Oral microbiology/ rotary endodontic courses by indian dental academyDocument Transcript
AQUSITION OF ORAL FLORA
THE ORAL FLORA
MICROBIAL ECOSYSTEMS OF THE ORAL CAVITY
BENEFITS OF ORAL FLORA
FACTORS DETERMINING ULTIMATE COMPOSITION
MICROBIOLOGY OF DENTAL CARIES
MICROBIOLOGY OF PERIODONTAL DISEASE
MICROBIOLOGY OF ENDODONTIC INFECTION
STUDY OF MICROBES
You get up in the morning and brush your teeth with the best available tooth paste and
think you are clean. You rinse, you gargle a millions times, you floss religiously, use
mouth washes and get professional prophylaxis done and you think you are safe……but,
unfortunately, you are not. You can run and you can hide but you can never escape from
these tiny, yet amazing, inhabitants of your mouth…….. the oral micro flora.
A multitude of organisms are present in the mouth because of a wide range of sites with
different environmental characteristics.
Some organisms are almost always present in nos: > 1% and constitute the normal,
constant, indigenous flora. These organisms do not comprise the host’s survival
Eg: S. Sanguis, Neisseria
Some organisms are present in low numbers (<1%) and are called supplemental flora.
These become indigenous (i.e increase no:>1%) if the environment changes. A classic
example is that of lactobaccilus sp, which becomes indigenous only when plaque pH
drops and becomes acidic in a carious lesion. Thus, an environmental change has led to a
supplemental flora to become indigenous.
Certain microbes stay for a short time and comprise organisms that are just “passing
through” a host. These constitute the transient flora and may be those organisms present
in food/drink that temporarily get established in the mouth.
ACQUISITION OF NORMAL FLORA:
An infant’s mouth at birth is microbiologically sterile. Within a few hours of birth,
streptococci (especially S. Salivarius) establish themselves in the mouth. These come
from the mother or the environment. The streptococci alter the oral environment in such a
way that it makes it more hospitable for other microorganisms.
During infancy and early childhood other species of streptococci, staphylococci, neisseria
and veillonella occupy their habitats. Lactobacilli, actinomyces and fusobacteriumalso
colonize in most children. With the eruption of teeth and the availability of a solid
surface, the microbial flora undergoes dramatic changes.
S.mutans, S.sanguis and A.viscosus establish themselves on dental hard tissues.
Simultaneously, significant increase in the number of obligate anaerobes (strict O2
avoiding organisms) takes place. Due to the eruption of permanent teeth, gingival sulcus
depth also increases. With adolescence spirochaetes and bacteroides tend to appear in the
mouth. Adulthood brings in complex and varied organisms, which vary among different
people, at different times and at different sites.
The loss of normal teeth brings about another drastic change in microbial
flora, which reverts back to the pre-eruptive days.
Interestingly, use of artificial dentures yet again invites those bacteria, which are
fond of hard dental tissue. The number of fungi can also increase.
Bacteria are by for the most predominant type of microorganisms present in the human
oral cavity. These bacteria can be aerobic/ anaerobic depending on O2 requirement
bacteria ,Gram-positive / Gram negative according to the gram stain and bacilli / cocci /
treponemes / mycoplasmas according to shape.
Gram +ve bacteria
Cocci: Of all the bacteria present in the oral cavity Streptococci constitute the single
largest group. A large number of its species are encountered in the mouth including
S. faecalis S, Sanguis, S, mitis, S.mutans, S.milleri and S.salivarius. The other cocci
include peptostreptococcus, micrococcus and staphylococcus. The last one is a transient
member of the oral flora. S.mutans has been shown to be the most important organism in
the initiation of caries.
Bacilli: Lactobacilli are widely present in the human body and frequently found in the
oral cavity. 27 recognized species of lactobacilli are known with important ones being
L. acidophilus, L.salivarius and L.casei They are known to be involved in the progression
Other G + ve bacilli include actinomyces, Arachnia, Eubacterium, clostridium etc.
Gram -ve bacteria:
Cocci: Veillonella are the most numerous G -ve bacteria accounting for 10% of
cultivable salivary and tongue flora. Neisseria and moraxella are also seen.
Bacilli: Bacteroides, Fusobacterium, leptotrichia are the significant gram -ve bacilli
Bacteroid species and F. nucleatum are most commonly occurring bacilli in gingival
Treponemes: Spirochaetes such as T.denticola, T.orale, T.vincenti are common
inhabitants of gingival crevices and are often associated with periodontitis
Mycoplasma: These pleiomorphic organisms are regularly seen in plaque, calculus,
periodontal pockets etc. M. salivarium is the most predominant species while others like
M. pneumonione, M.orale, M. hominess have also been isolated.
Fungi: Candida albicans is the most common fungus isolated from the oral cavity & is
detected in high numbers in gingival flora, periodontal abscess, infected root canals etc.
Other fungi isolated are Pencillium, Aspergillus, Hemispora.
Protozoa: Are present in periodontal diseases Eg: Entamoeba
Virus: EBV, mumps virus, measles and influenza virus can be observed during the active
stage of the disease.
MICROBIAL ECOSYSTEMS IN THE ORAL CAVITY
The mouth provides a congenial environment for the organisms to grow and survive.
Variation in the different ecological niches causes differences in the number
(quantitative) and in the type (qualitative) of the organisms. These variations are due to
the complex interactions between the microbe and the host. The establishment, survival
and persistence of the flora in the oral cavity is determined by:
Microbial Factors Host Factors
- Adherence Anatomical
- Production of anti- microbial agents Saliva
- Sensitivity to anti microbial agents Crevicular fluid
- Metabolic capability Diet
- Nutritional requirement Oral hygiene
Oral and systemic disease
Oral immune defenses
Adherence: is a very important property for a bacteria to become a part of the oral flora.
If it cannot be retained and subsequently multiply, it will be eliminated from the oral
cavity by the washing action of the saliva. Adherence occurs reversibly initially via weak,
non-specific electrostatic attraction forces and later becomes permanent involving
specific bridging between host and normal flora. These 2 phases occur continuously. The
factors influencing adherence are
- Pilli and fimbriae
- Cell wall components
- Extra cellular polymers
Salivary factors like IgA lectins
Once adherence occurs via pilli or fimbriae, aggregation of homotypic and heterotypic
organisms occur via extra cellular polymers, IgA, lectins etc.
Antimicrobial Agents:Certain chemicals and biologically active substances are
produced by one microorganisms against another. For eg: Bacteriocins are produced by
certain organisms against other species. Certain metabolic end products like H2O2 can be
antagonistic to unrelated species. Thus, the vulnerability of an organism to sucuumb to
these antimicrobial factors can determine its survival in the oral cavity.
Metabolic Capability: Organisms, which have the metabolic machinery to utilize the
available nutrients and neutralize the deleterious chemicals produced in the oral cavity,
have the capacity to survive and propagate
Nutritional Requirements: Organisms use microbial sources such as intra cellular
storage granules or extra cellular metabolic end products and host sources such as
directory sucrose, salivary proteins and minerals and crevicular proteins for their growth.
Aquired Pellicle: Salivary glycoproteins form a layer on the tooth surface within minutes
of cleaning the tooth. The organism attach first to this pellicle and not directly to the
Salivary Factors: saliva with its composition of 99% water and 1% solids influences the
oral ecology in a number of ways:
Mechanical washing: prevents the overgrowth of microbes. Swallowing of saliva
causes ingestion of aggregates of microbes.
Pellicle formation: salivary Glyco proteins serve as nutrition and also promotes /
inhibits bacterial adhesion.
Inhibitory Action: - Lysozyme, lactoferrin, lactoperoxididase kills exogeneous
Buffering capacity: to maintain pH at 6.7 prevents overgrowth of microbes which
need a high or a low pH for growth.
IgA usually inhibits adherence and also alters microbial metabolism.
Anatomical Factors: Untreated malocclusion, irregular teeth and failing or poorly
contoured restorations can serve as stagnation sites for microbes to proliferate. Pits and
fissures serve as natural stagnation areas.
Crevicular Fluid: Is similar to serum in composition. It contains albumin,
immunoglobulins (IgG,IgA,IgM) and transferin which aids is the host defense. Apart
from electrolytes, it also contains enzymes such as proteinase, collagenase etc which
helps to combat microbes.
There are many other factors which significantly influence the ecology the oral cavity
⇒ A low redox potential supports the growth of anaerobic bacteria in the depth of
the dental plaque.
⇒ Administration of antibiotics/ local antiseptics depresses the number of some
species while allowing some other species to proliferate.
⇒ Smoking, tobacco chewing, poor oral hygiene, carbohydrate rich diet, systemic
diseases conditions can influence the oral micro flora in an unpredictable manner.
The transit of air through the mouth seems to preclude the possibility of any
anaerobic organisms thriving in it. However, culturing procedures revealed that 50-70%
of organisms cultured from the mouth were anaerobes. The factors determining the oral
a) Oxygen tension: is the measure of the amount of oxygen in a gas. For air it is 21%. On
the tongue it is 12-14%, in the buccal mucosa it is 1% and in a periodontal pocket it is 1-
2%. This indicates that the dento-gingival surface is mainly anaerobic especially where
sub-gingival plaques form. The supra-gingival plaque on the labial, lingual and occlusal
surface exhibited an O2 tension of 1-20%, thus producing an ideal niche for facultative
anaerobes and microaerophillic species.
b) Oxidation- reduction potential (Eh) = is the tendency for a medium or compound to
oxidize or reduce a molecule by the removal or addition of electrons. Microorganisms
that need a +ve Eh are termed aerobes and those needing a –ve Eh are anaerobes. A low
Eh would be expected in a microbial community which performs a fermentative
metabolism as fermentation results in formation of reduced end products. Thus, Eh
eventually becomes negative in a site where microbes accumulate. Portions of plaque
keeps changing from aerobic to anaerobic environment with corresponding drop in Eh.
This change coincides with a shift in the flora from a facultative microaerophillic to
c) Super-oxide radical and presence of super-oxide dismutase : The super oxide
radical is the most lethal form of O2 in biologic system as it causes both alteration is cell
membrane and inactivation of enzymes. Aerobes contain the enzyme super-oxide
dismutase which can destroy this super-oxide radical while anaerobes lack it. Hence,
aerobes predominate on tongue and supra-gingival plaque while anaerobes are in sulcus
The oral habitat can either be a non-tooth habitat or a tooth habitat
The non-tooth habitat includes:
Oral Mucosa (lip, cheeks, palate)
Dorsum of the tongue
Saliva and tonsillar areas
While the tooth habitats include:
Sub- gingival areas
Pit and fissure areas
Smooth surfaces – gingival to proximal contact and gingival 1
/3 of facial and
lingual surface of clinical crown.
The oral mucosa: harbors organisms that can overcome abrasive forces of food, tongue
and teeth for retention. Due to the washing effects of saliva, these organisms should be
able to reproduce in great numbers to ensure survival by reattachment.
The dorsum of the tongue: with its surface papilla provides additional shelter to
organisms. Streptococcus salivarius and Micrococcus mucilaginous are commonly found
here and rarely on teeth.
Saliva: has a wide variety of microbes as most of the microbes which get detached from
the tissue / tooth surface will appear in saliva. All species of streptococcus especially
S. oralis and S. salivarius are found in the saliva.
The tooth surface: is unique as it is not protected by surface shedding mechanisms,
which occur in other tissues. It is stable and gets covered by pellicle, which is ideal for
streptococcal attachment. Tooth habitats favorable for harboring pathogenic plaque
Pits and Fissures: provides excellent shelters for organisms especially S.Sanguis and
other streptococci. S. mutans can also be isolated at these sites even in the absence of
caries. Obligate anaerobes and gram –ve species are infrequently isolated or are absent.
Smooth surface: The proximal area immediately gingival to the contact area is protected
physically and is relatively free from the effects of mastication, tongue movements or
salivary flushing. The composition of the microflora varies and is complex but is
predominantly actinomyces and streptococci. Tooth topography (such as a rough surface
due to defective/ poor restoration), the size and shape of gingival papilla (apically
migrated papilla) and oral hygiene can predispose the tooth to caries or periodontal
Root surface: The proximal root surface near the CEJ is usually unaffected by flossing
due to roughness / fluting. This favors formation of mature, isolated cariogenic plaque.
Mainly, gram -ve obligate anaerobes and actinomyces are present here.
Sub-gingival areas: The initial occupants of the sub- gingival area are an extension of the
community from the adjacent tooth surface. Metabolites released from the plaque induce
a strong inflammatory response in the sulcus leading to vascular changes and release of
Ig, PMNL etc. This leads to a variation in the local environment by removal of some
species and introduction of newer ones. Thus, progressive changes from the cocci in the
supra-gingival plaque to filamentous bacteria and spirochetes in the sub-gingival plaque
is seen. Pathogenic Bacteroid melaninogenicus can exploit this habitat and cause
destruction of the gingival epithelium.
Factors determining the presence of different organisms in different anatomic sites:
Nutrient Availability: Micro organisms like B melaninogenicus, B. gingivalis and
capnocytophaga need hemin, certain bacteroids need vit. K, estradiol and progesterone
while T. denticola need spermin as nutrients. As these nutrients are present in serum and
not is saliva / food; these organisms take refuge in the gingival crevice where the
crevicular fluid sustains them. Thus, an organism’s nutritional requirement decides on
which niche they will occupy.
Inhibitory factors: Specific antibodies, lyzozymes, lactoperoxidase and lactoferrin,
which are present in saliva will inhibit bacteria from growing.
Microbial acid production which reduces pH, their reduced end products with reduces Eh,
production of H2O2 which oxidizes enzymes and presence of bacteriocins can prevent the
growth of certain species while promoting the growth for another.
Thus, interplay of these ecological determinants will decide which organism
will reside where.
BENEFITS OF THE ORAL MICROBIOTA
Production of vitamins and co-factors =oral bacteria like the intestinal flora produce
certain vitamins- the vit K, biotin and riboflavin- as well as co-factors required for our
normal systemic functioning.
Production of digestive enzymes= Small quantities of amylase, lipase and protease are
produced by the oral microflora.
Prevention of colonization of exogenous organisms to establish themselves and
produce disease= This may cause sensitivity to antimicrobial substances produced by the
It can be defined as a tenacious microbial deposit which forms on hard surfaces within
the mouth and consists of microbial cells and their products along with host compounds
mainly derived from saliva or crevicular exudates.
Composition of Plaque:
It mainly consists of proliferating micro organisms along with a scattering
of epithelial cells, leukocytes and macrophages in an adherent intercellular matrix.
Bacteria may be up 70-80% of this material. 1mm3
of plaque weighing 1mg
contains more than 108
bacteria. 200-400 different species of micro organisms can be
present in an extremely complex arrangement at one site alone. Other than bacteria,
mycoplasma, protozoa and fungi can also be present.
The material among the bacteria is termed “inter microbial matrix” and
accounts for 25% of the plaque volume. It consists of microbial substances, salivary
material and gingival exudates. The organic portion aids in adherence and co-
aggregation. These include poly saccharides, glyco proteins. The inorganic components
include calcium, P, K, Mg and Na which increase on calculus formation.
Types of plaque: According to site
Supra gingival: Fissure
Formation of plaque:
Stage I Acquired pellicle formation: It involves the adsorption of salivary proteins
to apatite surfaces via electrostatic ionic interactions
Stage 2: Transport and Adherence of pioneering organisms: The transition
between pellicle to plaque is rapid. The first constituents are cocci with
small numbers of epithelial cells and PMNL’s which initially adhere via
electrostatic interactions these long range interactions facilitate a reversible
adhesion. A little later specific short range interactions occur between
adhesins on microbial surface and receptors in the pellicle and results in
irreversible adhesions. S. mutans and S. Sanguis produce glycans in the
presence of sucrose which aids in adherence to pellicle and also promotes
adherence to other micro organisms
Within 8-10 hours, about 10,000 cells/ mm2
are deposited. The pioneer ent.
An extra-cellular matrix develops consisting of polysaccharides,
salivaryorganisms multiply to produce micro colonies which with time
become conflu glycoproteins.
Stage 3: Co-Aggregation (Within 1-3 days):
The metabolic products of the pioneering organisms alter the immediate
environment such as creating conditions with a low redox potential suitable
for anaerobes. Other organisms become incorporated into the plaque with a
resulting gradual increase in microbial complexity, biomass and thickness
Stage 4: Multiplication: The proliferation of the attached micro-organisms and
further aggregation produces a confluent growth and a biofilm.
Stage 5: Seeding: Detachment of cells form this bio film into the saliva results in
colonization of fresh sites.
Growth And Accumulation Of Supra-Gingival Plaque:
The total plaque mass that develops is mainly determined by the multiplication of
the attached bacteria as well as cohesion of bacterial cells. Thus, growth is by:
Factors which determine the ultimate composition and pathogencity of plaque are:
o Extra-cellular products: eg.glucans produced by S.mutans are sticky and
help in co- aggregation
o Bacterial interactions: are important for bacteria that cannot attach
directly to the tooth.Eg. Violonella which of incapable of direct
attachment accumulates on A.viscosis.
o Plaque ecology: formation and growth of plaque is an orderly sequence of
‘replacement community’ with each community modifying the local
environment of that site. This process of mutual change of community and
its environment is called ‘ecological succession’.
⇒ Oral cleansing mechanism such as salivary flow, movements of
tongue and cheek control the plaque formation rate.
⇒ Saliva influences
• The plaque pH by its buffering action and acid neutralization.
• Inhibition of adherence by coating the surface receptors
• Inhibition of adherence via promotion of bacterial
⇒ Immune response: Main sources of immune components in oral
• IgA and antibodies in saliva which compete with bacterial
adherence and influences their metabolism, growth and
• Crevicular fluid which contains antibodies, leukocytes,
complement factors etc.
Structure of plaque:
1) Bacterial cells near to the enamel surface tend to have a reduced cytoplasm:cell
wall ratio, indicating metabolic inactivity.
2) In some areas (esp. outer surface) cocci attach and grow on the surface of
filamentous microorganisms giving a ‘corn-cob’ appearance.
3) There is a tendency for filamentous bacteria to orient themselves at right angles to
the enamel surface producing a ‘palisade effect’.
4) Bacteria containing glycogen-like storage granules intra-cellularly are seen
indicating that these bacteria are relying on themselves for nutrition.
Sub gingival plaque: Supra-gingival plaque can induce inflammatory changes in the
gingiva, which leads to edematous enlargement of the gingiva. This leads to an increase
in the capacity of the sub-gingival area for bacterial colonization. Since this area is
protected from normal cleaning mechanisms, has increased crevicular fluid and has
desquamated epithelial cells, it favors a new ecological environment favoring anaerobic
bacteria. Thus, Gram +ve filaments, Gram –ve cocci, rods and spirochetes predominate
here. This flora is associated with root caries periodontitis, gingivitis etc.
MICROBIOLOGY OF DENTAL CARIES:
Over the years, there has been a debate whether one or more specific bacteria are
principally involved in the initiation of caries or if this disease is caused by a non –
specific mixture of bacteria.
Given the variations found in the microbial composition, it seems unlikely that the
initiation and progression of all carious lesions are associated with identical or even
However, there is evidence that some bacteria – S. mutans, Lactobacilli and actinomyces-
are more important than others.
S. mutans: The feature that supports its role as cariogenic organisms are its:
- Rapid generation time
- Acidogenic nature
- Production of extra cellular polysaccharides from sucrose which aids on
adherence and acts as a nutrient
- Isolated in high numbers in caries active mouth in incipient lesions
- Capacity to attain critical pH for enamel demineralization more rapidly than other
S.mutans is able to utilize dietary sucrose to enhance colonization. S. mutans can produce
glucan by using glucosyl transferase enzyme.It is this glucans which help in the
attachment of the bacteria to the tooth surface.
S. Mutans also produces dextran using enzyme dixtransucrase which helps in clumping
Once caries initiates, the local environment alters and other bacteria arrive and take up
the process of progression of the caries. Notable among these are the lactobacilli.
Lactobacilli for many years was believed to be the causative agent of dental caries as
- High numbers were obtained in most enamel caries
- Production of caries in germ-free rats
- Able to synthesis extra cellular and intra cellular polysaccharides form glucose.
- They produces lactic acid at pH < 5
Although these properties seemed valuable to a cariogenic organism, it was also seen that
their affinity for the tooth surface and their number prior to development of caries was
low. They were, in fact, absent from incipient lesions while present is significant numbers
in developed caries. Thus, they were categorized as secondary invaders which caused
progression of caries due to their acidogenic and aciduric properties.
Other Cariogenic micro organisms:
Actinomyces species: A.viscous, some other Gram +ve bacilli are involved in the
initiation of lesions on root surface.
Role played by a large number of other bacteria isolated from caries such as Arachnia,
Eubacterium, Rothia etc are not yet clearly known.
Veillonella: Is a species which is supposed to be antagonistic to the caries process. Its
protective role is contributed to its ability to decrease the acidity within the plaque.
MICRO BIOLOGY OF PERIODONTAL DISEASE:
PD disease occurs in all parts of the world and few individuals live out their natural life
span without getting affected.
The main PD diseases believed to have a microbial etiology are:
AUG(Acute Ulcerative Gingivitis)
Early onset -juvenile
- rapidly progressive
Acute herpetic stomatitis
The microbial composition during health is mainly Gram +ve cocci and few
In AUG, treponemes and bacteroide are present while in chronic gingivitis
actinomyces and bacteroides are present.
In LJP, 65% are Gram –ve especially A.actinomycetemcomitans while in chronic adult
periodontitis 75% are gram –ve with predominant species being spirochactes eg. B.
Periodontal abscess cultures reveal colonies of B. gingivalis, Fusobacterium and
Dental calculus: Are calcifying or calcified deposit on teeth which can be either supra-
gingival or sub-gingival. Calculus is preceded by dental plaque which subsequently gets
calcified. The plaque matrix gets calcified first followed b the bacteria themselves. The
degenerating bacteria can act as seeding agents for mineralisation.
The outer surface of calculus is covered by a thin viable dental plaque. It is
this plaque which has toxic and irritant potential rather than the calculus; of course,
calculus does cause mechanical trauma and its rough surface serves as a nidus for
continuous accumulation of plaque and keeps the latter in close proximity to the gingiva.
MICROBIOLOGY OF ENDODONTIC INFECTIONS:
A large number of bacteria and some fungi can cause infection in the dental pulp as well
as periapical tissues.
Dental pulp is a unique formative organ with limited capacity to withstand
bacterial, mechanical and chemical onslaughts. When bacteria enter the pulp it is unusual
for the host defenses to completely eliminate them. Healing is uncommon and necrosis
With pulpal degeneration, antigens collect within the root canal system and move to the
periapical tissues. Inflammatory or immunological responses ensue and can lead to
formation of abscess, granulomas or cysts.
Sources Of Infection:
- Infected carious lesion / iatrogenic exposure
- Via periodontal tissues through exposed dentinal tubules, lateral accessory canals
or apical foramen.
- By the lymphatic / hematogeneous route i.e anachoresis which is defined as the
localization of transient bacteria in the blood into an inflamed area, such as a
traumatized / inflamed pulp.
- Traumatic displacement / fracture can also become a route for microbial ingress to
Once the disease sets in, these tissues can act as source for spreading infection
to various organs of body through blood resulting in septicemia.
Disease occurring secondary to endodontic treatment include
- Infective endocarditis
- Cavernous sinus thrombosis
- Bacterial myocarditis
- Cerebral abscess
Various lesions result as sequelae to pulp necrosis such as granulomas, abscesses,
actinomycosis, cellulites, osteomylitis etc.
The organisms most commonly encountered in endodontic infections include –
• Bacteroides especially B.endodontalis and B. gingivalis.
• Streptococci mainly S.milleri
It is interesting to note that flare-ups during endodontic therapy are associated with
facultative Gram +ve cocci especially, S. salivarius, S. mutans and enterococci and no
anaerobes are present.
Dento-alveolar infections are associated with mainly streptococci, Bacteroids,
STUDY OF MICROBES:
- Direct examination under Microscopy using:
• Phase Contrast
• Gram Stain
• Acid fast stain
Antigenic structures study
Antibiotic sensitivity test
SUMMARY AND CONCLUSION:
“PREVENTION IS BETTER THAN CURE”
A thorough knowledge concerning the microorganisms is essential for not only providing
safe and aseptic means of treatment for the patient but also for keeping ourselves safe
from any communicable diseases. Some new organism is discovered almost everyday.
So, the next time you decide to treat a patient without a mouth mask or a pair of gloves,
remember that there is a microorganism just waiting round the corner to make you its