAL MICROORGANISMS:
1. Early colonizers:
- are either independent or defined complexes.
- Member of Yellow (Streptococcus spp.) or purple
complexes (Actinomyces odontolyticus).
2. Secondary colonizers:
- Members of Green complexes (Eikenella corrodens,
Actinobacillus actinomycetemcomitans serotype a, and
Capnocytophaga species) and Red complexes.
GROWTH DYNAMICS OF DENTAL PLAQUE
Important changes within first 24 hours.
First 2 to 8 hours – Pioneering streptococci saturate the
salivary pellicular binding sites and thus covering 3% to
30% of the enamel surface.
Next 20 hours – a short period of rapid growth is
After 1 day – the term ‘Biofilm’ is fully deserved
because organization takes place within it.
The further growth of the plaque mass occurs
preferably by the multiplication of already adhering
microorganisms rather than by new colonizers
DE NOVO SUPRAGINGIVAL PLAQUE FORMATION; CLINICAL ASPECTS
Clinically it follows an exponential growth curve.
First 24 hours – negligible plaque, covering <3% of the
vestibular tooth surface.
During next 3 days – plaque growth increases at a rapid
rate, then slows down.
After 4 days – average of 30% of the total tooth crown
area will be covered with plaque and no more increase
substantially with time.
There will be an ecological shift within of the biofilm
there is a transition from the early aerobic environment to
a highly oxygen-deprived environment.
BENEFITS OF DENTAL PLAQUE:
Dental plaque is part of the natural resident
microflora of the human body. Play a critical role in the
normal development of the physiology of the host.
Germ-free animals have altered mucosal surfaces,
poor nutrient absorption, suffer from nutrient deficiencies,
and have impaired host defences.
The resident microflora also reduces the risk of
infection by acting as a barrier to colonization by exogenous
(and often pathogenic species (termed ‘colonization
ASSOCIATION OF PLAQUE MICROORGANISMS WITH PERIODONTAL DISEASES
The current concept on the etiology of periodontitis
considers three groups of factors that determine whether
active periodontitis will occur in a subject:
A susceptible Host.
The presence of pathogenic species.
The absence, or a small proportion , of beneficial
PATHOGENS STRENGTH OF RELATIONSHIP WITH DISEASE:
MICROBIAL SPECIFICITY OF PERIODONTAL DISEASE
NONSPECIFIC PLAQUE HYPOTHESIS:
This hypothesis maintains that periodontal disease results
from the “elaboration of noxious products by the entire plaque
SPECIFIC PLAQUE HYPOTHESIS:
The specific plaque hypothesis states that only certain
plaque is pathogenic, and its pathogenicity depends on the
presence of or increase in specific microorganisms.
ECOLOGICAL PLAQUE HYPOTHESIS:
A change in the nutrient status of a pocket or chemical
and physical changes to the habitat are thus considered the
primary cause for the overgrowth by pathogens.
The periodontal microbiota is a complex community of
microorganisms, many of which are still difficult or impossible to
isolate in the laboratory.
Studies have established that disease progresses at
different rates, with alternating episodes of rapid tissue
destruction and periods of remission.
Currently periodontitis is considered a mixed infection,
which has a significant impact on both its diagnosis and its
treatment. Recent microbiologic tests clearly indicate that the
presence of periodontal pathogens by itself is not sufficient for
the development of periodontitis.
MICROORGANISMS ASSOCIATED WITH SPECIFIC PERIODONTAL DISEASES
At periodontally healthy sites the microbial load is low with
only 102 to 103 bacteria.
In health, approximately 75% to 80% of the recoverable
microflora is gram-positive with most of the remainder belonging
to gram-negative species of the Veillonella and Fusobacterium
Protective or beneficial to the host, including S. sanguis,
Veilonella parvula, and C. ochraceus.
Finally, the microflora in health is mostly of a nonmotile nature.
The microbial load at diseased sites is greater, with
approximately 104 to 106 bacteria.
The microbiota of chronic gingivitis (plaque induced)
consist of app. Equal proportions of gram positive (56%) and
gram negative (44%) species, as well as facultative (59%) and
anaerobic (41%) microorganisms.
Pregnancy associated gingivitis is accompanied by
increases in steroid hormones in crevicular fluid and dramatic
increases in levels of P. intermedia, which uses the steroid as
Cultivation of plaque microorganisms from sites of
chronic periodontitis reveals high percentages of
anaerobic (90%) and gram negative (75%) bacterial
Certain gram-negative bacteria with pronounced
virulence properties have been strongly implicated as
etiologic agents (e.g. P. gingivalis and Tannerella
LOCALIZED AGGRESSIVE PERIODONTITIS
The microflora of subgingival biofilms from patients with
LAP is similar to that of patients with chronic periodontitis and is
predominantly composed of gram negative, capnophilic, and
On a percentage basis, the most numerous isolates are
several species from the genera Eubacterium, A. naeslundii, F.
nucleatum, C. rectus, and Veillonella parvula.
Actinobacillus actinomycetemcomitans plays a causative
role in LAP, especially in cases in which patients harbor highly
leukotoxic strains of the organism.
However, some populations of patients with LAP do not
harbor A. actinomycetemcomitans, and in still others P.
gingivalis may be etiologically more important.
GENERALIZED AGGRESSIVE PERIODONTITIS
The subgingival flora in patients with generalized
aggressive periodontitis resembles that in other forms of
The predominant subgingival bacteria in patients
with generalized aggressive periodontitis are P. gingivalis,
T. forsythensis A. actinomycetemcomitans, and
REFRACTORY CHRONIC PERIODONTITIS
The microflora taken from progressing sites in some
of these patients is unusually diverse and may contain
enteric rods, staphylococci, and Candida.
In other patients, persistently high levels are found
of one or more of the following bacteria: P. gingivalis, T.
forsythensis, S. intermedius, P. intermedia,
Peptostreptococcus micros, and Eikenella corrodens.
Persistence of Streptococcus constellatus has also
NECROTIZING ULCERATIVE GINGIVITIS/PERIODONTITIS
The majority of the spirochetes (treponemes)
associated with necrotizing ulcerative gingivitis are
uncultivable, but it is clear that they constitute a very
large and diverse group.
More than 50% of the isolated species were
strict anaerobes with P. gingivalis and F. nucleatum
accounting for 7-8% and 3.4%, respectively.
The bacteria isolated from abscesses are similar to those
associated with chronic and aggressive forms of periodontitis.
An average of approximately 70% of the cultivable flora in
exudates from periodontal abscesses are gram-negative and
about 50% are anaerobic rods.
Periodontal abscesses revealed a high prevalence of the
following putative pathogens: F. nucleatum (70.8%), P. micros
(70.6%), P. intermedia (62.5%), P. gingivalis (50.0%), and T.
Enteric bacteria, coagulase-negative staphylococci, and
Candida albicans have also been detected.
Only bacilli isolated from oral cavity- Actinobacillus actinomycetem comitans
Habitat : sub gingival
0.4 -1 um
Straight or curved rods with rounded ends.
Grow as white, translucent. Smooth & non- hemolytic colonies on blood agar
Growth is promoted by CO2.
Aclinobaciltus" refers to the internal star-shaped
morphology of its bacterial colonies on solid media and to the
short rod or bacillary shape of individual cells
selective media: tryptone-soy-serum-bacitracin-
vanomycin agar (white translucent colonies with * shaped
This species was first recognized as a possible
periodontal pathogen in lesions of localized juvenile
ACTINOMYCETES True bacteria with long branching filament. Important genera: actinomyces (microaerophilic / anaerobic) Nocardia (aerobic)
Actinomyces - soil organism Found in dental plaque. Species : A israelli, A odontolyticus, A naeslundii, A myeri, A georgiae Gm + filamentous branching rod. Non-motile, non- sporing, non-acid fast.
Culture :grows slowly under anaerobic condition (blood/serum
glucose agar at 37 deg C) Produces small, creamy white adherent colonies on blood
A israeliiA israelii
It is a Gram negative, anaerobic, non motile,
asaccharolytic rods non-motile that usually exhibit coccal to
short rod morphologies.
Found in sub gingival sulcus.Culture: grow anaerobically with dark pigmentation on media containing lysed blood.
Does not ferment carbohydrate.
Second consensus periodontal pathogen. P. gingivalis is a member of the much investigated “black pigmented Bacteroides” group. Initially grouped into a single species, B. melaninogenicus
Third consensus periodontal pathogen, B. forsythus, was
first described in 1979 as a “fusiform” Bacteroides.
The organism is a Gram negative, anaerobic, spindle
shaped, highly pleomorphic rod.
B. forsythus was detected more frequently and in
higher numbers in active periodontal lesions than inactive
This species has been shown to produce trypsin like
proteolytic activity and induce apoptotic cell death
These are Gram negative, anaerobic, helical shaped, highly
motile microorganisms that are common in many
Clearly, a spirochete has been implicated as the
likely etiologic agent of acute necrotizing ulcerative
The organism has been considered as possible
periodontal pathogens since the late 1800s and in the
Difficulty in distinguishing individual species. At
least 15 species of subgingival spirochetes have been
T. denticola was found to be more common in
periodontally diseased than healthy sites
These “were the most frequently detected
spirochetes in supra and subgingival plaques of
Pathogen related oral spirochetes” (PROS) were
also shown to have the ability to penetrate a tissue barrier
in in vitro systems
PREVOTELLA INTERMEDIA/PREVOTELLA NIGRESCENS
P. intermedia is the second black pigmented
Bacteroides to receive considerable interest in pathogenesis
of chronic periodontitis.
It is a Gram negative, short, round-ended anaerobic
rod have been shown to be particularly elevated in acute
necrotizing ulcerative gingivitis and also in certain forms of
This species appears to have a number of virulence
properties exhibited by P. gingivalis and was shown to
induce mixed infections.
It has also been shown to invade oral epithelial cells
Strains of P. intermedia that show identical
phenotypic traits have been separated into two species, P.
intermedia and P. nigrescens.
F. nucleatum is the type species of the genus
Fusobacterium, which belongs to the family
Bacteroidaceae. The name Fusobacterium has its
origin in fusus, a spindle; and bacterion, a small rod: thus,
a small spindle-shaped rod.
Gharbia and Shah (1990) divided Fusobacterium
species into four subspecies: subspecies nucleatum,
polymorphum, fusiforme, and animalis.
F. nucleatum is a Gram negative, anaerobic, spindle
shaped (cigar shaped) rod that has been recognized as part of
the subgingival microbiota for over 100 years.
This species is the most common isolate found in cultural
studies of subgingival plaque samples comprising app. 7-10% of
The species can induce apoptotic cell death in
mononuclear and polymorphonuclear cells.
Acts as “microbial bridge” facilitating coaggregation
between early and Late colonizers.
THE MILLERI STREPTOCOCCI:
Cultural studies of the last two decades have also
suggested the possibility that some of the streptococcal
species are associated with and may contribute to disease
At this time, evidence suggests that the milleri
streptococci, Streptococcus anginosus, S. constellatus and S.
intermidius might contribute to disease progression in
subsets of periodontal patients.
These species was found to be elevated at sites which
demonstrated recent disease progression.
Obviously all periodontal pathogens have not yet been
Interest has grown in groups of species not commonly
found in the subgingival plaque as initiators or possibly
contributors to the pathogenesis of periodontal disease,
particularly in individuals who have responded poorly to
Emphasis have been placed on enteric organisms,
staphylococcal species as well as other unusual mouth
Slots et al (1990) found Enterobacter cloaceae, K.
pneumoniae, Pseudomonas aeruginosa, Klebsiella oxytoca and
Enterobacter agglomerans which constitute more than 50% of
strains isolated from Chronic periodontitis patients.
More recently, viruses including cytomegalo, Epstein
Barr, Papilloma and herpes simplex have been proposed to
play a role in the etiology of periodontal diseases, possibly
by changing the host response to the local subgingival
HSV-1 and EBV are significantly associated with
destructive periodontal disease including chronic and
aggressive periodontitis. HSV-1 detected sites is associated
with severity and progression of destructive periodontal
Hannula J, Dogan B, Slots (2001) showed
geographical differences in the subgingival distribution of C.
albicans serotypes and genotypes and suggested
geographic clustering of C. albicans clones in Subgingival
samples of Chronic Periodontitis patients.
Reynaud AH (2001) found a weak correlation
between yeasts in periodontal pockets and E. saburreum.
At the pathogenic end of the spectrum, it is conceivable
that different relationships exist between pathogens.
The presence of two pathogens at a site could have
no effect or diminish the potential pathogenicity of one or
other of the species.
Alternatively, pathogenicity could be enhanced
either in an additive or synergistic fashion.
It is not clear whether the combinations suggested in
the experimental abscess studies are pertinent to human
MICROBIAL DIAGNOSTIC TESTING
Histopathological & ultra-structural changes in
experimental gingivitis & periodontal diseases
have suggested 4 stages of development:
Initial Early Established Advanced lesion
May be in response to generation of chemotactic substances by bacterial plaque Ags
Difficult to differentiate b/w normal & pathological tissue reaction.
In relatively plaque free subjects small No of leucocytes migrate through JE towards gingival crevice.
Develops within 2 – 4 days of plaque accumulation.
Lesion is localized to gingival sulcus & JE & CT.
Bld vsls dilate with exudation of fluid with Igs (IgG),
complement, fibrin & PMNs
Few lymphocytes & macrophages – in JE & adjacent CT.
Systemic immunity Serum ABs
Variety of immune complexes
Classical C pathway
C3a & C5a
Increased vascular permeability & are chemotactic for PMNs
Develops within 4 – 7 days.
Dense lymphoid cell infiltration.
Often seen in normal gingiva – when plaque control is
not practiced efficiently.
Lymphocytes constitute 75%.
Only few plasma cells.
Most of them – T-cell series with small No of B-cells.
Fibroblasts adj to lymphocytes show degenerative changes
Localized loss of collagen fibers.
Exudation of serum Igs, C, fibrinogen & leucocytes is increases.
GCF & leucocytes reach maximum.
Lymphoid cells – seeded into gingival focus of inflm
Leukocyte migration inhibition
Localization of leucocytes & proliferation of lymphocytes
Develops within 2 – 3 weeks.
Prominent plasma unfiltration.
It is probable that B lymphocytes found in early Lesion have been stimulated by plaque Ags to differentiate into plasma cells.
Still confined to small site adj to gingival sulcus but consists of numerous plasma cells. However they are also found among bld vsls &
collagen fibers. These produce IgG, some IgA & few IgM. There are also some T lymphocytes. JE & oral epithelium proliferate apically into CT &
there is loss of collagen. Gingival sulcus deepen & JE is converted into
Proliferative response of lymphocytes become evident at
14 – 21 days of plaque accumulation.
Potent activator – LPS, dextran & levan as well as some bacteria ( A.viscosus)
Both T & B lymphocytes stimulate blast cells which are seeded into inflm foci & there will be continuous supply of these cells.
A slight increase in salivary IgA & serum IgA, IgG or IgM can be detected.
Established lesion persists for years & change into advanced lesion marks the transition from a chronic & successful defense reaction to destructive immunopathological mechanism
There are 2 principal schools of thought:• That the host immune responses may be involved• That some specific microorganism in dental plaque may
be responsible for development of the advanced lesion.
Bacteroides gingivalis, AA and some spirochetes –potentially significant organisms.
This stage recognized clinically as periodontitis with pocket formn, ulcern of pocket epim, destrun of collagenousPDL & bone resorpn.
Which will lead to tooth mobility & eventually loss of tooth.
The pathological changes extend apically & laterally with dense infiltration of plasma cells, lymphocytes ¯ophages.
Breakdown of epithelial barrier --- permits direct access of plaque Ags & metabolites.
Essential feature – irreversible loss of PDL & bone with progressive increase in pocket formation. Gingival fluid contains high conc if IgG, IgA, IgM &
Cas well as predominantly PMNL infiltration.
Role of macrophages
IL-1 production which activates - T cells to proliferation. - Collagenase production by fibroblasts. - Bone resorption.
Release PGs – which affects immune responses.
Imp role in phagocytosis & bacterial killing.
Release lysosomal enzymes which enhances local damage.
Type IV HSR
Stimulates T& B cells to proliferate.
This leads to expansion of cells involved in delayed HSR (not adequately investigated)
Dental plaque as well as microorganisms induce lymphoproliferative responses & release cytokines.
Immunopathology & mechanism of adult periodontitis
Immunocytochemical investigation from tissue from adult periodontitis ---- proportion of CD4 (helper induced)to CD8 (cytotoxic-suppressor) cell subsets (2:1 to 1:1) inperiodontitis.
Furthermore both CD4 & CD8 appear to be activated atthe site of the lesion, & some of them express HLA- class2 Ag.
There is decrease in lymphoproliferative responses to stimulation with oral microorganisms (Actinomyces, Veilonella or Bacteroides) is due to the activity of CD8-suppressor T cells& their soluble products.
Significance of polyclonal B cell activators
Elicit non-specific B cell proliferation & AB production.
This accounts for large Nos of B cells & plasma cells.
However the regulatory function of T cells extends to Polyclonal B cell activation.
Hence in adult periodontitis whilst quantitatively B cell activity may predominate, the immunological changes are
underT cell control.
Indeed it is possible that the lesion develops as a result of
imbalance in T cell immunoregulation.