References

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.

Terms used

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

edentulous mouth.

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.

Habitat

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

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

viruses.

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

anaerobic environment.

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

periodontitis.

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

oxygen.

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.

LAYERS

Lower plaque layers: these are dense, microbes are bound

together in a polysaccharide matrix with other organic and

inorganic materials.

Loose layer: on top of the lower layer. Appears that is often

irregular in appearance; it can extend into the surrounding

medium.

Fluid layer: bordering the biofilm has a rather stationary

sublayer and a fluid layer in motion.

Nutrient components penetrate this fluid medium by

molecular diffusion.

The intercellular matrix consists or organic and

inorganic materials derived from saliva, gingival

crevicular fluid, and bacterial products.

INTERCELLULAR MATRIX:

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

serum transudate).

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

surface.

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

surface.

This pellicle consists of;

Glycoproteins (mucins)

Proline-rich proteins

Phosphoproteins (statherin)

Histidine-rich proteins

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

hydrogenbonding).

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

Ultrastructural Aspects:

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

observed.

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

resistance’)

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

bacteria.

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

flora”

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.

COMPLICATING FACTORS

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

PERIODONTAL HEALTH

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

genera.

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.

GINGIVITIS

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

growth factors.

CHRONIC PERIODONTITIS

Cultivation of plaque microorganisms from sites of

chronic periodontitis reveals high percentages of

anaerobic (90%) and gram negative (75%) bacterial

species.

Certain gram-negative bacteria with pronounced

virulence properties have been strongly implicated as

etiologic agents (e.g. P. gingivalis and Tannerella

forsythensis).

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

anaerobic rods.

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

periodontitis.

The predominant subgingival bacteria in patients

with generalized aggressive periodontitis are P. gingivalis,

T. forsythensis A. actinomycetemcomitans, and

Campylobacter species.

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

been reported

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.

PERIODONTAL ABSCESSES:

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.

forsythensis (47.1%).

Enteric bacteria, coagulase-negative staphylococci, and

Candida albicans have also been detected.

PARVOBACTERIA

Actinobacilli

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.

ACTINOBACILLUS ACTINOMYCETEMCOMITANS

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

internal structure)

This species was first recognized as a possible

periodontal pathogen in lesions of localized juvenile

periodontitis.

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

agar.

A israeliiA israelii

PORPHYROMONAS GINGIVALIS

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

P gingivalis

TANNERELLA FORSYTHIA

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

lesions.

This species has been shown to produce trypsin like

proteolytic activity and induce apoptotic cell death

SPIROCHETES

These are Gram negative, anaerobic, helical shaped, highly

motile microorganisms that are common in many

periodontal pockets.

Clearly, a spirochete has been implicated as the

likely etiologic agent of acute necrotizing ulcerative

gingivitis

The organism has been considered as possible

periodontal pathogens since the late 1800s and in the

1980s.

Difficulty in distinguishing individual species. At

least 15 species of subgingival spirochetes have been

described.

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

periodontitis patients.

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

periodontitis

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

in vitro

Strains of P. intermedia that show identical

phenotypic traits have been separated into two species, P.

intermedia and P. nigrescens.

FUSOBACTERIUM NUCLEATUM:

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

total isolates.

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

progression.

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.

OTHER SPECIES

Obviously all periodontal pathogens have not yet been

identified.

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

periodontal therapy.

Emphasis have been placed on enteric organisms,

staphylococcal species as well as other unusual mouth

inhabitants.

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.

VIRUSES

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

microbiota

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

disease

FUNGI

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.

MIXED INFECTIONS:

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

periodontal diseases

MICROBIAL DIAGNOSTIC TESTING

IMMUNOLOGY

Local immunopathology

Histopathological & ultra-structural changes in

experimental gingivitis & periodontal diseases

have suggested 4 stages of development:

Initial Early Established Advanced lesion

Initial 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.

Local immunopathology

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

forms

activates

generate

induce

Early lesion

Develops within 4 – 7 days.

Dense lymphoid cell infiltration.

Often seen in normal gingiva – when plaque control is

not practiced efficiently.

Local immunopathology

Lymphocytes constitute 75%.

Only few plasma cells.

Most of them – T-cell series with small No of B-cells.

Some macrophages

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.

Systemic immunity

Lymphoid cells – seeded into gingival focus of inflm

Leukocyte migration inhibition

Localization of leucocytes & proliferation of lymphocytes

releases

augment

Established lesion

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.

Local immunopathology

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

pathological pocket.

Systemic immunity

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.

Advanced lesion

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.

Local immunopathology

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 &macrophages.

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.

Systemic immunity

Role of macrophages

Antigen processing.

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.