PMS COLLEGE OF DENTAL SCIENCE AND RESEARCH

Golden Hills, Venkode PO, Vattapara

Thiruvananthapuram, Kerala, India - 695028

DEPARTMENT OF PEDODONTICS & PREVENTIVE DENTISTRY

SEMINAR

MICROFLORA OF THE ORAL CAVITY

Presented By:
Arjun sreenivas
1sy Year PG Student
Presented On:

Signature of Guide:

Signature of HOD:

CONTENTS
SECTION 1: INTRODUCTION

The human microflora

 The oral microflora in health and disease
 Microbial ecology
 The oral microflora and general health

SECTION 2: THE MOUTH AS A MICROBIAL HABITAT

The mouth as a microbial habitat

 Mucosal surfaces
 Teeth
 Saliva
 Gingival crevicular fluid

Factors affecting the growth of microorganisms in the oral cavity

SECTION 3:THE RESIDENT ORAL MICROFLORA

Principles of microbial classification

 Principles of conventional microbial identification

 Gram positive cocci
 Gram positive rods and filaments
 Gram negative cocci
 Gram negative rods
 Fungi
 Mycoplasma
 Viruses
 Protozoa

SECTION 4: ACQUISITION, ADHERENCE, DISTRIBUTION AND METABOLISM

OF THE ORAL MICROFLORA

 Acquisition of the resident oral microflora

  Ageing and the oral microflora
 Distribution of the resident oral microflora
Functions of the climax community: colonization resistance

SECTION 1: INTRODUCTION
Mouth is gateway of the body to the external world, represents one of the most
biologically complex and significant sites in the body. Disease that is localized
elsewhere in the body can be reflected in the mouth. As a result, saliva is becoming
increasingly recognized as a key diagnostic fluid

The human microflora

It has been estimated that the human body is made up of over 1014 cells of which only
around 10% are mammalian. The remainder are the microorganisms that comprise the
resident microflora of the host. The resident microflora does not have merely a
passive relationship with its host. This contributes directly and indirectly to the normal
development of the physiology, nutrition and defence systems of the organism.These
natural microfloras live in harmony with humans and both parties benefit from the
association. Loss of this resident microflora can lead to colonization by exogenous
(pathogenic) microorganisms, & predisposes sites to disease.

The oral microflora in healthand disease

The mouth is similar to other sites in the body in having a natural microflora with a
characteristic composition exist in a harmonious relationship with the host. More
commonly than elsewhere in the body, this relationship can break down in the mouth
and disease can occur.
This is usually associated with major changes to the biology of the mouth from
exogenous sources (eg: frequent intake of fermentable carbohydrates in the diet) or
from endogenous changes (eg: alterations in the integrity of the host defences
following drug therapy, which disturb the natural stability of the microflora). The
presence of microorganisms at sites not normally accessible to them.

eg; When oral bacteria enter the blood stream following tooth extraction or other
traumas and are disseminated to distant organs, they can cause abscesses or
endocarditis

Bacteria with the potential to cause disease in this way are termed ‘opportunistic
pathogens’, and many oral microorganisms have the capacity to behave in this
manner.

Most individuals suffer at some time in their life from localized episodes of disease in
the mouth caused by imbalances in the composition of their resident oral microflora.

The commonest clinical manifestations of such imbalances are dental caries and
periodontal diseases

Microbial Ecology

Most diseases of the mouth have a polymicrobial (multiple species) etiology. The
ability of bacterial groups to cause disease depends on the outcome of interactions
between the microbes themselves, and between these microorganisms and the host.
The activity and behaviour of these microbes is intimately linked to other biological
systems in the mouth.
Thus, the composition and metabolism of bacteria at a site will be influenced by:
 the flow rate and properties of saliva
 the life-style of an individual (presenceof a tobacco habit, nature of the
diet, and exposure to medication)
 the integrity of the host defences.

Eg:, caries may occur not only because of the frequent intake of fermentable
carbohydrates in the diet, but also as a consequence of long-term medication for an
unrelated medical complaint, since a side-effect of such treatment can often be a
reduced saliva flow.
A common adverse effect of certain drugs is a reduction in the production of saliva,
which in turn reduces its protective properties.
Similarly, smoking tobacco can impair the functioning of the host defenses, leading to
a failure to control the growth of potentially pathogenic microorganisms.

Oral fungal infections arise following the wearing of dentures, suppression of the host
defenses, or antibiotic therapy that removes competing indigenous bacteria.

The oral microflora and general health

The significance of oral diseases is generally considered only in the context of the
health of the mouth, but evidence suggests that they can also have an impact on the
general health of an individual.

In periodontal diseases, for eg, large numbers of Gram negative bacteria accumulate
around the roots of the teeth, and produce virulence factors such as:

 lipopolysaccharide (LPS)
 cytotoxic metabolites
 immunoreactive molecules.

The host mounts an inflammatory response to the microbial ‘insult’, and

prostaglandins and pro-inflammatory cytokines are produced.

These bacterial and host factors can enter the blood stream due to the high vascularity
of the periodontium and affect distant sites in the body.

Inflammatory changes associated with periodontal microorganisms can predispose to

diabetes or affect glycaemic control. Oral microorganisms, including periodontal
pathogens, can enter the blood stream during transient bacteraemias.

Here they may play a role in the development and progression of atherosclerosis
increasing the risk for coronary heart disease.

The mouth may also affect general health by acting as a reservoir for opportunistic
pathogens.

Oral hygiene is poor among patients in intensive care, and dental plaque from these
patients contains large numbers of potential respiratory pathogens.
Aspiration of these pathogens associated with periodontal disease, into the lower
respiratory tract can increase the likelihood of serious lung infection, especially in
immunocompromised or elderly people.

Helicobacter pylori is also detected in dental plaque , and this organism is strongly
associated with chronic gastritis and peptic ulcers, and is a risk factor for gastric
cancer.

Helicobacter pylori is not a normal bacterial inhabitant of the mouth, and its presence
may be associated with gastro-oesophagal reflux.
Its intermittent persistence in the mouth is linked with the presence of deep periodontal
pockets, and this carriage may aid its transmission from person-to-person.
This pathogen may be retained in dental plaque by selective adherence to already
attached bacteria, namely Fusobacterium spp., by a process called coadherence or
coaggregation .

Section 2: The mouth as a microbial habitat

The properties of the mouth make it ecologically distinct from all other surfaces of the
body, and dictate the types of microbe able to persist, so that not all of the
microorganisms that enter the mouth are able to colonize. Moreover, distinct habitats
exist even within the mouth
each of which will support the growth of a characteristic microbial community
because of their particular biological features.

Habitats that provide obviously different ecological conditions include :

 mucosal surfaces (such as the lips, cheek, palate, and tongue)

 teeth
 The eruption of teeth provides a unique, hard non-shedding surface
which enables much larger masses of microorganisms (dental plaque) to
accumulate as biofilms;
 in addition, gingival crevicular fluid (GCF) is produced which can
provide additional nutrients for subgingival microorganisms.

The ecology of the mouth will change over time due to

 eruption or extraction of teeth

  insertion of orthodontic bands or dentures
 any dental treatment including scaling and
restorations..

Transient fluctuations in the stability of the oral ecosystem may be

induced by :
 frequency and type of food ingested
 variations in saliva flow (for example, certain medications
impair saliva flow)
 and courses of antibiotic therapy

Four features that help to make the oral cavity distinct from other areas of
the body are:
 specialized mucosal surfaces
 teeth
 saliva
 gingival crevicular fluid.

Mucosal surfaces

The mouth is similar to other ecosystems in the digestive tract in having mucosal
surfaces for microbial colonization. The microbial load is relatively low on such
surfaces due to desquamation. The oral cavity have specialized surfaces which
contribute to the diversity of the microflora at certain sites.
Papillary structure of the dorsum of the tongue provides refuge for many
microorganisms which would otherwise be removed by mastication and the flow of
saliva. Such sites on the tongue can also have a low redox potential , which enable
obligately anaerobic bacteria to grow. The tongue can act as a reservoir for some of the
Gram negative anaerobes that are involved in the aetiology of periodontal diseases ,
and are responsible for malodour .The mouth also contains keratinized as well as non-
keratinized stratified squamous epithelium which may influence the intra-oral
distribution of some microorganisms.
Teeth
 non-shedding surface enabling large masses of microbes to accumulate
(dental plaque biofilms)
 teeth have distinct surfaces for microbial colonization;
 each surface (e.g. fissures, smooth surfaces, approximal, gingival
crevice) will support a distinct microflora because of their intrinsic biological
properties.

Saliva
Plays a major role in maintaining the integrity of teeth, by clearing food, by buffering
the potentially damaging acids produced by dental plaque following the metabolism of
dietary carbohydrates.

Bicarbonate - major buffering system in saliva, but phosphates, peptides and proteins
are also involved.
Mean pH of saliva is between pH 6.75 and 7.25, although the pH and buffering
capacity will vary with the flow rate.
Within a mouth, the flow rate and the concentration of components such as proteins,
calcium and phosphate have circadian rhythms, with the slowest flow of saliva
occurring during sleep.
Thus, it is important to avoid consuming sugary foods or drinks before sleeping
because the protective functions of saliva are reduced.

The major organic constituents of saliva are proteins and glycoproteins, such as
mucin.

They influence the oral microflora by, adsorbing to the tooth surface to form a
conditioning film (, which determines which microorganisms are able to attach acting
as primary sources of nutrients (carbohydrates and proteins) for the resident microflora
aggregating exogenous microorganisms facilitating their clearance from the mouth by
swallowing inhibiting the growth of some exogenous microorganisms.

Other nitrogenous compounds provided by saliva include urea and numerous amino
acids.
Oral microorganisms require amino acids for growth but not all of these are present
free in saliva
They are obtained from salivary proteins and peptides by the action of microbial
proteases and peptidases.

The concentration of free carbohydrates is low in saliva and most oral bacteria
produce glycosidases to degrade the side-chains of host glycoproteins .

 Antimicrobial factors, including lysozyme, lactoferrin, and the

sialoperoxidase system, are present in saliva and play a key role in controlling
bacterial and fungal colonization of the mouth.

 Antibodies have been detected, with secretory IgA (sIgA) being the
predominant class of immunoglobulin

 IgG and IgM are also present but in lower concentrations.

 A range of peptides with antimicrobial activity, including histidine-rich

polypeptides (histatins), cystatins and defensins are also present in saliva.

 The properties of saliva are fundamental to the maintenance of a

healthy mouth; consequently, it is often referred to as the ‘defender of the oral
cavity’.

Gingival crevicular fluid (GCF)

Serum components can reach the mouth by the flow of a serum-like fluid through the
junctional epithelium of the gingivae. The flow of GCF is relatively slow at healthy
sites, but increases by 147% in gingivitis and by up to 30-fold in advanced periodontal
diseases, as part of the inflammatory response to the accumulation of plaque around
the gingival margin.

 GCF can influence the microbial ecology of the site in a number of

ways:
remove non-adherent microbial cells & also introduce components of the host
defences, especially IgG and neutrophils.
 an additional and novel source of nutrients for the resident
microorganisms

 Many bacteria from subgingival plaque are proteolytic and interact

synergistically to break down the host proteins and glycoproteins to provide
peptides, amino acids and carbohydrates for growth.
 Essential cofactors for growth, including haemin for black-pigmented
anaerobes, can also be obtained from the degradation of haeme-containing
molecules such as transferrin, haemopexin, and haemoglobin.

 The increased production of GCF during disease is associated with a

rise in the pH of the periodontal pocket.
 The mean pH during health is approximately 6.90 and this can rise
during inflammation in gingivitis and periodontal disease to between pH 7.25
and 7.75.

 Even such a small change in pH can alter the competitiveness of

individual bacteria, which can affect the proportions of bacteria
 ….. some of the putative periodontal pathogens are favoured by an
alkaline environment

 Also, the activity of some proteases associated with the virulence of

these opportunistic pathogens is enhanced at alkaline pH (pH 7.5–8.0).
 GCF contains components of the host defences which play an
important role in regulating the microflora of the gingival crevice in health and
disease.

 In contrast to saliva, IgG is the predominant immunoglobulin; IgM and

IgA are also present.

 GCF contains leukocytes, of which 95% are neutrophils, the remainder

being lymphocytes and monocytes.
 The neutrophils in GCF are viable and can phagocytose bacteria within
the crevice.
 A number of enzymes can be detected in GCF, including collagenase
and elastase, which are derived both from phagocytic host cells and
subgingival bacteria.
  These enzymes can degrade host tissues and thereby contribute to the
destructive processes associated with periodontal diseases.

Factors affecting the growth of microorganisms in oral cavity

1. Temperature
2. Redox potential/anaerobiosis
3. pH
4. Nutrients
5. Host defences
6. Host genetics
7. Antimicrobial agents and inhibitors

1. Temperature

This has an effect on bacterial metabolism, enzymes, and habitat. Parameters like pH,
ion activity, aggregation of macromolecules and the solubility of gases are dependent
in temperature.

Human mouth temperature is kept relatively constant (35-36OC), which provides stable
conditions suitable for the growth of a wide range of micro organisms.

Periodontal pockets with active disease has higher temperature (39OC), which –

(Carranza, 9th ed.)

a). Affect gene expression of Porphyromonas gingivalis

b). Down regulate: fimbriae and major proteases

c). Up regulate: superoxide dismutase (neutralizes toxic oxygen metabolite)

2. Redox potential/ anaerobiosis

Mouth has a ready access to atmospheric oxygen concentration which is nearly 20%.
Still most of the oral habitat is facultative anaerobe or obligate anaerobe and very few
are capnophilic and microaerophilic. This happens because its actually the degree of
oxidation and reduction at a site which governs the survival of these microorganisms.
This oxidation and reduction level is usually expressed as the Redox potential (Eh).
Oxygen is the only one of the many interacting components influencing the Eh of a
habitat and its inhibitory action is usually attributed to its ability to raise the redox
potential.

With plaque accumulation the Eh tends to decrease, making sites suitable for survival
and growth of a changing pattern of microorganisms.

Reduced Eh seen at

Periodontal pockets

Surface under plaque

Approximal areas

Variations on oxygen tension in oral cavity

 Anterior surface of tongue: 16.4%

 Posterior surface of tongue: 12.4%

 Buccal fold of upper and lower jaw: 0.3-0.4%

Initial redox potential of plaque is around +200mV which after 7 days decreases to
-141mV. Early colonizers of plaque utilize O2, produce CO2, while late colonizers
produce H2 and other reducing agents- more suitable for anaerobes.

3. pH

This in maintained by saliva around 6.75 – 7.25. Microorganisms require pH around

neutrality for growth, and are sensitive to extreme of acid or alkali. Many
microorganisms can stand small fluctuations of pH but might get inhibited when these
changes happen frequently and for longer times. For example frequent consumption of
sugar leads to growth of aciduric species, which hence forth disposes to dental caries.

On the other hand, in areas of inflammation the pH might raise, for example in
periodontal pockets the mean pH is found to be around 7.8. This leads to proliferation
Porphyromonas gingivalis and from being less than 1 % of microbial community starts
to predominate the culture. (fig. no.8)

4. Nutrients
Populations within the microbial community are dependent solely on the habitat for
the nutrients essential for their growth. These can be of two types

a) Endogenous nutrients

These are provided by the host. Main source is saliva. Gingival crevice acts as a minor
source. No single species has the full enzyme complement to metabolize these
nutrients totally. Individual organisms posses overlapping patterns of enzyme activity,
so that they co – operate to achieve complete degradation.

b) Exogenous (dietary) nutrients

Fermentable carbohydrates are the major influencing nutrients falling in this category.
It gets broken down into glucans and fructans which help in attachment and
extracellular nutrient storage compounds, respectively.

Dairy products (milk, cheese) also influence the ecology of the mouth. They cause
buffering via milk proteins and may cause decarboxylation of amino acids after
proteolysis, helping in prevention of caries. Milk proteins also adsorb on tooth surface
and reduce adhesion of resident microflora; they also sequester calcium phosphate and
enhance remineralization. Cheese has also been known to reduce salivary flow rate
and also reduces plaque pH changes.

Xylitol cannot be metabolised by oral bacteria, and also has said to have inhibitory
effect on growth of S. mutans.

5. Adherence and agglutination

Chewing and natural flow of saliva detaches microorganism not firmly attached to an
oral surface. Salivary components can aggregate certain bacteria which facilitates their
removal from mouth by swallowing. Microorganisms are lost faster in saliva than they
can multiply.

6. Antimicrobial agents and inhibitors

Toothpastes and mouthwashes regularly challenge the oral microflora. Sodium lauryl
sulphate, fluoride, metal ions, phenolic compounds and plant extracts are the major
antimicrobials in tooth pastes. Chlorhexidine, essential oils (thymol, menthol) are the
mojor antimicrobials in mouth washes.
Antibiotics given orally or systemically reach oral cavity via saliva or GCF and show
their antimicrobial effect.

7. Host defense

Depends on integrity of mucosa and enamel which prevents penetrance of

microorganism.

a) Non specific factors

Flow of saliva removes microorganism by swallowing. Lysozyme has potential to

hydrolyse peptidoglycan which confers rigidity to bacterial cell wall. Lactoferrin
causes iron sequestration and affects growth of microorganisms that require iron for
their growth. Apo – lactoferin is iron free lactoferin said to inhibit growth of S.mutans
under anaerobic conditions. The salivary peroxidase enzyme (sialoperoxidase) can
generate hypothiocynate at neutral pH or hypothiocyanous acid at low pH in presence
of H2O2, and both can inhibit glycolysis by plaque bacteria..Histidine – rich proteins are
capable of killing S.mutans and Candida albicans. It also regulates the levels of yeasts
in the oral cavity.

b) Specific factors

Components of the specific host defences (intraepithelial lymphocytes and Langerhans

cells. IgA and IgG) are found within the mucosa, where they act as a barrier to
penetrating antigens. Secretory IgA can agglutinate oral bacteria, modulate enzyme
activity, and inhibit the adherence of bacteria to buccal epithelium and to enamel.
Salivary antibodies have been detected with activity against a range of oral bacteria,
including streptococci.

The antimicrobial factors described above not necessarily act in isolation.

Combinations of specific and non specific host defence factors can function in
synergism for ex. Lysozyme and sIgA can react with salivary agglutinins and so be
presented directly to immobilize cells, another ex. sIgA and sialoperoxidase.

Despite all these mechanisms microorganism are still found in oral cavity which can
be attributed to

 Antigenic variation

Continuous but subtle changes in antigenic make up

  Antigenic masking

By capsules, slime- layers or by the adsorption of host macromolecules onto this cell
surface.

 Antigen sharing

Organisms have some antigens similar antigens to those of hosts or they adsorb host
molecules on this surface

 Inactivation of host defence factors

By production of specific protease

The resident oral microflora

Oral microflora is diverse because of varied habitats possible in oral cavity and its
associated structures. It consists of

 Fungi
 Candida
 Viruses
 CMV
 Coxsackie A2,4,5,6,8,9,10 and 16
 Hepatitis
 HIV
 Protozoa
 Trichomonas tenax
 Entamoeba gingivalis
 Mycoplasma
 M. salivarius
 M. pneumoniae
 M. hominis
The various bacteria present can be divided according to their morphology and
staining technique as -

o Gram positive cocci

 Streptococcus
 Enterococcus
 o Gram positive Rods and filament
 Actinomyces
 Eubacterium
 Lactobacillus
 Propionibacterium
o Gram negative cocci
 Neisseria
 Veillonella
o Gram negative rods
 Haemophillus
 Eikenella
 Capnocytophagia
 Actinobacillus
 Porphyromonas
 Fusobacterium

Streptococci (fig. no.8)

It was isolated by Clarke in 1924, but the real proof of its presence was proven by
artificially in 1960 by experiments. The term mutans explains its ability to change its
Coccal morphology to some other morphology (small rod shaped) frequently. It is
divided into eight serotypes”a-h” depending on serological specificity of carbohydrate
antigen located in cell wall. The common isolates from mouth are mutans and sobrinus
which can be found in more in fissures and posterior regions of oral cavity. It consists
of 4 groups of gram positive cocci, which can be divided as -

 mutans
S. mutans
S. sobrinus
S. rattus
s. cricetus
s. ferrus
s. macacae
s. downei
 salivarius
S.salivarius
S.vestibularis
 Milleri
S.constellatus
S.intermedius
 S.anginosus
 mitis
S. sanguis
S. gordonii
S. oralis
S. mitis
Metabolism of streptococcus mutans

The most important substrate for the involvement of s. mutans in the caries process is
the disaccharide sucrose. Different pathways by which s. mutans may dissimilate
sucrose are by conversion of sucrose to adhesive extracellular carbohydrate polymers by cell
bound and extracellular enzymes. The transport of sucrose into the interior is accompanied by
direct phosphorylation for utilization through the glycolytic pathway, leading to lactic acid
production and degradation of sucrose to free glucose and fructose by invertase. The
intermediary metabolites from sucrose enter the glycolytic cycle or may be utilized in
intracellular polymer synthesis in order to provide a reservoir for energy.
Most of the sucrose metabolized by s. mutans is utilized for its energy requirements and results
in the production of lactic acid. Sucrose, which does not enter the cell, may be used for the
extracellular synthesis of carbohydrate polymers. The ability of S. mutans to form adhesive
plaques could explain its specific dependence on sucrose rather than other dietary carbohydrates.
it must BE emphasized that S. mutan polymerize A GLUCOSE and the fructose moieties of
sucrose to synthesize glucans and fructans, which are two TYPES of extracellular polymers.
Glucans are more significant as they promote the accumulation of S. mutans on teeth. Two
homopolymers of glucans, namely dextran and mutan are synthesized by S mutan. Mutan is
an important constituent, of fibrillar plaque matrix and is less soluble and more resistant to
enzymatic attack than dextran. Fructans, on the other hand, unlike the mutan homopolymer of
glucan, are generally highly soluble and can be degraded by plaque bacteria, thus serving at
reservoir of fermentable sugars for oral bacteria. The enzyme responsible for the synthesis of
extracellular glucans and fructans are called glucosyl and fructosyltransferase respectively.

Lipoteichoic acid is another extracellular polymer that is found in cultures of s.

mutatis. These highly negatively charged compounds might contribute to the
adhesiveness of bacteria. In addition to this, S, mutans strains have an ability to store
intracellular glycogen amylopectin type polysaccharide, which provides a reservoir of
substrate and enable prolonged periods of increased metabolic activity .
Intracellular glycogen and extracellular polysaccharides serve as substrate
RESERVOIRS , Which the organism may uTILIZE metabolized carbohydrate are
depleted. In this fashion, both types of polysaccharides may play a role in the
survival of is and in their potential to prolong acid production via glycolysis well
beyond mealtime.

Actinomyces (fig. no.9)

It is commonly found at the approximal sites where caries are seen, common resident of
healthy gingival crevice. Its counts are said to increase during Gingivitis and root surface
caries. Actinomyces viscosus is said to have to have more numbers of Extracellular slime and
fructans, while Actinomyces israelli is often called as Opportunistic pathogen which causes
actinomycosis. These forms granules which contain these microbes and prevent them from

antimicrobial actions. Actinomyces georgiae has been recently isolated from healthy gingival
crevice , while a recent discovery, Actinomyces odontolyticuswhich produces characteristic
Red brown pigmentand found to be involved in earliest stages of enamel demineralization.

Lactobacilli (fig. no.10)

Though it is found to be < 1 % of total cultivable microflora. Its numbers increases more in
advanced caries lesion in enamel and root surface. It is said to prefer the highly acidic
environment created by already present s. mutans in lesion. Few species found are -

 Lactobacilli rhamnose , Lactobacilli casei which are frequently found in oral

cavity

 Lactobacilli uli commonly associated with periodontal pocket

Its presence is also important in determining the caries activity through lactobacilli count
test.

Acquisition of the resident oral microflora

The fetus in the womb is normally sterile. During delivery it comes into contact with
the normal microflora of the mother's uterus and vagina, and at birth with the micro-
organisms of the atmosphere and of the people in attendance. Despite the widespread
possibility] contamination, the mouth of the newborn baby is usually sterile. However,
from the first feeding onwards, the mouth is regularly inoculated with micro-
organisms and the process of the acquisition of the resident oral microflora begins.

Acquisition depends on the successive transmission of microorganisms to the site of

potential colonization. Initially in mouth, this is by passive contamination from
mother, from food, milk and water, and from saliva of individuals in close proximity to
the baby. Acquisition of microorganisms from the birth canal itself may be of only
limited significance as studies as shown that acquisition of Candida and lactobacilli
from vagina of mother is only transient.

Pioneer community and ecologic succession

The first microorganisms to colonize the oral cavity are termed as pioneer species,
which continue grow and colonize until environmental resistance is encountered. This
resistance can be physical (shedding of epithelial cells, chewing and flow of saliva) or
chemical (Eh, pH, antibacterial properties of saliva). S. salivarius is the predominant
organism at this stage.
Pioneer community can influence the pattern of microbial succession by altering above
mentioned physical or chemical factors. Eventually a stable situation is reached with a
high species diversity, this is called as climax community.

The oral cavity of the newborn contains only epithelial surfaces for colonization. The
pioneer population consists of mainly aerobic and facultative anaerobic species. S.
salivarius isolated from infants mouth as early as 18 hours of birth. Streptococci are
the dominant pioneer species in the oral cavity over first year of life. Lactobacilli may
be transiently present during this period. The diversity of the pioneer oral community
increases during the first few months of life.

One year old infant will show streptococcus, staphylococcus, neisseria, veillonella.
Actinomyces, lactobacillus, fusobactrium can be isolated from more than 50% of
infants of this age.

Allogenic and autogenic succession

In allogenic succession, factors of non microbial origin are responsible for an altered
pattern of community development. Mutans streptococci and S. sanguis only appear in
mouth once teeth have erupted.

The increase in number of obligate anaerobes once teeth are present is an example of
autogenic succession in which community development is influenced by microbial
factors. As aerobic organisms utilize oxygen, the Eh is lowered in plaque and this
creates condition suitable for colonization by strict anaerobes. Another example would
be the development of food chains whereby the metabolic end product on one
organism becomes primary nutrient source for a second.

Ageing and the oral microflora

Following the tooth eruption, isolation frequency of spirochetes and black pigmented
anaerobes increases. Both of these presence markedly increases during puberty which
could be explained hormones entering the gingival crevice and acting as nutrient
source. Prevotella intermedia is also said to be increased in second trimester of
pregnancy which is ascribed to the elevated serum levels of oestradiol and
progesterone which can satisfy naphthaquinone requirement of this organism. Oral
contraceptives also said to rise the titer of black pigmented anaerobes.

In adults, the resident oral flora microflora remains relatively stable and coexists in
reasonable harmony with the host, due to a dynamic balance being achieved from
numerous inter bacterial and host bacterial interactions.

Upto 70 year old healthy people lactobacilli and staphylococcus were found to be
present in saliva, and yeasts were isolated more often in people aged more than 80
years.

Social habits also perturb the balance of oral flora for example in cases with high
cariogenic diet.

Methods of determining the resident oral microflora

Sample taking

The microflora can vary in composition over relatively small distances. Therefore,
large plaque samples or a number of smaller samples from different sites but which are
pooled together are of little value because important site differences will be obscured.
Consequently, small samples from discrete sites are preferable, but the method of
sampling will depend on site to be studied.
The oral mucosa can be sampled by swabbing, direct impression techniques, or by
removing epithelial cells by scraping or scrubbing with a blunt instrument into a
container. Data can then be related to a fixed area or to an individual epithelial cell.
Saliva can be collected by expectoration into a sterile container; the saliva flow can be
at a normal resting rate (i.e. unstimulated) or it can be stimulated by chemical means
or by chewing. Although a greater volume is collected by stimulation, such samples
will also contain many more organisms that have been dislodged from oral surfaces.
Transport to laboratory
All samples need to be transported to the laboratory for processing as quickly as
possible. Specially designed transport fluids help to reduce the loss of viability of
some of the more delicate organisms during delivery to laboratory. Reduced transport
fluid is used , thus helping to preserve the obligate anaerobes.
Dispersion
Clumps and aggregates of bacteria must be dispersed efficiently (ideally to single
cells) if the specimen is to be diluted and counted accurately. Plaque poses a particular
problem in this respect because, by definition, it is a complex mixture of a range of
micro-organisms bound tenaciously to one another. It is now accepted that mere vortex
mixing of a sample is inadequate. Mild sonication produces the maximum number of
particles from a specimen but it exerts a selective effect by specifically damaging
spirochaetes and some other Gram-negative bacteria, particularly Fusobacterium
species. One of the most efficient methods, particularly for sub-gingival plaque, is to
vortex samples with small glass beads in a tube filled with carbon dioxide.

Microscopy
It can be done just after sample collection or after dispersion. It is used to check the
motility of the organisms along with its morphology. Fluorescence techniques can be
used at this stage.
Serial dilution
Is done to dilute the colonies in medi , and prepare appropriate number and amount of
organisms required for further steps.
Colony counts
is done on selective or nonselective agar plates under aerobic or anaerobic
environments depending on type of microorganisms to be studies..
Sub-culture and identification
Done by sugar fermentation tests, identifying fermentation products, enzyme profiles,
membrane lipids and via antigen antibody reactions.

Distribution of the resident oral micro flora

The populations making up the resident microbial community of the oral cavity are not
found with equal frequency throughout the mouth. Many species are associated with
specific oral surfaces. It has been emphasized that the mouth has two distinct types of
surface for colonization - hard tissues (teeth) and soft mucosal surfaces. These latter
tissues are not homogeneous; the palate contains keratinized surfaces while the cheek
is composed entirely of unkeratinized cells. In contrast, the tongue is a specialized
structure having a highly papillated surface covered with sensory cells such as taste
buds. When these factors are considered along with known differences in the local
environment at particular sites, it is not surprising that variations occur in the
microbial community at each of these habitats.
In the following sections the predominant microflora from several different sites in
the oral cavity will be compared. Little information is available concerning mucosal
surfaces; because of its association with disease, most attention has been focused on
the microbial composition of dental plaque.

 Lips
 skin
 Staphlyococci
 Micrococci
 Corynebacterium
 Mouth
 Gram negative species
 Similar to skin
 Palate
 Streptococci
 Actinomyces
 Lactobacilli
 Cheek
 Streptococci mitis
 Actinomyces viscosus
 Haemophillus
 Tongue
 Streptococci mitis
 Haemophillus
 Streptococci salivarius
Saliva
 108 micro-organisms per ml
 Streptococci mitis
 mutans Streptococci
 Haemophillus

Dental plaque
DEVELOPMENT OF DENTAL PALQUE

As soon as tooth surfaces are cleaned, salivary glycoproteins are adsorbed forming the
acquired pellicle. Coccal bacteria are adsorbed to pellicle coated enamel within two
hours of cleaning. These pioneer species include Neisseria and streptococci,
predominantly S. sanguis, S. oralis, and S. mitis. Obligate anaerobes are detected only
rarely at this stage and usually in low numbers. These pioneer populations multiply,
forming micro-colonies which become embedded in bacterial extracellular slimes and
polysaccharides together with additional layers of adsorbed salivary proteins and
glycoproteins .Growth rates of bacteria are fastest during this early period with
doubling times ranging from 1- 3 hours. As plaque develops into a bio film so the
metabolism of the pioneer species creates conditions suitable for colonization by
bacteria with more demanding atmospheric conditions.

If plaque is allowed to accumulate undisturbed then there is a shift in the proportions

of the bacteria within the bio film. After 7 days, streptococci remain the dominant
group of organisms but by 14 days, they constitute only around 15% of the cultivable
microflora, and anaerobic rods and filaments predominate.

STRUCTURE AND FUNCTION

The development of plaque in terms mass will continue until a critical size is reached.
Shear forces will then limit any further expansion. However structural development
and re- organization may take place continually. From 7 to 14 days, the bulk layers
forms which show less orientation but a higher morphological diversity.

The structure and development of fissure plaque appears to be different to that of

smooth surfaces. The microflora of fissures is less complex, being composed
predominantly of rods and cocci. Palisading and branching filaments are absent,
although an interbacterial matrix can be observed.
Three structurally different types of plaque have been observed in the gingival crevice.
Plaque from the gingival margin is similar to smooth surface plaque, while within the
crevice two types, and have been recognized. Many bacterial associations can be
observed in which cocci are arranged along the length of filamentous bacteria. Such
associations are described as “corn cob” and “test tube brush”.

It can be argued that example three with the caries active strain directly over the
enamel is potentially a more pathogenic situation than example one, where a layer of
caries inactive organism is acting as a barrier

Either sharp or gentle gradients over small distances will exist in plaque for many of
the parameters influencing microbial growth and survival.

MECHANISM OF PLAQUE FORMATION

The development of a biofilm such as dental plaque can be divided into five stages. As
bacterium approaches surface, a number of interaction takes place which determine its
attachment. The interactions are –

1. Van der Waal attractive forces, which operate over relatively long
(>50nm) separation distances.
2. At the 10 – 20 nm distances, the interplay of Van der Waal attractive
forces and electrostatic repulsion produces a weak area of attraction that result
in reversible adhesion.
3. At these and shorter distances, the adhesion can become irreversible
due to specific short range interaction between bacterial adhesions and host
ligands.
4. The co-aggregation of bacteria to already attached cells.
5. The multiplication of the attached organisms to produce confluent
growth and a biofilm.

Micro-organisms are negatively charged due to their cell surface, while acidic proteins
present in the acquired pellicle would produce a net negative charge. The Derjaguin
and Landau and the Verwey and Overbeek theory is used to describe the interaction
between an inert particle and a substratum.

This theory states that the total interactive energy, Vt, of two smooth particles is
determined solely by sum of the Van der Waal attractive energy (Va), and the usually
repulsive, electrostatic energy (Vr).
MICROBIAL INTERACTION

In a biofilm such as dental plaque, micro-organisms are in close proximity to one

another and interact as a consequence, these interactions can be –

 Beneficial (fig. no.11)

Enzyme complementation, food chains, co aggregation

 antagonist

Bacteriocins, hydrogen peroxide, organic acids, low Ph, nutrient competition

Competition of nutrients will be one of the primary ecological determinants in

dictating the prevalence of a particular species in dental plaque. Individual species of
oral bacteria posses different but overlapping patterns of enzyme activity, so that the
concerted action of several species is necessary for the complete degradation of host
molecules.

In the above diagram organism A is able to cleave the terminal sugar of the
oligosaccharide side chain, which enables organism B or D to cleave penultimate
residue. A consequence of these interactions involving enzyme complementation is
that different species avoid direct competition for individual nutrient, and hence are
able to co-exist. This type of interaction is an example of proto-cooperation or
mutualism, whereby there is benefit to all participants that are involved in the
interaction.

Antagonism is also a major contributing factor in determining the composition on

microbial ecosystems such as a dental plaque. For example Bacteriocins produced by
many micro-organisms, Production of hydrogen peroxide by the members of the S.
oralis, S. salivarius can also produce an inhibitory substance such as enocin.

MICROBIAL HOMEOSTASIS

In spite of its microbial diversity, the composition of dental plaque at any site is
characterized by a remarkable degree of stability or balance among the component
species. The ability to maintain community stability in a variable environment has
been termed microbial homeostasis. It results due to a balance of dynamic microbial
interactions, including both synergism (such as proto-cooperation and commensalism)
and antagonism. This has a negative feedback mechanism, whereby change in one or
more organisms results in a response by others to oppose or neutralize such a change.
Factors responsible for the breakdown of microbial homeostasis. These can be –

 immunological factors

sIgA - deficiency, neutrophils dysfunction, chemotherapy induced myelosuppresion,

infections(AIDS)

 non - immunological factors

Xerostomia, antibiotics, dietary carbohydrates, increased GCF flow, oral

contraceptives

Dental caries
No single microbe is said to satisfy Koch’s postulates for caries, periodontal diseases
or any other opportunistic oral infection. So these are modified for any opportunistic
infections as –

1. A microbe should be present in sufficient numbers to initiate

diseases.
2. The microbe should have access to the affected tissue.
3. The microbe should be in an environment that permits its
survival and multiplication
4. Other micro-organisms that inhibit the growth of the microbe
should be absent or, if present, should not significantly affect it.
5. The host must be susceptible to the microbe.

Evidence for caries as an infectious disease

Miller (1890) suggested the presence of micro-organism in his famous chemico-

parasitic theory. Clarke isolated an organism (which he called streptococcus mutans)
from a human caries lesion in 1924, proof for causative role of bacteria came only in
1950s and 60s following experiments with germ free animals.

Evidence for the transmissibility of caries came from the studies on hamsters. Caries
inactive animals had no caries even when fed a highly cariogenic diet. Further proof
came when streptococci, isolated from caries lesions in rodents, caused rampant decay
when inoculated into the oral cavity of previously caries inactive hamster.
Immunization of rodents or monkeys with whole cells of S.mutans and S. sobrinus
leads to a reduction in the number of caries lesions of these organisms in plaque and a
decrease in the number of caries compared with sham – immunized animals.

ENAMEL CARIES

The plaque microflora is diverse, and disease is not due to exogenous species, which
would be easy to identify, but to changes in the relative proportions of members of the
resident microflora.

 SMOOTH SURFACE CARIES

Early studies shows higher proportions of mutans streptococci on white spot lesion on
smooth surfaces compared to sound enamel. Subsequent studies showed 10 – 100 fold
higher presence of mutans streptococci. Actinomyces and other species of streptococci
also make significant concentration of acids form carbohydrates.

 APPROXIMAL SURFACE CARIES

Mutans streptococci and lactobacilli are found to be more prevalent at these sites.
Especially S. sobrinus is present only in caries active sites.

 FISSURE CARIES

Most caries prone site on teeth. These sites shows strongest correlation between
Mutans streptococci, an inverse relation between Mutans streptococci and S. sanguis is
frequently observed. Lactobacilli are thought to be more responsible than Mutans
streptococci. Studies of distribution of Mutans streptococci have isolated S. mutans
more commonly than S. sobrinus from occlusal surfaces, where as S. sobrinus was
found more frequently on molars than anterior teeth.

 RAMPANT CARIES

Large increase in Mutans streptococci and lactobacilli is seen. Others associated with
sound enamel such as S. sanguis, neisseria and gram (-) anaerobes are seen to decrease
during this period. Mutans streptococci is said to be associated with initiations of these
caries while lactobacilli may be involved with lesion progression.

 EARLY SUBSURFACE DEMINERALIZTION

Lactobacilli, S. mutans and A. Viscosus were detected more commonly at day 14 than
at day 1. Demineralization was common in the presence of S. mutans.

 LESION PROGRESSION

Detection of lactobacilli and A. Odontolyticus at incipient lesion would indicate a site

with high risk of cavitation. Negative co relation with S. oralis and A. Naisulindii was
also found.

 RECURRENT CARIES

Micro-organisms penetrate around the margins of poorly fitting restoration to cause

such type of caries. In case of enamel, Mutans streptococci and in case of affected
dentine lactobacilli are reported to be present.

ROOT CARIES

Gram (+) filamentous bacteria, A. Naeslundii and A. Viscosus is believed to be the

major culprits in this type of caries. Other studies also demonstrated presence of
propionibaterium, bifidobacterium, lactobacilli, rothia in root caries.

BACTERIAL INVASION OF DENTINE AND ROOT CANALS

Dentine can be invaded by

a. Direct progression of an enamel caries lesion

b. From caries of the root surface
c. From the periodontal pocket via lateral or accessory canals
d. As a result of fracture of trauma during operative procedures.

Advancing front of a dentinal lesion is diverse and contains Actinomyces,

bifidobacterium, lactobacilli, and rothia. In vitro experiments have shown that Mutans
streptococci, A. Naeslundii, C. Gingivalis have the potential to invade dentinal tubules.
Porphyromonas endodontalis is found almost exclusively in infected root canals. P.
Gingivalis has also been isolated from endodontal abscesses.

PATOGENIC DETERMINANTS IN Mutans streptococci

 Sugar transport – happens even at low Ph, hence is can take place in
wide range of conditions
  Acid production – glycolytic pathway rapidly produces low terminal Ph
values in plaque
 Aciduricity – survive, metabolize and multiply in low Ph
 Extracellular polysaccharide production – contributes to the plaque
matrix. Consolidates attachment of cells, and may localize acidic fermentation
products.
 Intracellular polysaccharide production – utilization allows acid
production to continue in the absence of dietary sugars.

Periodontal diseases
Evidence of involvement of micro-organisms in periodontal diseases came from the
Gnotobiotic animal studies, Germ free mouths which rarely had periodontal diseases,
and Antibiotics which were found to lessen periodontal diseases.

Predominating micro-organisms commonly found in periopathologies are

 Obligate anaerobes

 Capnophilic gram negative rods

 Filaments, spiral shaped bacteria

Chronic marginal gingivitis

It is a Non specific inflammatory response, in which Microflora and plaque mass

increases 20 times the normal, with Streptococci as initial pathogen which is later
overcome by Actinomyces as the pathology progresses. With bleeding, black
pigmented anaerobes start appearing. Organisms usually found are Prevotella
intermedia, campylobacter, streptococci sanguis, wolinella, Peptostreptococci
anaerobius, Eubacteria.

Acute herpetic gingivitis(fig. no.12)

HSV – 1 is usually the culprit for the oral lesions. It presents as Ulcerated swelling of
gingiva which is often painful. Cytological smears and cytopathic effects confirms the
diagnosis by correctly identifying the etiologic pathogen.
Acute necrotizing ulcerative gingivitis(fig. no.13)

Spirochete and fusiform bacilli are the prominent organisms. Early invading micro-
organisms are large and medium size spirochete. Metronidazole is the drug of choice
for fusospirochetal complex. Usual pathogens are Prevotella intermedia, Actinomyces
viscosus, Capnocytophagia, Porphyromonas gingivalis, Streptococci sanguis.

Juvenile Periodontitis (fig. no.14)

It is said to be a Familial disease, with PMN dysfunction. Gram negative capnophilic

rods are mainly seen with “B” serotype of A. actinomycetemcomitans being the most
common. Leucotoxin production by this micro-organism is considered to be the most
important in pathology. Tetracycline (not Metronidazole) is the drug of choice.

Periodontitis in Children

If seen in Primary dentition it is called as prepubertal periodontitis, it has two forms -

Localized form - < 5 years, mild gingivitis

Generalized form – with tooth eruption, severe gingivitis

This is often associated with recurrent infectionswith abnormalities seen in PMN,

monocytes functions. Common pathogens are- Actinomycetemcomitans, Prevotella
intermedia, Capnocytophagia, e. Corrodens.

The role of oral bacteria in other infections

INFECTIVE ENDOCARDITIS/BACTREAMIA

Tooth tissue interface present in oral cavity makes it easy to bacteria to enter the blood
stream after minor treatments like Scaling, extraction, periodontal surgery. Few heart
conditions predispose to this infection –

 Previous episodes of infective endocarditis

 Prosthetic heart valve

 Mitral insufficiency

 Valvular stenosis

 VSD
  PDA

Due to these conditions the change in blood floe occurs and it becomes turbulent,
which leads to “eddy” formation. This eddy leads to thrombi or vegetation formation
which easily gets infected once bacteraemia occurs after minor oral treatment
procedures. This disease has got 30% mortality rate.

It’s not correct to call this as SUBACUTE BACTERIAL ENDOCADRITIS as its not
an sub acute condition, and not always caused by bacteria. Commonly seen pathogens
are Oral streptococci, Enterococci, Staphylococcus aureus, Actinomyces, Coxiella
burnetti, Candida.

Recently S. oralis has also been found to be a causative factor which gets attached to
thrombi through polypeptide in its cell wall which acts as adhesins .

CANDIDOSIS

Candida spp is the pathogen. It’s carriage rate is 40-60%. Most of these are found on
Dorsum of tongue, with species mainly find are - Candida albicans, Candida tropicalis,
Candida glabrata.

Calling this disease as candidiasis is wrong as the suffix “iasis” means a parasitic
infection and since Candida is normal pathogen so it cannot be candidiasis.

It can be classifies as

 Acute

 Pseudomebranous candidosis (fig. no.15)

 Atrophic candidosis

 Chronic

 Atrophic candidosis

 Hyperplastic candidosis

 Mucocutaenous candidosis

 Miscellaneous
Treatment of this is usually topical nystatin, and for severe systemic infections
systemic miconazole can also be prescribed.

Cross infection control

Before the AIDS epidemic in 1980s, kitchen cleanliness was considered more than
appropriate for dental operatory. But after 1980 strict infection control protocols
started getting applied in this field as well.

EXOGENOUS ENDOGENOUS

Atmosphere Saliva
Dust/dirt Blood
Water GCF
Instruments Skin
Materials/drugs Faeces
Cross infection differs from opportunistic infections in that its causative agents are
exogenous in origin while for opportunistic infections endogenous sources are often
blamed. E.g.

There can be four routes of transmission possible in a dental operatory –

1. Contact – herpes, staphylococci

2. Ingestion – hepatitis A

3. Inhalation – mycobacterium, influenza

4. Inoculation – hepatitis B, HIV

There are few vaccinations which are usually advised for a dentist and its supporting
staff like –

 Hepatitis B - 3 doses given intramuscular at 0, 1 month, 6

month time, with booster dose every 5 years

 Tuberculosis – intramuscular with booster dose every 5 years

  Poliomyelitis – intra oral given at birth, with booster dose every
5 years

 Tetanus – first dose given at 6 weeks, with booster dose every 5

years

 Rubella – only indicated for pregnant ladies with booster dose

every 15 years.

Micro-organisms can also be classified according to the risk of transmission - (Fariba

S, 1996)

Classification microorganism Level of risk Infection control

I Measles, mumps No risk if vaccinated Immunization

II Gonorrhea, syphilis Small risk Universal precautions

III Varicella zoster, EBV, Some risk Universal precautions

CMV
IV Hepatitis B, C, D, HIV High risk Universal precautions

V tuberculosis High risk Respiratory precautions, isolation

Sucrose Metabolism of Streptococcus mutans (- nikiforuk)

The most important substrate for the involvement of S. mutans caries process is the
disaccharide sucrose. Sucrose not only serves as a primary energy source but it also
permits the initiation of additional biochemical events which are responsible for the
cariogenic potential of this organism. The three pathways involved are:

1. The conversion of sucrose to adhesive extracellular carbohydrate

polymers by cell bound and extracellular enzymes.
2. The transport of sucrose into the cell interior accompanied or followed
by direct phosphorylation for energy utilization through the glycolytic pathway
leading to lactic acid production.
 3. The degradation of sucrose to free glucose and fructose by invertase.
The intermediary metabolites from sucrose enter the glycolytic cycle or may be
utilized in intracellular polymer synthesis in order to provide a reservoir for
energy.
Synthesis of Carbohydrate Polymers

Most of the sucrose metabolized by S. mutans is utilized for its energy requirement
and results in the production of lactic acid However, the sucrose which does not enter
the cell may be used for the extracellular synthesis of carbohydrate polymers. Several
investigators [reviewed by Gibbons and van Houte, 1975] observed that in the
presence of sucrose, s. mutans formed adhesive colonies which adhered to the surfaces
of culture flasks or to hard objects such as a tooth suspended in the culture medium.
The ability of S.mutans to form adhesive plaques could explain its specific dependence
on sucrose either than other dietary carbohydrates.

It is known that S. mutans can polymerize glucose and the fructose moieties of sucrose
to synthesize two types of extracellular polymers, glucans and fructans, respectively.
Of the two classes of polysaccharides, the glucans are of more importance in
promoting s. mutans accumulation on teeth. Two types of glucose homopolymers
(glucans) are formed by S. mutans: one Type is called dextran and contains
predominantly a (1-6) core linkages with lesser proportions of branches of a(l-2), a(1-
3), and a(l-4) linkages. A second type of glucan, called mutan, has a core a(l-3) linkage
with branches at a(1-4) and a(l-6) positions. Mutan is an important constituent of the
fibrillar plaque matrix and is less soluble and more resistant to enzymatic attack than is
dextran. The a(l-6) polymers (dextrans) tend to be more water-soluble and degradable
by enzymes produced by some other plaque bacteria than a(l-3) polymers (mutan).
These two classes of glucans, while tending to be rich in a(l-6) or a(l-3) linkages, also
contain mixtures of the two linkage types and thus present difficulties in precisely
defining the compounds chemically.

The major component of the polysaccharide known as fructan or levan is fructose.

Fructans, unlike mutans, are generally highly soluble and can be degraded by plaque
bacteria. Fructan therefore does not persist in plaque after its synthesis. It serves as
reservoir of fermentable sugars for oral bacteria.

Lipoteichoic acid is another extracellular polymer that is found in cultures of S.

mutans. This compound contains a glycolipid covalently linked with a glycerol
teichoic acid to which may be attached a carbohydrate moiety. These highly negatively
charged compounds may contribute to the adhesiveness of bacteria but experimental
evidence is incomplete.

Many s. mutans strain also produce an idiopathic intracellular polysaccharide which is

branched glyocogen – amylopectin like glucan with a(1-4) and a(1-6) linkages and
which is susceptible to amylase. Cells of gram-positive cocci and other bacteria in the
deep regions of the plaque often contain numerous intracellular polysaccharide
granules visible by electron microscopy.

Intracellular glycogen and the extracellular polysaccharides (fructans and glucans)

serve as substrate reservoirs which the organism may utilize for energy production as
the exogenous supplies of readily metabolized carbohydrate are depleted. In this
manner both types of polysaccharides may play a role in the survival of organisms and
in their potential to prolong acid production via glycolysis well beyond meal time.
Also, as has been previously noted extracellular polymer production by s. mutans is
limited at low sucrose concentrations in the growth medium, and under these
conditions most of the carbon catabolized is converted to lactic acid. Sucrose-adapted
S. mutans also possess significant levels of invertase activity which permits the
organism to metabolize the disaccharide via its hexose moieties.

Enzymes Involved in Synthesis of Glucans and Fructans

The enzymes responsible for the synthesis of extracellular glucans and fructans are
called glucosyl and fructosyltransferases, respectively. The enzymes are formed
constitutively (i.e. the presence of enzyme is independent of the presence of the
principal substrate, sucrose) and they act by transferring glucosyl or fructosyl moieties
from sucrose to primer molecules. The enzymes are highly specific for sucrose and
have a wide pH optimum of 5.2-7.0 and a low Km indicating a high affinity between
enzyme and substrate. No phosphorylated intermediates are involved. The energy
required is derived from the energy-rich disaccharide bond of sucrose, and this
explains why this sugar is the essential substrate. In the case of glucan synthesis, the
glucose is incorporated into the polymer and the fructose is a reactant product.
Accumulation of fructose inhibits this reaction. However, fructose is rapidly
transported into the cell and utilized by the cell for energy or synthesis of intracellular
polysaccharide. Separate glucosyltransferases are likely to be involved in the synthesis
of the a(l-3) and a(l-6)-linkages and it is probable that other transferases are involved
in the synthesis of branch point linkages.
Lactic Acid Production

All S. mutans strains studied have been demonstrated to be homolactic fermenters

converting over 90% of hexose to lactic acid [Brown, 1974], The production of lactic
acid from hexose by S. mutans proceeds strictly by the glycolytic pathway. At low
concentrations of sucrose, both the fructosyl and glucosyl moieties are converted to
lactic acid. Colonies, but not necessarily broth cultures of S. mutans, attain pH values
lower than those produced by other common streptococci. The low pH produced in
plaque, in the range of 4.2-5.7, creates conditions favoring de-mineralization of
adjacent enamel . When different bacteria from caries-associated plaques are grown on
5% sucrose-containing agar the colonies with the lowest terminal pH are
predominantly S. mutans.

Under slow growth conditions sugar transport by S. mutans is mediated by a

phosphoenolpyruvate-dependent phosphotransferase system

(PTS), specific for sucrose, as follows:

Sucrose + phosphoenolpyruvate GIVES sucrose-6-phosphate + pyruvate.

The phosphorylated sucrose is then cleaved by a sucrose-6-phosphate hydrolase (SPH)

that yields glucose 6-phosphate and fructose as follows:

Sucrose-6-phosphate GIVES gIucose-6-phosphate + fructose.

SPH has a high affinity for its substrate, sucrose-6-phosphate; therefore, low levels of
sucrose-6-phosphate can be rapidly hydrolyzed.

The synthesis of SPH is constitutively regulated (independent of substrate) whereas

PTS activity may be induced by growth of cells in a sucrose-containing medium. The
activities of both PTS and SPH are greatly repressed when fructose, but not glucose, is
used as a growth substrate [Martin and Wittenberger, 1979]. The sucrose PTS activity
of sucrose-adapted cells may be completely inhibited by raffinose and lactose [Slee
and Tanzer, 1979]. Low levels of fluoride have been shown to inhibit sugar
translocation in cell membranes thus reducing the rate of carbohydrate metabolism
[Hamilton, 1977].

Recent work indicates that S. mutans has at least one other sugar transport mechanism
operative under higher growth rate, more abundant sucrose, and lower pH conditions
than the PTS system. This system may operate somewhat like a pump, being linked to
the expulsion of protons from the cell interior [Hamilton and St. Martin, 1982]. This
ability to switch transport systems in response to substrate availability demonstrates S.
means' efficient adaptation in a sucrose-dependent environment.

Invertase Activity in Streptococcus mutans

It is known that sucrose-adapted S. mutans strains possess significant levels of

invertase activity [Tanzer et al., 1972]. This enzyme hydrolyzes sucrose intracellularly
to free glucose and fructose. Invertase in these organisms is subject to control by the
cell and is activated by inorganic phosphate. Since phosphate accumulation is coupled
to acid production it is probable that one of several mechanisms by which sucrose
degradation is regulated in S. mutans is the activation of invertase by inorganic
phosphate.

A detailed knowledge of the cellular control mechanisms that play a role in the
utilization of sucrose and other fermentable sugars by S. mutans is lacking.
Furthermore, a complete picture of the metabolic characteristics which account for the
high cariogenic potential of S. mutans is not available. However, some important
metabolic traits can be summarized as follows:

1. S. mutans has the metabolic potential to produce a low pH (acidogenic)

and to survive in a low pH environment (aciduric). The terminal pH of S.
mutans grown in glucose or sucrose-enriched media is well below pH 4.5.
Surface colonies of S. mutans also attain a lower pH than colonies of other
common plaque streptococci. Transport systems operative at low pH may
contribute to its relative aciduricity.
2. S. mutans is known to utilize sucrose at faster rates than other
organisms such as S. mitior, S. sanguis and A. viscosus.
3. S. mutans can synthesize complex mixtures of extracellular glucans and
fructans from sucrose, and the glucans permit cells of the organism to
accumulate on teeth.
4. S. mutans has the ability to store intracellular glycogen-amylopectin
type polysaccharides which provide it with a reservoir of substrate and enable
prolonged periods of increased metabolic activity.
5. The major fermentation product of S. mutans is lactic acid. At low
glucose concentrations formic and acetic acids and ethanol may also be
produced.
Principles influencing the development of the oral microflora
(S.Socransky,1971)

1. Variability
 Variability as related to host species
Microbiota of different species is qualitatively and quantitatively different.the minkey
has a microbiota generally similar to the human except they have much lower counts
of facultative “diphtheroids” and “veillonella”. The rice rat differs from the rat by the
presence of numerous Enterococci and actinobacilli.
 Variability within species
Quantitative differences in the types of the microorganisms can be demonstrated in
samples from the gingival crevice, dental plaque, and dorsum of the tongue and in
saliva of different individuals.
 Variability between sites in the same oral cavity
S. salivarius appears to reside primarily on the tongue; S. mutans appears to reside
primarily on the tooth of hamsters and humans, where as spirochetes and bacteroids
melaninogenicus are found in highest numbers in gingival crevice of the human oral
cavity.
 Variability within the same site in same individual
Repeated culturing of the same site within an individual’s mouth, reveals that a
marked variability exists in the microbial composition of that site on different
occasion.
2. Retention
Can be of two types
A. Adhesive retention
Gibbons and Nygaard divided the phenomena of adhesion into two types
i. Adhesion to the tooth or epithelial surfaces
ii. Adhesion of organisms to each other
There can be three mechanisms involved in interbacterial adhesion
I. Due to production of extracellular polymers by the bacteria –
e.g. dextran for s. mutans, hyaluronidase for a. naeslundii
II. Attachment involving the mediation of substances produced by
the host – e.g. agglutination in s. sanguis, a. naeslundii and a. viscosus
III. Attachment between the surface coating of bacteria of different
species
B. Non adhesive retention
Organisms unable to adhere to epithelial or tooth surfaces with any degree of avidity
may also be found in oral cavity. These organisms must be capable of being retained in
the mouth by some mechanisms other than adhesion. E.g. microorganisms may be
retained mechanically in the pits and fissures of teeth, in carious lesions, in the
gingival crevice or periodontal pocket. E.g. lactobacilli, spirochetes, yeasts.
3. Physiological consideration
A. Nutrition
Provide the growth requirements of microorganisms via host’s diet, secretions or
microorganisms living in same vicinity.
B. Diet
It affects both quantity and quality of microflora. E.g. sucrose affects S.mutans and
their density in plaque. Soft diet leads to more cariogenic micro-organisms
accumulation than hard diet.
C. Host
Saliva, GCF, and mammalian tissues can also lead to bacterial accumulation. B.
melaninogenicus requires heme and T. denticola requires alpha 2 globulin compounds
whose sole source in the oral cavity is mammalian tissues or secretion. L. arabinosus
was found to penetrate into dentine in seeking niacin which is found in the human
pulp.
D. Bacteria
Certain essential metabolites for some bacteria are supplied only by other bacteria like,
the requirement of B. melaninogenicus fro vitamin k can only be supplied by certain
other bacteria. T. microdentium could only exist in oral cavity because of presence of
isobutyrate and polyamines supplied by other bacteria.
E. Redox potential
Obligate anaerobes, microaerophiles, aerobes, capnophilic all kind of micro-organisms
are found in oral cavity due to variable redox potentials in oral cavity. It seems likely
that different balances are achieved between the reducing activities of the organisms
ans the oxidising capacities of environmental oxygen.

4. Transmissibility
Although all the factors governing the implantation of micro-organisms in the mouth
are not known, it is likely that the number of organisms introduced the frequency of
introduction and the nature of the specific conditions existing at the times of
introduction play a significant role. Frequently it is necessary for the host diet to be
conducive for the establishment of certain micro-organisms. E.g. for the implantation
of S.mutans sucrose is essential.
Implantation may also be depended on the nature of micro-organisms already present.
It appears to be more difficult to establish micro-organisms in the oral cavity of older
animals than younger ones.
Window of infectivity (Caufield, 1993 )
Caufield (1993) monitored oral cavity levels from birth up to 5 ye ars, He noted the
initial acquisition m ut ans S, and designated the time period AS 'window of infectivity.
A S the teeth (primary teeth) erupt into the oral cavity they provide a virgin habitat
which enables MS to colonize the oral cavity avoiding competition with other
indigenous bacteria. Thus in the window period in deci duous teeth the MS i s
established by 19-31(mean – 26 months) months of age und may have di ffi cul t y in
establishing later because i t would need to compete with other indigenous bacteria.

Krass et a l (1 9 6 7), Edrman et a l (1975) reported that at 2-o yr of age the child is less
susceptible to acquiring MS. The “Second Window of Infectivity” is present in
permanent dentition between 6-12 years of age (Klock and kroske 1977). 90% of

teenagers have MS colonization while others found only 3% of adults (mothers).

Few terms (Fejerskov)

Habitat - A specific area which supports a bacterial flora.
Fugitive habitat - A localized habitat which will support a population for periods of
time when it is eliminated from its main habitat
Population - A group of bacteria with common characteristics, defined at a given
taxonomic level of, eg. genus, species, sub-species (biovar, serotype, phage type etc.)
oranalysis of their DNA.
Community - A collection of more than one population of bacteria in a specific
habitat.
Climax community - A community of bacteria which has developed to be in balance
with the environment of its habitat.
Niche - The function of a bacterial population within a community.
Primary succession - Growth and changes in the bacterial populations in a habitat
when it is initially colonized by microorganisms

Secondary succession - Growth and re-development of a disrupted bacterial

community within a habitat
Allogenic - Describes effects on bacterial communities arising from changes external
to the community.
Autogenic - Describes effects on bacterial communities resulting from activities of the
community itself.
Ecosystem - Describes the habitat its environment and its bacterial flora.
Competition - Interaction between one or more bacterial populations having
characteristics that require them to occupy the same or similar niches in the
community.
Mutualism - Two populations benefit from an interaction that is essential to both.
Synergism - Two populations benefit from interactions that are not essential
Commensalism - One population benefits from another population that is unaffected
Neutralism - No interaction between two populations.
Amensalism - One population inhibits or eliminates another.

Ecological plaque hypothesis (P.Marsh, 1996)

Cariogenic bacteria may be found naturally in dental plaque, but at neutral pH, these organisms
are weakly competitive and are present only as a small proportion of the total plaque community.
In this situation, with a conventional diet, the processes of de- and remineralization are in
equilibrium. However, if the frequency of fermentable carbohydrate intake increases, then
plaque spends more time below the critical pH for enamel demineralization (approximately pH
5.5). The effect of this is two-fold. Conditions of low pH favor the proliferation of mutans
streptococci and lactobacilli, while tipping the balance towards demineralization. Greater
numbers of mutans streptococci and lactobacilli in plaque would produce more acid at even
faster rates, thereby enhancing demineralization still further. The rise in lactic acid production
would also select for the growth of Veillonella spp. Other bacteria could also make acid under
similar conditions, but at a slower rate. If these aciduric species were not present initially, then
the repeated conditions of low pH coupled with the inhibition of competing organisms might
increase the likelihood of colonization by mutans streptococci or lactobacilli. This sequence of
events would account for the lack of total specificity in the microbial aetiology of caries and
explain the pattern of bacterial succession observed in many clinical studies. This theory might
be termed the ecological plaque hypothesis.

Treponema denticola (Carranza, 9th ed)

Seen to be increased in aggressive periodontitis patients. It produces chymotrypsin like
protease can activate matrix matello protienases and hence can be considered as
etiological agent for periodontitis.
 Relationship between s mutans count of mother and child (berkowitz.
1981)
When mothers harbored greater than 105 cfu/ml of saliva – 58% child had caries
When mothers harbored greater than 103 cfu/ml of saliva – 6% child had caries

Probiotic (taber’s)
It is having favorable or health promoting effect on living cells and tissues. E.g.
lactobacillus inhibits growth of salmonella.

Lactobacilli count test

No. of organisms Degree of caries activity suggested
0- 1000 None
Less than 10.000 Slight
Less than 1.00.000 Moderate
Less than 1,000,000 Marked

Bacterial factors implicated in aetiology of periodontal disease (Marsh)

1. Attachment to host tissue - Surface components, e.g. 'adhesins' Surface
structures, e.g. fimbriae, fibrils
2. Multiplication at a susceptible site - Protease production to obtain nutrients
Development of food chains Inhibitor production, e.g.Bacteriocins
3. Evasion of host defences - Capsules and slimes PMN-receptor blockers
Leucotoxin Immunoglobin-specific proteases Complement-degrading proteases
Suppressor T cell induction
 Tissue damage (a) direct –
 Enzymes
 Trypsin-like' protease
 Collagenase
 Hyaluronidase
 Chondroitin sulphatase
Bone resorbing factors
 Lipoteichoic acid
 Lipo polysaccharide
  Capsule
Cytotoxins
 Butyric and propionic acids Indole Amines Ammonia
Volatile sulphur compounds
b) indirect
Inflammatory response to plaque
antigens Interleukin-1 production and
proteinase synthesis in response to plaque antigens

Nosocomial Infections (taber’s)

An infection acquired in a hospital, nursing home, or other health care setting. Patient in burn
unit and ICU has highest chances of getting it.
Enterobactor, pseudomonas, c. difficile and fungi are common causative pathogens.

Flora of root canals (Grossman, 11th ed)

The bacterial flora of the root canal has been studied over many years. Earlier
papers described a flora consisting predominantly of aerobic and facultative
anaerobic microorganisms. The differences in flora, as reported by different
investigators over the past 5 years, are the result of improved technology in sampling,
such as new anaerobic culturing techniques, new and improved culture media, and
more sophisticated methods of isolation and identification of microorganisms. One
obvious factor in the reports of changing flora is frequently overlooked; that is, the in-
terest of the investigator. If one seeks to isolate and identify microorganisms from an
environment substantially anaerobic, using anaerobic sampling techniques, and
culturing in media and an environment that encourages growth of anaerobic types of
microorganisms, it is not surprising to find a flora predominantly of anaerobes. The
same is true of aerobic sampling. Henrici and Hartzell found a predominance of
Streptococcus viridans (63%). followed by Staphylococcus albus (17%),
Staphylococcus aureus, Bacillus proteus. Streptococcus hemolyticus, and B. coli, all in
the pulps of pyorrhetic teeth.

Naidorf compiled a list of generalizations regarding organisms isolated from root

canal as follows –

1. Mixed infections are common than single-organism isolates.

 2. The wide variety of organisms found in root canals by different
investigators can be partially related to the principal interests and culture
techniques of these investigators.
3. The invasion of dentin from the pulp has been described, but the types
of organism, growth rate, and viability are poorly understood.
4. pulpal isolates are similar to oral flora, with gram-positive cocci
predominating.
5. Approximately 25% of the isolated organisms are anaerobes.
6. Organisms associated with flare-ups do not differ from asymptomatic-
canal isolates.
7. Organisms cultured from infected canals elaborate a variety of invasive
enzymes, but this capability cannot always be equated with pathogenicity.
8. The present practice of treating the obvious source of infection, the root
canal, and not the periapical tissue conforms to the findings of Hedman, as well
as those of Melville and Birch.

CONCLUSION

A good knowledge about oral microbiology will open a gateway leading to more
specific and practive therapeutic approaches in combating dental caries and
periodontal diseases.
We as dentist should know the microbiology of the diseases we are dealing with,
which helps us in understanding the pathogenesis of the disease. Microbiology also
helps us in various diagnostic and prognostic tools like caries activity test. It also
guides us in deciding the right antibiotic and other medicines to be prescribed along
with understanding that why infection control is required in our practises.

Reference

 Oral microbiology : Philip Marsh, Michael V. Martin; 3RD edition

 Carranza’s Clinical Periodontolgy – Michael G. Newman; 9th ed.

 Textbook of Pediatric Dentistry: Nikhil Marwah

 Essential Microbiology for Dentistry by L. Samaranayake