Microbiology of Oral Cavity: Resident Oral Flora & Streptococci

 Introduction & History
Classification
Resident oral flora
Bacterial changes from birth to adolescence
o Acquisition of normal flora
o Pioneer community and ecological succession
o Window of infectivity
o Climax community
CONTENTS

Benefits of oral microflora
Host bacteria interrelationships
Normal microbial flora at different sites of mouth.
Develepment of plaque and oral microbiota
Importance of biofilm
Plaque ecology
Localization of flora related to caries and periodontal
disease
Clinical implications
Factors that affect oral microflora
Oral microbial ecology

INTRODUCTION
The oral flora comprises a diverse group of organisms and
includes bacteria, fungi, mycoplasmas, protozoa and
possibly a virus flora.
Bacteria are the predominant group of organisms. The
diversity of oral flora is due to the fact that the mouth is
composed of varied habitats supplied with a range of
different nutrients.
MICROBIOLOGY : is the science of living organisms
that are only visible under the microscope .
Scientific development of microbiology was ushered by LOUIS
PASTEUR, perfection on microbiological studies by ROBERT
KOCH

Important contributions of Louis
Pasteur in microbiology
• Development of methods and techniques of bacteriology .
• Introduction of sterilisation techniques and development of steam steriliser , autoclave and hot air
oven
• LIVE VACCINE: He introduced live vaccine for prophylactic use. An accidental observation that
chicken cholera bacillus cultures left for several weeks lost their pathogenicity but retained their
ability to protect the birds against subsequent infection by them led to the concept of attenuation
and development of live vaccines .

Koch’s postulates
• He is also known as the father of bacteriology
• According to kochs postulates, a microorganism can be
accepted as the causative agent of an infectious disease
only if the following conditions are fulfilled :
• The organism should be constantly associated with the
lesions of the disease..
• It should be possible to isolate the organism in pure
culture from the lesions of the disease .
• The isolated organism when inoculated in suitable
laboratory animals should produce a similar disease.
• It should be possible to re isolate the organism in pure
culture from the lesion produced in experimental
animals

MICROFLORA
• Is the aggregate of microorganism that reside on
the surface and in deep layers of skin, in the
saliva and oral mucosa , in the conjuctiva, and
in the gastrointestinal tracts.
Oral microflora
 Oral microflora refers to the community of
microorganisms coexisting in the oral cavity
as its primary habitat

The first microbes observed :
Anton van leewenhoek ( 1632-
1723)
Developed the microscope and was
the first to discover oral bacterial
flora in the material that had lodged
in small amounts on the gums and on
the teeth …
He found few living animalcules

TYPES OF ORAL
FLORA
• Indigenous flora : it refers to the organisms present in
greater than 1 % of total viable count in a particular site
such as surface of tongue or supragingival plaque . They
are in a compatable relationship of the host do not
compromise the survival of host . The common indigenous
organisms present in the oral cavity are streptococcus,
actinomyces, and Neisseria .
• Supplement flora : it refers to the organism less than 1 %
in certain individuals . Most commonly include
lactobacillus. They are influenced by environmental
changes . In carious lesion ,as ph of plaque becomes acidic
,lactobacillus which is acid tolerant multiplies and becomes
the dominant microorganisms

• Transient flora : this flora comprises of
organisms that may be present in the oral cavity
for short period of time ,these organisms may be
temporarily established due to exogenous
factors like food or drinks and are not harmful
to the host . This flora may flourish and
becomes opportunistic in conditions where the
host is immunocompromised

Structure of gram positive and gram
negative bacterial cell wall

Classification of microbial flora
.
• 1.Depending upon staining
• Gram positive – Resist
decolourisation and retain the
primary stain appearing violet.
They have thicker cell wall
that prevents the
decolourisation by organic
acid.
• Gram Negative –
Decolorized by organic
solvents and take up the
counter stain appearing red.

Difference between cell
wall of gram positive
and gram negative
bacteria

Depending upon shape bacteria classified into several types
• Cocci these are oval or spherical cells. These cocci may
arranged in pairs (diplococcic) , chains ( streotococci), and
clusters (staphylococci)
• Bacilli these are rod shaped cells . Some of these bacilli
may be having peculiar arrangements or shape as follows:
• 1 coccobacilli : length of bacteria is approximately same as
its width e.g. brucella.
• 2 streptobacilli : these bacilli are arranged in chain e,g
streptobacilli
• 3 Chinese letter or cuneiform pattern : arranged at angles
to each other e.g. corynebacterium.
• 4 comma shaped : curved appearance e.g. vibrio
• 5 spirilla : rigid spiral forms e.g. spirillum

• Spirochaetes these are slender , flexous spiral
forms e.g. treponema
• Actinomycetes these are branching filamentous
bacteria resembling fungi.
• Mycoplasmas : these bacteria are cell wall
deficient and hence do not posses a stable shape

• 3.Depending upon influence of oxygen
on growth and viability.
• 1.Aerobes: Required O2 for growth.
• 2.Anaerobes:
• Obligate: Grows in the absence of O2.
• Facultative: Grow in the presence or absence of O2.
• 3.Microaerophilic

Main groups of microorganisms found in
the mouth
GRAM POSITIVE COCCI
STREPTOCOCCUS
Group Species
MUTANS GROUP
S. mutans, serotypes c,e,f
S. sobrinus, serotype d, g
S. cricetus, serotype a
S. rattus, serotype b
S. ferus
S. macacae
S. downei, serotype h
SALIVARIUS GROUP
S. salivarius
S. vestibularis
ANGINOSUS GROUP
S.constellatus
S.intermedius
S. anginosus
MITIS GROUP
S. sanguis
S. gordoni
S. parasanguis
S. oralis
S. mitis
S. crista
Strep. have been isolated from all sites in the
mouth and comprise a large proportion of
resident oral microflora.

• Alpha haemolytic : produces a greenish
discoloration around the colonies . This
is due to partial haemolysis . This zone
lysis is small with the presense of
unlysed erythrocytes . Alpha
haemolysis is seen in the viridian group
of streptococci and pneumococcus .
• Beta haemolytic : the streptococci
produces a clear colourless zone of
complete haemolysis around the
colonies erythrocytes are completely
lysed due to production of 2 types of
streptolysin by the organisms :
streptolysin o and streptolysin s e.g
streptococcus pyogens
• Gamma haemolytic : they produces
no haemolysis and streptococuss
faecalis

Human streptococcal pathogens
• S. Pyogenes
• S. viridians : it has six species groups s. mutans ,s.oralis, s.salivarus, s.sanguis,
s.milleri , s.mitis
• S. pneumonia
• S. faecalis : enterococci

Streptococci in general are gram positive cocci arranged
in chains of varying length.
Morphology:
Individual cocci are spherical or ovoid, 0.5 – 1 mm in
diameter and are arranged in chains.

General charecteristics of
streptococci
• Gram positive spherical / ovoid cocci arranged in long chains commonly in pairs .
• Non spore forming , non motile
• Facultative anaerobes
• Small non pigmented colonies
• Sensitive to drying , heat , and disinfectants

S.mutans group (Mutans Streptococci)
• S.mutans was originally isolated from carious human teeth by
Clarke (1924) and later recovered from a case of infective
endocarditis.
• Epidemiological studies have implicated S.mutans as the
primary pathogen in the etiology of enamel caries in children
and young adults and nursing caries in infants.
• Species of mutans strep. comprise the major microorganisms
implicated in the etiology of dental caries occurring on
smooth surfaces (Kristofersson et al. 1985).

• Characteristics of strep. Mutans: They are non-motile,
catalase negative, gram positive cocci in short or medium
chains.
• mitis – salivarius agar they grow as highly convex to
pulvinate (cushion shaped) colonies.

• When cultured with sucrose, they form
extracellular polysaccharides that are
insoluble.
• This property is regarded an important
characteristic contributing to its caries
induciveness.
• Strep. also ferment mannitol and usually
sorbitol.
S.mutans exhibits several important properties :
I. It synthesizes insoluble polysaccharides
II. Homofermentative lactic acid former
III. Colonizes on tooth surface
IV. More aciduric than other strep.

• Although these are not unique characteristics that can be
correlated to cariogenicity , cariogenic strains of S. mutans
contain lysogenic bacteriophage
• Noncariogenic mutants of S.mutans are unable to adhere to
tooth surface and have decreased ability to form insoluble
polysaccharide.
• If these are infected with lysogenic phages, they are
transformed, acquiring ability to adhere and form abundant
insoluble polysaccharide.

Biochemical composition of
S.mutans cell wall
S. mutans possesses:
• outer capsule of glucan/levan when grown in presence of
sucrose.
• cell wall polysaccharide composed of rhamnose, glucose and
galactose.
• cell wall also possesses peptidoglycan and glycerol teichoic
acid.
Many of these cell wall macromolecular components are also
present at the cell surface and are of potential importance in any
attempts at developing anti-S.mutans vaccines.

Ecology of S. Mutans
• Accumulated evidence of ecology of S. Mutans indicates
that this organism can survive in the mouth only when solid
surfaces such as teeth or dentures are present (Carlsson et
al, 1969).
• Unlike S. salivarius, whose preferred habitat is the tongue,
which establishes itself early in the mouth of the newborn
(Carlson et al, 1970).
• Although the favourite habitat of S. mutans is the tooth
surface, it does not uniformly colonize all tooth surfaces
but localizes on certain surfaces.

• S.mutans contains tightly and covalently bound polypeptide molecules that
may serve as factors in attachment of bacteria to smooth surfaces (Nesbitt
et al, 1980).

Physiology of S.mutans:
• Fermentation
• The S.mutans group, ferment many sugars.
• These are alpha- haemolytic strep. which ferment mannitol
and sorbitol
• Also, these organisms show quantitative differences in the
amount of acid produced from glucose.
• S.mutans accumulates more acid and causes a larger drop in
pH on solid media than do S.sanguis strains.

Sucrose metabolism
Dextran :
• Many oral strep. can synthesize carbohydrate polymers
from sucrose, this is done by utilizing cleavage of high
energy glycosidic bond as energy source.
• In S. mutans gp., glycosyltransferase predominates and
the major carbohydrate produced is a dextran (glucan).
• dextran produced by S.sanguis, the polymer produced by
S.mutans.

• This polymer much less soluble and so distinctly
different in properties from the usual dextran so that it
has been called a mutan.
• This mutan is relatively insoluble and its presence on
the bacterial surface causes it to become sticky so that
bacteria do not separate easily.
• This property is believed important in holding plaque
together (cohesion) than initiating attachment via
salivary pellicle.

SCREENING TESTS FOR MUTANS GROUP
OF STREPTOCOCCI
Plaque/toothpick method
Plaque samples are collected from gingival third of buccal tooth
surfaces (one from each quadrant) and placed in Ringer’s solution.
The plaque suspension is streaked across a mitis salivarius agar
plate.
After aerobic incubation at 37oC for 72 hours, the cultures are
examined under low power microscope and total colonies in 10
fields are recoded.
Grade Colonies/10 fields
1 None
2 < 8
3 >8

Saliva/tongue blade method
• The subjects are advised to chew paraffin wax for 1 minute to
displace plaque microorganisms,
• They are then given a sterile tongue blade, which they rotate in
their mouth ten times, so that both sides of the tongue blade are
thoroughly inoculated by the subjects’ flora.
• Excess saliva is removed by withdrawing the tongue blade
through closed lips.
• Both sides of the tongue blade are then pressed onto an MSB
agar which is incubated at 37oc for 48 hours.
. Counts of more than 100 cfu are proportional to greater than
1000000 CFU of S. mutans per ml of saliva.

S.Mutans adherence method
Unstimulated saliva (0.1ml) is inoculated in MSB broth.
Inoculated aerobically at 37oC for 24 hours.
After growth has been observed, the medium is removed and
cells adhering to glass surface are examined macroscopically and
scored as follows :
- (no growth expressed)
+ (few deposits : 1-10)
++ (scattered deposits of smaller size)
+++ (numerous minute deposits with >20 large size deposits)
When the adherence score is +++, S.mutans is present at a level
higher than 105 cfu/ml of whole saliva.

S.sobrinus
The next most commonly isolated sp. is
S.sobrinus, which has also been associated
with human dental caries.
However, there is less known about the role
of S.sobrinus in disease.
S.cricetus and S.rattus are mainly related to
dental caries in animals.
S. ferus does not related to dental caries.

Scardovia wiggsiae is a Gram-positive bacillus found in oral
cavities and thought to be a causative agent for early childhood
dental caries, also known as bottle rot.
This new pathogen, S. wiggsiae, has been discovered in children
who have tooth decay, but where S. mutans has been absent in their
oral culture.
S. wiggsiae is a non-motile, non-spore forming bacillus, and
measures 0.6–0.7 μm wide and 1.6–4 μm long.
The cells arrange singularly or in pairs, some even colonize in short
chains.
The shape of the cells can vary in response to fluctuating conditions
of the environment and can range from straight to slightly curved.
Cell color can differ from off-white, cream or gray
Scardovia wiggsiae

S. salivarius group
• This group comprises S.salivarius,
S.vestibularis and S.thermophilus
• S.salivarius preferably colonizes mucosal
surfaces, esp. tongue upper respiratory tract .
• Strains produce an extracellular fructan
(polymer of fructose with levan structure) and
this gives rise to characteristically large mucoid
colonies when grown on sucrose containing
agar.
• It is seen in humans just a few hours after after
the birth . The bacteria considered to be the
opportunistic pathogen rarely finding its way
into bloodstream ,it has been implicated in case
of sepsis

S.milleri (anginosus) group
This group is readily isolated from dental plaque and mucosal
surfaces, but are an important cause of purulent disease in humans
eg. abscesses of internal organs such as brain, liver,
appendicitis, peritonitis, endocarditis.
The unique characteristic of them from other pathogenic
streptococci is their ability to cause abscesses
These strep are potential opportunistic pathogens.

S.mitis (oralis) group
• The strains originally designated have been subdivided into
S.sanguis and S.gordoni.
• Both produce extracellular soluble and insoluble glucans
from sucrose. S.sanguis produces a protease that can cleave S
IgA, while S.gordoni can bind alpha - amylase enabling these
strains to break down starch.
• Amylase binding may also mask bacterial antigens and allow
the organism to avoid recognition by the host defences.

Enterococcus
• S.Faecalis
• It is a gram positive bacteria
• Identified by maconkey agar .colonies are magenta in
colour and pin point
• It can grow in the range of 10 to 45 degree celcius and
survive at temperature of 60 degree for 30 min
• It is non motile facultative anaerobic bacteria
• It ferment glucose They have been recovered in low
numbers from several oral sites.
• The most frequently isolated species is Enterococcus
buccalis. Enterococci can be isolated from the mouth of
immuno- and medically-compromised.
• Some strains can induce dental caries in while others
have been isolated from infected root canals and from
periodontal pockets.
• Aminoglycosides are drug of choice

Streptococcus pneumonia
• These are commonly seen in nasopharynx of healthy
persons . It will not cause any illness itself unless a
viral infection or other factors provoke
• These are gram positive cocci
• Measures 0.5 – 1.25 micrometre
• Non motile
• Small oval shaped cells arranged in pairs and short
chains

Streptococcus pyogenes
• Most serious streptococcus pathogen . It
inhabits throat, nasopharynx,
occasionally skin
• Gram positive
• Spherical /ovoid cocci arranged in long
chains.
• Nonmotile

Staphylococcus, Micrococcus
• Staphylococci & Micrococci are not commonly isolated in large
numbers from the oral cavity, although the former have been
reported in plaque samples from subjects with dentures,
immunocompromised patients, infections.
• Although these bacteria are not usually considered to be
members of resident oral microflora, they may be present
transiently and they have been isolated from some sites with
root surface caries, and periodontal pockets that fail to respond
to conventional therapy.

Gram positive rods and filaments
Gram positive rods and filaments are commonly isolated
from dental plaque.
ACTINOMYCES
• Major portion at approximal sites and gingival crevice
• Root surface caries, numbers increase in gingivitis
• Actinomyces is a good plaque former, forming tenacious
deposits on teeth.
• Short rods ,but often pleomorphic

• Actinomyces sp. found in the oral cavity include :
Facultative anaerobic
A. naeslundii A. israelii
A. viscosus A. meyeri
A. odontolyticus
• All sp. ferment glucose, producing mostly lactic acid, some
acetic, succinic and formic acid.

• A. georgiae is a facultatively anaerobic organism that is found
occasionally in the healthy gingival crevice.
• Other species include A. odontolyticus, of which about 50%
of strains form colonies with characteristic red brown
pigment.
• This species has been correlated with the earliest stages of
enamel demineralisation, and with progression of small caries
lesions.

• Some strains of A. naeslundii produce an extracellular slime and a
fructan from sucrose, some strains also produce a urease that may
modulate pH in plaque.
• Two types of fimbriae may be found on the cell surfaces of
A.naeslundii.
• Each type serves a specific function, and is implicated either in
cell to cell contact (co-aggregation) or cell to surface interactions
• A naeslundii predominates in tongue, salivary flora and
plaque of young children; while plaque from teenagers and
adults has higher proportion of A. viscosus (Ellen, 1976).

Eubacterium
• This is a poorly defined genus which contains a variety of
obligatory anaerobic
Asaccharolytic Eubacterium species can comprise over
50% of anaerobic microflora of periodontal pockets.

• Among these are E.brachy, E.timidum, E.nodatum and
E.saphenum, and are strongly implicated in advanced forms of
periodontal disease.
• Saccharolytic strains such as E.saburreum, E. yurii can also be
found in subgingival plaque in both health and disease.
• These organisms can be frequently isolated from
infections of the head, neck and lung, carious dentin and
necrotic pulp.

Lactobacillus
• Their proportions and
prevalence increase in
advanced caries lesions both
of the enamel and root
surface.
• A number of homo- and
hetero-fermentative sp. have
been identified, producing
either lactate or acetate from
glucose.

• Lactobacilli has been isolated from periodontal pockets.
• These Lactobacillus (LB) sp. are highly acidogenic organisms,
associated more with carious dentine and advancing front of carious
lesions.
The following LB are those most commonly encountered in the mouth
:
Homofermentative Heterofermentative
L.casei L.fermentum
L. acidophilus L.brevis
L-plantarum L.buchneri
L. salivarius L.cellobiosus

The most common sp. are:
L. casei, L. fermentum, L. acidophilus, L.salivarius, L.plantarum,
L.brevis, L.cellobiosus, L.buchneri. L.oris.
L.rhamnosus L.paracasei.

• The idea that LB played the major role in the carious process
dominated dental literature earlier.
• This was because they are both acidogenic and aciduric and could
multiply in low pH of plaque and caries.
• As more information on microbial composition of plaque became
available, it was found that LB constitute only a minor fraction of
the plaque flora (1/10,000) (Gibbons, 1964).
• Also, the amount of acid that can be formed by this small number
is insignificant in comparison with that produced by other
acidogenic oral organisms. (Strafours, 1950).

Propionibacterium
Several sp. of propionibacteria have been reported from
the mouth, including P.acnes in dental plaque. These
bacteria are obligatory anaerobic.
P.propionicus is morphologically indistinguishable from
A.israelii but can be differentiated by production of
propionic acid from glucose.

Gram negative cocci
Neisseria and Moraxella
• Neisseria are isolated in low numbers from most sites in the
oral cavity, and are among the earliest colonizers of a clean
tooth surface.
• The most common species is N.subflava, which is
saccharolytic and polysaccharide producing.

Other neisseriae are asaccharolytic and non-polysaccharide
forming.
Some strains metabolise lactic acid. Neisseria are rarely
associated with disease.

Veillonella
• These are strictly anaerobic gram negative cocci.
• 3 recognised sp. are V.parvula, V. dispar, V.atypica.
• Veilonellae have been isolated from most surfaces of the
oral cavity, although they occur in highest numbers in
dental plaque.

• Lactic acid is the strongest acid produced in quantity by oral
bacteria and therefore implicated in dissolution of enamel.
• Veillonella may reduce this harmful effect by metabolising
lactic acid and converting it to weaker acids (predominantly
propionic).

Gram negative rods
Facultatively anaerobic
• The majority of facultatively anaerobic gram – negative rods
in the mouth belong to genus Haemophilus.
• H. Parahemolyticus is
isolated from soft tissue
infections of the oral cavity,
but is probably not a regular
member of the oral
microflora.
• Strains have been isolated
however from jaw infections
and cases of infective
endocarditis.

Other facultatively anaerobic gram negative rods include
Eikenella corrodens.
Have been isolated from a range of oral infections, causing
abscesses, and has been implicated in periodontal disease.

Capnocytophaga are CO2 dependent gram negative
rods, with a gliding motility and found in sub-gingival
plaque and increase in gingivitis. Eg Capnocytophaga
gingivalis.

Actinobacillus actinomycetemcomitans
• (A.a) has been implicated in the etiology of
particularly aggressive forms of periodontal disease
in adolescents (localized juvenile periodontitis).
• It has been described as being microaerophilc or
facultatively anaerobic, although it appears to grow
best in an aerobic atmosphere enriched with 5-10%
CO2.

• It possesses cell surface layers
that contain molecules that
stimulate bone resorption.
• A.a produces a range of
virulence factors, including a
powerful leukotoxin,
collagenase, immuno-
suppressive factors and
proteases capable of clearing
IgG strains and can also be
invasive.

Obligatory anaerobic gram negative rods
• Asaccharolytic and saccharolytic oral organisms were placed
in the genera Porphyromonas and Prevotella.
• Some organisms produce colonies with a brown or black
pigment when grown on blood agar.
• This pigment may act as a defence mechanism helping to
protect the cells from the toxic effect of oxygen.
• In black pigmented anaerobes, haemin is an essential growth
factor.

Porphyromonas gingivalis is found almost solely at
sub-gingival sites (esp. in advanced periodontal lesions)
,tongue and tonsils, also routinely in infected root canals.
It has fimbriae on its cell surface that mediate adherence to
oral epithelial cells, and to saliva coated tooth surfaces.

Virulence factors
include proteases, with
specificity for arginine
that can degrade host
molecules such as IgA,
complement, and iron
and haeme sequestering
proteins and
glycoproteins.
It also produces a
haemolysin, collagen
degrading enzymes,
cytotoxic metabolites
and a capsule.

PREVOTELLA
• The new definition of Bacteroides has meant that
many strains have been placed in the genus
Prevotella.
• Species within this group are moderately
saccharolytic, producing acetic acid, succinic acid
and other acids from glucose.
• This new genus includes pigmented species
P.intermedia, P.nigrescens, P.melaninogenica,
P.loescheii, P.corporis

• P. intermedia have greater peptidase
activity and are associated more
with periodontal disease, abscesses.
• P.nigrescens is isolated more often
and in higher numbers from healthy
sites.

• Another major group of obligately anaerobic gram negative
bacteria belongs to genus fusobacterium.
• Cells are characteristically in the form of long
filaments (5-25 micronm in length) and include the
following sp. F.alocis and F.sulci from the normal
gingival crevice, F.periodonticum from sites with
periodontal disease.
FUSOBACTERIUM

F.nucleatum is capable of removing sulphur
from cytosine and methiomine to produce
ammonia, butyrate, hydrogen sulphide and
methyl mercaptan.
The last 2 compounds are highly
odorous and are implicated in
odour associated with halitosis.
Fusobacteria are able to co-aggregate
with most other oral bacteria and are
believed to be an important bridging
organism between early and late
colonizers during plaque formation

Spirochetes are numerous in subgingival plaque.
The numbers of spirochetes are raised in periodontal disease,
SPIROCHETES

Oral spirochetes fall within the genus Treponema and a
number of sp. have been identified, including T.denticola,
T.macrodentium, T.oralis, T.skoliodontium, T.socranskii,
T.malto-philum, T.amylovorum, T.vincentii.
T.denticola appears to be
proteolytic, possesses
proline aminopeptidase
and an arginine specific
protease, can degrade
collagen

FUNGI
• The main fungal species causing oral infection are
Aspirgillus, Geotrichium and Mucor sp.
• These when seen in healthy individuals may be transient
rather than resident members of oral microflora.

In contrast, Candida sp. are
distributed evenly throughout the
mouth.
The most common site of isolation
is dorsum of tongue, esp. posterior
area near circumvallate papillae.
Their incidence increases in the
presence of intra oral devices such
as plastic dentures on orthodontic
appliances, esp. in the upper jaw
on the fitting surface.
This is because Candida sp. attach
tenaciously to acrylic.

VIRUSES
The commonest
virus detected in the
oral cavity is Herpes
simplex, both type 1
& 2 of which 1 is
most common. This
is the cause of cold
sores.
.

PROTOZOA
• Entamoeba gingivalis is the most common protozoan.
• It has been isolated from periodontal tissues, esp. in
patients who have received radiotherapy
• Trichomonas tenax ,a flagellated protozoan, has been
isolated from the oral cavity of healthy patients.
• It may cause salivary gland swellings esp. in the
parotid.

 Bacterial changes from birth to adolescence
o Acquisition of normal flora
o Pioneer community and ecological succession
o Window of infectivity
o Climax community
CONTENTS
Benefits of oral microflora
Host bacteria interrelationships
Normal microbial flora at different sites of mouth.
Develepment of plaque and oral microbiota
Importance of biofilm
Plaque ecology
Localization of flora related to caries and periodontal
disease
Clinical implications

Development of the oral flora
At birth
Infancy and early childhood
Adolescence
Adulthood

Birth
• Bacteria begin to colonize on infant’s oral cavity during
birth, and succession of bacteria in the mouth continues
throughout life.
• The earliest microorganisms which can be isolated from the
mouth during the first few weeks after birth are
predominantly streptococci and include mitis, oralis and
salivarius.
• These are able to become established in the mouth. Other
bacteria appear as transients eg. lactobacillus of fecal origin
and S.mutans prior to tooth eruption.

• Several streptococcal and staphylococcal species may be
isolated either with lactobacilli, bacillus spp, neisseria spp
and yeasts.
• Streptococcus salivarius is the most common isolate from
the mouth of young babies together with staphylococcus
albus.
• Occasionally candida albicans multiply rapidly in the
mouth and in low pH it prevents the normal growth of
other commensals ,and this overgrowth of yeasts produces
what is known as oral thrush.

• The foetus in the womb is normally sterile.
• During delivery the baby comes into contact with
normal microflora of mother’s uterus and vagina, and
at birth with microorganisms of atmosphere and
people in attendance.
• Despite this, the mouth of the newborn baby is usually
sterile.

• From the first feeding onwards the mouth is regularly inoculated
with microorganisms and the process of acquisition of resident
oral microflora begins.
• Acquisition depends on successive transmission of
microorganisms to the site of potential colonization.
• Initially in the mouth, this is by passive contamination from the
mother; from food, milk and water; from saliva of individuals in
close proximity to the baby.

Infancy and early childhood
• With increase in number of teeth and changes in the diet
the overall properties of organisms in the mouth will
change.
• During the first months of life the flora becomes more
complex with anaerobic bacteria such as Veilonella and
Prevotella (Bacteroides) increasing in number.
• Tooth eruption has a major impact on oral flora
composition: colonization by species that adhere and
grow on tooth surfaces.
S. mutans, sobrinus, sanguis and actinomyces species will
predominate.

QUALITATIVE CHANGES IN ORAL FLORA ASSOCIATED
WITH ERUPTION OF TEETH

PIONEER COMMUNITY AND ECOLOGICAL
SUCCESSION
• The first to colonize are termed pioneer species. These
continue to grow and colonize until environmental
resistance is encountered, in the form of physical or
chemical factors.
One species is usually predominant during development of
pioneer community: ie streptococci, esp. S. salivarius,
S.mitis, S.oralis, many of these possess IgA protease
activity, which enables them to evade the effects of IgA.

With time, the metabolic activity of the pioneer community
modifies the environment, providing conditions suitable for
colonization by a succession of other populations by :
 Changing local pH.
 Modifying or exposing new receptors for attachment
 Generating novel nutrients as end products of metabolism
(lactate, succinate, etc.) or as breakdown products
(peptides, haemin, etc) which can be used as primary
nutrients by other organisms as part of a food chain.

.
• S. salivarius has been isolated from infants 18 hours after birth
and from 75% of infants. (Igarashi et al. 1998).
• S. salivarius is dominant among S.oralis or S.mitis and may
make up 98% of total oral flora until appearance of teeth (6-9
months).
• The diversity of the pioneer oral community, including
streptococci, increases during first few months of life and
several gram negative anaerobic species appear.

Window Of Infectivity
• The acquisition of some bacteria may occur optimally only at certain ages.
• Studies of the transmission of mutans streptococci (MS) to children have identified a
specific ‘window of infectivity’ between 7 - 31 months (median age = 26 months).
• According to studies by Caufield et al, 46 mother-child pairs from birth to 5 years were
studied, 38 out of 46 acquired MS at age of 26 months.
• According to mohan et al (1998) 20% children were infected by 14 months, karn et al –
10 months. And children aged 2-6 year showed less succeptible unless prior acquisition.

• According to Straetesman et al, 2nd window of infectivity is speculated at =6 years
of age when 1st molars are erupting and found 75% uninfected at age 5 were
infected at 11.
• According to li and caufield the vertical colonization of MS from mother to infant
is well documented. In 71% cases genotype of mother and infant MS strain
matched.
• The significance of that was due to :
• High caries rate runs in family
• Children of mothers with high caries rates are at higher risk
• According to wan et al 50% of preterm infants and 60% of full term infants
among 172 infants were harboured with MS.
• The factors associated were:
• Incraesed frequency of sugar , sharing foods.
• Breast feeding, sharing utensils contaminated with saliva
• Sucking adults fingers.

• According to kohler and Andreen ,MS in mothers during the emergence of
primary teeth in her child had long term influence on colonization and caries
caries experience in child.
• Salivary contamination off cups , glasses and eating utensils such as spoons may
account for transmission of MS from parent to child.
• According to Reneta et al, children in brazillian nursery school had presecence of
matching genotypes among children attending same school suggesting of
horizontal transmission by sharing pacifies and toys.
• In a study of 78, 4-year-old children, Kohler and coworkers showed that 89 % of
children colonized with mutans streptococci at 2 years of age had caries, with a
decayed, filled tooth (dft) index of 5.

Window of infectivity is a combination of
• frequent and close maternal contacts,
• cessation of lactation with its protective antibodies,
• an immature immune response
MS colonization occurs between 7 – 31 months of age, but
has been seen as early as 10 months is some
populations/studies.

• Mothers with infected infants had more plaque and calculus and
periodontal disease, brushed less and snacked more.
• They also had less education and came from lower socio-
economic classes.
• By 1 year of age, the predominant sp. isolated are streptococci,
Neisseria, Veionella, Staphylococci.
• Less frequently isolated are lactobacillus, actinomyces,
prevotella and fusobacterium.
• McCarthy et al (1998) reported that a marked increase in the
number of microorganisms can be observed in children after 1
year of age from both a qualitative and quantitative aspect, with
streptococci continuing to be numerically dominant.

• Following tooth eruption, the isolation frequency of spirochetes
and black pigmented anaerobes increases, especially in teenagers.
• It has been proposed that increased prevalence of these 2 groups
during puberty might be due to hormones entering the gingival
crevice and acting as a novel nutrient source.
• In adults, the resident oral microflora remains relatively stable
and coexists in reasonable harmony with the host.
• This stability (microbial homeostatis) is due to a dynamic balance
among members of resident flora due to numerous inter bacterial
and host-bacterial interactions.

• Eventually a stable situation is reached with a high species diversity, this is
termed the climax community.
• Succession is associated with a change from a site possessing few niches to one
with a multitude of potential niches.
Eg: Streptococci esp. S.salivarius, which binds to epithelial cells are usually the
first to colonize.
This S.salivarius produces extracellular polymers from sucrose to which other
bacteria eg. actinomyces sp. can attach.

Climax Community: Colonization Resistance
• Colonization resistance: It is the ability of resident microflora to
exclude exogenous organisms by preventing their colonization
which are pathogenic for the host.
 Competition for receptors for adhesion
 Competition for essential endogenous nutrients and co-factors
Creation of micro-environments that discourage
the growth of exogenous species
 Production of inhibitory substances

Adolescence
• These teeth have deep tissues in their surfaces,
inter proximal spaces are much larger in these
than in deciduous dentition.
• The gingival crevice is deeper and allows for a
great increase in anaerobic organisms
• The lesion of dental caries will create a new
environment for organisms especially
streptococci which will furnish.

• A study conducted by SUMMERSGIL KS et al in 2001
suggested the evidence of Human papilloma virus to be 5.2
% in healthy adolescents.(ie 5/97 adolescents)
• Protozoa s rate of colonization increases with age and being
more frequent between 11 and 19 years than in young
children.

Puberty is the time of major hormonal changes
• This is accomplished by nutritional enrichment of oral environment.
• This phenomenon lead to increase in some group of oral microorganisms
including gram negative anaerobes and spirochetes.
Substance abuse
• Smoking and alcohol consumption-streptococcus- salivary acetaldehyde-
carcinogenic

• Adolescents are more concern about esthetics .So
orthodontic treatment rates are high.
• In 2006 Naranjo et al reported an increase in
Porphyromonas gingivalis,Provetella intermedia,Provetella
nigrescens,Taneralla forsythia and fusobacterium species
after bracket placement.
• In a recent study by Andruceoli et al show the presence of
orange complex to be 40% followed by vellionella parvula to
be 22%

Adulthood
• There is increase in bacteroids spp and spirochetes .
• Actinomycetes are also regularly isolated.
• Edentulous patients harbour few spirochetes or bacteriodes
but their carriage of yeasts increases.

• Recent studies by Aas et al showed 10% of children and young adults
with dental caries-absence of S mutans-presence of Lactobacilus,
Veillonella, Bifidobacterium have been detected.

Chromogenic bacteria
• Black stains in primary teeth
• Black tooth stain is a characteristic extrinsic discoloration, which occurs
along the third cervical line of the buccal and/or lingual surfaces of teeth,
particularly in the primary dentition
• Dental discoloration is primarily of two types:
• Intrinsic discolouration (change to the structural composition) and
extrinsic discolouration (discolouration on the tooth surface or pellicle).
• Teeth discoloration may be a result of metabolic diseases (alkaptonuria,
hyperbilirubinaemia, enamel hypoplasia, etc.), local injury, physiological
ageing, lifestyle habits (smoking, caffeine consumption) or bacterial
colonization (Watts and Addy, 2001).

• Chromogenic bacteria were proposed as an etiological factor in the production of black pigment.
• Periodontal pathogens such as Porphyromonas gingivalis, Prevotellaintermedia, and Prevotella nigrescens are
reported to be black-pigmented anaerobes inoral cavity. Former studies assumed Prevotella melaninogenica was
closely related to black tooth stain.
• Examinations have implicated Actinomycetes as the predominant cultivable microorganisms found in black stain.
However, almost 50% of oral bacteria are non-culturable.
• Significantly higher prevalences of Actinomyces and Aggregatibacteractinomycetemcomitans were observed in
black stain .
• Enterobacter sp are also responsible for tooth blackening (wint et al )

Mechanism
• Black stain is a form of dental plaque. It’s different from other types by insoluble
iron salt and high calcium and phosphate composition.
• Bacteria-induced teeth discoloration is caused due to the hydrogen sulphide
produced by certain kind of bacteria in the oral cavity which interacts with
salivary iron content resulting in chromogenous insoluble ferrous deposition and
blackening of teeth (Bandon et al., 2011).
• Some hypothesis concerning the association between black stains and some
bacterial strains (actinomyces, lactobacillus sp, prevotella melaninogenica) has
been reported
• The attraction of materials to the tooth surface is important to the formation of
extrinsic dental stain. These attractive forces include electrostatic, van der Waals,
hydration forces, hydrophobic interactions and hydrogen bonds. However, the
mechanisms that determine the strength of adhesion are not perfectly understood.

Extrinsic tooth discoloration: The causes of extrinsic staining can be divided into
two categories;
a) Direct extrinsic tooth staining: Those compounds which are incorporated into
the pellicle and produce a stain as a result of their basic color.
b) Indirect extrinsic tooth staining: Those which lead to staining caused by
chemical interaction at the tooth surface.
Direct extrinsic tooth staining has a multi-factorial aetiology with chromogens
derived from dietary sources or habitually placed in the mouth .
These organic chromogens are taken up by the pellicle and the color imparted is
determined by the natural color of the chromogen.

Diagnosis and Classification
• Koch et al. [3] considered that the presence of dark dots (diameter less than
0.5 mm) forming linear discoloration (parallel to the gingival margin) at dental
smooth surfaces of at least two different teeth without cavitation of the enamel
surface.
• Shourie [3] recorded the presence or absence of pigmented plaque as:
• (1) no line;
• (2) incomplete coalescence of pigmented dots;
• (3) continuous line formed by pigmented spots
• Gasparetto et al. [4] added another criteria based on the area of the tooth
surface affected:
• First degree corresponds to the presence of pigmented dots or thin lines with incomplete
coalescence parallel to the gingival margin;
• second degree indicates the presence of continuous pigmented lines, which are easily observed and
limited to half of the cervical third of the tooth surface;
• third one equals the presence of pigmented stains extending beyond half of the cervical third of the
tooth surface.

Factors responsible for extrinsic
strains
• Diet: Brown stains on the surface of the teeth could be due to the
deposition of tannins found in tea, coffee and other beverages .
• Oral hygiene: Accumulations of dental plaque, calculus and food
particles cause brown or black stains .
• Chromogenic bacteria have also been suggested as an etiological factor
in the production of stains typically at the gingival margin of the tooth .
• Habits: Tobacco from cigarettes, cigars, pipes, and chewing tobacco
causes tenacious dark brown and black stains that cover the cervical one
third to midway on the tooth . Chewing of pan results in the production
of blood red saliva that results in a red-black stain on the teeth, gingiva
and oral mucosal surfaces .

• Medication factors: Cationic antiseptics such as chlorhexidine, cetylpyridinium chloride
and other mouth washes can cause staining after prolonged use . Chlorhexidine, for
example, produces brown to black discoloration. Most evidence indicates that the likely
cause of staining is the precipitation of anionic dietary chromogens onto the adsorbed
cations .
• Some systemic medications (e.g. minocycline ,doxycycline , co-amoxiclav , linezolid ) are
also shown to cause extrinsic staining. Metallic compounds are also implicated in dental
discoloration (e.g. Iron containing or al solutions , mouth rinses containing metal salts ).
• Occupation and environmental factors: Industrial exposure to iron, manganese, and silver
may stain the teeth black. Mercury and lead dust can cause a blue-green stain; copper and
nickel, green–to–blue-green stain and chromic acid fumes may cause deep orange stain .
There is a positive correlation between dental extrinsic stains and the concentration of
trace elements, especially iron in the water sources.

Examination
• The patient's history of tooth discoloration provides useful
information regarding the etiology . The history should
include the following:
• Dental history (previous dental treatment, oral hygiene
practices, use of mouthwashes, amount and scheduling of
fluoride intake, history of dental trauma )
• Medical history (history of maternal or childhood
diseases, use of medications)
• Family history (genetic disorders) § Diet history
(nutritional deficiencies, diet that can cause staining of the
teeth)
• Social history (occupational exposure to metals, use of
tobacco)

Clinical examination
• The scratch test is usually used to distinguish between extrinsic and intrinsic discoloration .
• Discolored tooth surfaces are scratched with care by using a dental explorer, scaler, or similar
sharp instrument to assess surface texture.
• Light scratching with a dental instrument removes weakly adherent plaque that causes extrinsic
discoloration. If the discoloration requires removal with a sharp dental scaler, the discoloration is
considered to be tenacious.
• Intrinsic discoloration cannot be removed by using the scratch test.
• Extrinsic staining of a single tooth is unusual. The distribution is usually generalized. The stains
are usually found on surfaces with poor tooth brush accessibility.
• Whereas in case of intrinsic discoloration distribution is either generalized to all teeth or
localized to certain teeth or tooth surfaces.
• An intrinsic etiology usually exists when a single tooth is discolored. Teeth with extrinsic tooth
discoloration usually demonstrate no signs of pulp disease, usually associated with intrinsic
discoloration.

Treatment
• The black stain particularly poses an aesthetic problem. Daily Tooth brushing is not
enough to remove this external stain.
• The professional cleaning is necessary to remove stains and resolve this aesthetic
problem. Although a simple scaling and tooth brushing with pumice powder are usually
sufficient, frequently black stain is recurrent.
• The ultrasonic cleaning is not recommended, this modality can lead to enamel removal;
therefore, their repeated use is undesirable. Nevertheless, it can challenge the dentist,
especially when it is deposited on roughened or pitted areas of the tooth.
• In a professional dental hygiene appointment, removal through polishing with rubber cup
and fluoride pumice is possible. If the staining is resistant, the excess water can be blotted
from the pumice and the tooth should be dried before the polishing procedure is
performed Also, sharp scaling instruments are of use against firmly attached deposit.
Black stain tends to reform again despite good personal oral care, but quantity may be
less when biofilm control procedures are meticulous

Factors affecting the development of the oral
flora

In order to become established in
the mouth an organism must
• Be introduced
• Be retained
• Be able to multiply in the
conditions present in the mouth

Adherence:
• Some bacteria have the ability to
adhere to soft tissues.
• Streptococcus salivarius can adhere to
the mucosa of dorsum of tongue.
• Other in particular streptococcus
mutans, mitior and sanguis to enamel
as the result of production of
extracellular polysaccharide.
• Some oral actinomycetes adhere
through a hyaluronic acid mediated
mechanism.

Protected sites:
• Dental plaque will provide a
protected environment for bacteria.
• The largest protected site is
gingival crevice where species such
as melaniogenicus and sphirochetes
can survive
Detachment forces
• Salivary flow.
• The movement of tongue.
• Abrasive action of diet

PH:
• The metabolism of microorganisms is often dependent on pH
• Bacteroids melaninagericus and veillanella spp are inherent if
pH below 5.5.
• Lactobacillus spp and candida albicans can tolerate very low
pH values.
• Members of the oral flora grow best in vitro at about 7.

Concept of Critical pH
• Indicates the PH at which saliva no longer saturated with
respect to Ca & Ph ions--- hydroxyapatite dissolves.
• Plaque fluid & saliva, both cease to be saturated at PH range 5-
6 , Avg 5.5.

• Acid neutralizing activity in plaque Eg.Veillonella
• Veillonella --- metabolic conversion of lactic acid to
weaker organic acid i.e.., propionic & volatile acid.
• Strain of Veillonella – often found in plaques
associated with strong lactic acid producers
• Amines in plaque: alpha-amino butyric acid, putrescine,
histamine, & cadavarine.
• These have a significant neutralizing effect in plaque
under conditions of moderate sugar uptake.

Diet as a nutrient source
Three factors influences the
effectiveness of the diet as a microbial
nutrient sources.
These are the
• Chemical composition of the diet
• The physical consistency of its
components
• The frequency of its presentation.

• Both consistency and frequency influence the length of
time that food remains in contact with the plaque and
thus is available for bacterial use.
• When snacks are interposed between meals they
augment the time of nutrient availability.
• The consistency of food also influences the plaque flora
liquid foods such as fruit juices and tonics are usually
swallowed quickly and for this reason they are not
readily available to oral flora.

Saliva as a nutrient source
Saliva is hameostatic fluid that buffers the plaque, saliva
can provide nutrients to flora it contains about 1% solids,
which include glycoproteins inorganic salts, aminoacids,
glucose.
Gingival crevice fluid
Gingival crevice contain serum transudate that contains
tissue and serum proteins as well as free amino acid,
vitamins, glucose.

Bacteria
• The bacteria themselves can
provide nutrients for each other.
• One well-documented interaction is
the relationship between lactic-
acid-producing bacteria such as the
streptococci and a lactate utilizing
species such as Veillonella
alkalescens
• V. alkalescens has lost the enzyme
hexose kinase, so it cannot produce
phosphorylate glucose

• Many organisms in plaque, including the streptococci,
form lactic acid.
• This would suggest that the Veillonella parasitize the
lactate producers.
• However, the relationship between the Veillonella and
organisms such as streptococci may be symbiotic, as
the lactic acid is converted to propionate, acetate, and
carbon dioxide, with a resultant elevation in pH.
• This shift away from low pHs would favorable for
acid sensitive streptococci.

Microbial Interactions
• The complexity of communities of
microorganisms is the result of a
mucosa of microbial interactions.
• Some of these are nutritional such
as provision of para amino benzoic
acid by streptococcus sanguis for
streptococcus mutans in reduced
conditions.

Commensal activity of the oral flora

Host bacteria
interrelationships:
Symbiosis
• The living together or interaction of
dissimilar organisms.
• The smaller organism is generally
the symbiont.
• The larger organism is generally the
host.

Antibiosis
• An antibiotic relationship is the
opposite of a symbiotic relationship.
• When bacteria cause an infection that is
combatted by the defense systems of the
host, the relationship is said to be
antibiotic.

• This antibiotic relationship is very unstable for both
the host and the pathogenic bacteria.
• If the host is killed by the pathogen, the pathogen also
dies unless it is able to make its way to another host.

Amphibiosis
• Recent medical history has shown a decreased
virulence for all the classic pathogens, such as the
tubercle bacillus, Treponema pallidum and others.
• This means that these organisms have entered into a
new, more stable relationship with the host.
• Rosebury introduced the term "amphibiotic" to
describe an intermediate state in which the host and
its flora exist in a form of stable balance with each
other.

Colonization of microenvironments

• The tooth can be described as a non shedding hard surface
that offers many different sites for colonization by bacteria
below (subgingival) and above (supragingival) the gingival
margin.

In contrast, the oral mucosa is characterized by a continuous
desquamation of its surface epithelial cells, which allows rapid
elimination of adhering bacteria.
The mucosa that covers the cheek, tongue, gingiva, palate, and
floor of the mouth varies according to the anatomical site.

The tongue, with its papillary surface, provides sites of
colonization that are protected from mechanical removal.
Gingival crevice, also provides a unique colonization site that
includes both hard and soft tissues.

• The oral surfaces are also constantly bathed by two important
physiological fluids, the saliva and the gingival crevicular
fluid.
• These fluids are essential for the maintenance of the oral
ecosystems by providing water, nutrients, adherence, and
antimicrobial factors.
• The supragingival environment is bathed by saliva, while the
subgingival environment (gingival crevice) is bathed mainly
by the gingival crevicular fluid.

Ageing and the oral
microflora

Saliva
• Saliva is present as a proteinaceous film covering all surfaces of the oral cavity.
• The thickness of this film is the major factor responsible for distributing and eliminating
material within the oral cavity.
• When sleeping an individual may produce ~ 0.25 ml saliva per minute but whereas in
vigorous chewing and lively conversation may produce as much as 10ml per minute.
• Since saliva is normally supersaturated, dental hard tissues do not dissolve spontaneously
under the pH values prevailing under normal conditions.

• Numerous cellular elements are also found in saliva;
predominantly desquamated surface epithelial cells from
-mucous membrane,
-neutrophils from gingival pockets
-millions of microorganisms
• Several antibacterial factors are present in saliva like
lysozyme, lactoferrin and sialoperoxidase system etc.
• Antibodies have also been detected, with secretory IgA being the
predominant class of immunoglobulin.
• IgG & IgM are also present but in lower concentrations.
• Peptides with antimicrobial activity, eg. Histidine- rich peptides
(histatins) are also present.

• The major organic constituents of saliva are proteins and glycoproteins, such as mucin, it is
influenced by oral microflora by :
• Adsorbing to tooth surface to form conditioning film (acquired pellicle) to which microorganism
can attach.
• Acting as primary sources of nutrients (carbohydrate and protein) for resident microflora.
• Aggregating microorganisms and thereby facilitating clearance from mouth by swallowing.
• Inhibiting growth of exogenous microorganisms.

Microorganisms in saliva
• The surfaces of the oral cavity are constantly colonized.
• One ml of whole saliva may contain more than 200 million organisms representing
more than 250 different species.
• These constitute a complex microbiota which does not result in disease as they exist in
equilibrium with the host.
• Streptococci constitute an essential part of the microflora which constantly colonize
mucosal surfaces and teeth.

Gingival Crevicular Fluid
• Serum components can reach the mouth by the flow of serum like fluid
through the functional epithelium of the gingiva.
• The flow of GCF is relatively slow at healthy sites (0.3 ml/tooth/hour) but
increases during inflammation.
• Provides a novel source of nutrients
• flow will remove non adherent microbial cells
• Many bacteria from subgingival plaque are proteolytic and they interact
synergistically to break down host proteins and glycoproteins which helps
to provide peptides, amino acids and carbohydrates for their growth.

• IgG is the predominant Ig, IgM & IgA are also present.
• GCF contains leucocytes: 95% neutrophils, remainder lymphocytes and monocytes.
• Enzymes such as collagenase, elastase, trypsin, etc. derived from both
phagocyte host cells and bacteria can be detected in GCF.
.

Normal Microbial Flora of Different Sites of the
Mouth
• On the lips there is a transition from skin to oral
mucosa and also changes in the bacterial
population.
• Staphylococcus albus with large number of
streptococci are typical of the mouth.
• If the commisures are moistened by saliva, angular
cheilitis may develop from candida albicans.
Lips:

Cheek:
• Predominant cheek bacterium is streptococcus mitis or with
streptococcus sanguis and salivarius
• Yeasts may be isolated from the cheeks.
Palate:
• Hard palate supports a streptococcus flora, haemophili are found
regularly and lactobacilli are common.
• Few anaerobes found in exposed mucosal surface.
• Yeasts and lactobacilli will increase dramatically in some denture
wearers.
• The soft palate will harbour respiratory tract bacteria such as
hemophillus, cornybacterium, Neisseria.

Tongue:
• The keratinized dorsal surface of the tongue is an ideal site for the
retention of microorganisms, streptococcus salivarius is the predominant
organisms.
• Streptococcus mitior and Hemophillus spp are also common.
• Dorsum of the tongue is frequently colonized by small number of
candida albicans

Specific and non-specific host defence factors
of the mouth

ORAL FLORA IN PERIDONTAL DISEASES

DEVELOPMENT OF PLAQUE leading to
periodontal conditions

FORMATION OF THE PELLICLE
• Within nanoseconds after a vigorously polishing the teeth, a thin, saliva derived
layer called the acquired pellicle, covers the tooth surface.
• Consists of more than 180 peptides, proteins, glyco proteins, including keratins,
mucins, proline – rich proteins, and other molecules can function as adhesion
sites( receptors) for bacteria.

ULTRA STRUCTURE OF DENTAL PELLICLE
 2 hr pellicle: Granular structures which form
globules, that connect to the Hydroxyapatite
surface via stalk like structures.
 24 hrs Later: Globular structures get covered up
by fibrillar particles : 500 - 900 nm thick
 36 hrs Later: The pellicle becomes smooth,
globular

• Studies of early (2-hour) enamel pellicle reveal
that its amino acid composition differs from that
of saliva, indicating that the pellicle forms by
selective adsorption of the environmental
macromolecules.
• Mechanism involved are:
Electrostatic forces *
Van der waals *
Hydrophobic forces*

Initial Adhesion & Attachment of
Bacteria
• This concept approaches microbial adhesion to
surfaces in aquatic environment as 4 stage sequence:
DENTAL PAQUE
Transport
to surface
Initial
adhesion
Attachment
Colonization
of surface &
biofilm
formation

Clean
substratum
Molecular
adsorption
(Phase 1)
Single
organisms
(Phase 2)
Multiplication
(Phase 3)
Sequential
adsorption
of organisms
(Phase 4)

• The initial bacteria colonizing the pellicle-coated tooth
surface are predominantly gram-positive facultative
microorganisms such as Actinomyces viscosus and
Streptococcus sanguis.
• These initial colonizers adhere to the pellicle through specific
molecules, termed adhesins, on the bacterial surface that
interact with receptors in the dental pellicle.
• For example, cells of A. viscosus possess fibrous protein
structures called fimbriae that extend from the bacterial cell
surface.
• Protein adhesins on these fimbriae specifically bind to
proline-rich proteins that are found in dental pellicle, resulting
in the attachment of the bacterial cell to the pellicle-coated
tooth surface.

• The plaque mass then matures through the
growth of attached species, as well as the
colonization and growth of additional species.
• In this ecologic succession of the biofilm, there
is a transition from the early aerobic
environment characterized by gram positive
facultative species to a highly oxygen-deprived
environment in which gram-negative anaerobic
microorganisms predominate.

SECONDARY COLONIZATION AND PLAQUE
MATURATION.
• Secondary colonizers are the microorganisms that do not initially
colonize clean tooth surfaces, including Prevotella intermedia,
Prevotella loescheii, Capnocytophaga spp., Fusobacterium
nucleatum, and Porphyromonas gingivalis.
• These microorganisms adhere to cells of bacteria already in the
plaque mass. Extensive laboratory studies have documented the
ability of different species and genera of plaque microorganisms
to adhere to one another, a process known as coaggregation.
• This process occurs primarily through the highly specific
stereochemical interaction of protein and carbohydrate molecules
located on the bacterial cell surfaces, in addition to the less
specific interactions resulting from hydrophobic, electrostatic,
and van der Waals forces.

• The significance of coaggregation in oral colonization has been
documented in studies of biofilm formation in vitro' as well as
in animal model studies .
• Well-characterized interactions of secondary colonizers with
early colonizers include the coaggregation of F. nucleatum with
S. sanguis, P. loescheii with A. viscosus,and Capnocytophaga
ochracea with A. viscosus.
• Most studies of coaggregation have focused on interactions
among different gram-positive species and between gram-
positive and gram-negative species.
• In the latter stages of plaque formation, coaggregation between
different gram-negative species is likely to predominate.

Primary colonizers
12/27/2011 DENTAL PAQUE 183

Secondary colonizers
12/27/2011 DENTAL PAQUE 184

S.mitis
S.oralis
S.sanguis
Streptococcus sps
S.gorondi,
S.intermedius
DENTAL PAQUE
EARLY
COLONIZERS
V.parvula
A.odontolyticus
P.intermedia
P.nigrescens
P.micros
F.nucleatum
C.rectus
E.nodatum
C.showae
E.corrodens
Capnocytophaga sps
A.actinomycetocomitans
P.gingivalis
T.forsythus
T.denticola
CLOSELY ASSOCIATED
COMPLEXES IN THE
ORAL CAVITY
LATE COLONIZERS

PLAQUE AS A BIOFILM
• The term biofilm describes the relatively undefinable
microbial community associated with a tooth surface or
any other hard, non-shedding material (Wilderer &
Charaklis 1989)
• Biofilms have an organized structure.
• They are composed of micro colonies of bacterial cells non
randomly distributed in a shaped matrix or glycocalyx.
• In lower plaques layers microbes are bound together in
polysaccharide matrix with other organic & inorganic
materials.
• On top of lower layer, a loose layer appears that is often
irregular in appearance; it can extend into surrounding
medium.

Genetic expression is different in biofilm bacteria when
compared to planktonic (free floating) bacteria.
Biofilm cells can coordinate behavior via intercellular
"communication“ using biochemical signaling
molecules.

• Involves the regulation of expression of specific genes
through the accumulation of signaling compounds that
mediate intercellular communication
• Dependent on cell density and mediated through signaling
compounds
• Quorum sensing gives biofilms their distinct properties

Quorum sensing is involved in the regulation of
• genetic competence
• mating
• bacteriocin production
• sporulation
• stress responses
• virulence expression
• biofilm formation

Microbial Specificity of
Periodontal Diseases
Non Specific Plaque Hypothesis
Specific Plaque Hypothesis

NON SPECIFIC PLAQUE HYPOTHESIS
• The nonspecific and specific plaque hypotheses were delineated in 1976 by
Walter Loesche
• The nonspecific plaque hypothesis maintains that periodontal disease results
from the "elaboration of noxious products by the entire plaque flora.
• According to this thinking, when only small amounts of plaque are present,
noxious products are neutralized by the host.
• Similarly, large amounts of plaque would produce large amounts of noxious
products, which would essentially overwhelm the host's defenses.
• Nonspecific plaque hypothesis is the concept that control of periodontal
disease depends on control of the amount of plaque accumulation.
• Treatment of periodontitis by debridement (nonsurgical or surgical) and oral
hygiene measures focuses on the removal of plaque and its products and is
founded in the nonspecific plaque hypothesis.

SPECIFIC PLAQUE HYPOTHESIS
• The specific plaque hypothesis states that only certain plaque is pathogenic, and its
pathogenicity depends on the presence of or increase in specific microorganisms.
• This concept predicts that plaque harboring specific bacterial pathogens results in
periodontal disease because these organisms produce substances that mediate the
destruction of host tissues.

Ecological plaque hypothesis
• In 1990, Marsh et al developed the ecologic plaque
hypothesis
• According to this, both the total amount. of dental plaque
and the specific microbial composition of plaque may
contribute to the transition from health to disease.
• A change in the nutrient status of a pocket or chemical and
physical changes to the habitat are thus considered the
primary cause for overgrowth by pathogens

Ecological plaque hypothesis (Marsh 1991)

SOCRANSKY'S CRITERIA FOR
PERIODONTAL PATHOGENS
• ASSOCIATION: A pathogen should be found more
frequently and in higher numbers in disease states than in
healthy states.
• ELIMINATION: Elimination of the pathogen should be
accompanied by elimination or remission of the disease.

• HOST RESPONSE: There should be evidence
of a host response to a specific pathogen which
is causing tissue damage.
• VIRULENCE FACTORS: Properties of a
putative pathogen that may function to damage
the host tissues should be demonstrated.
• ANIMAL STUDIES: The ability of a putative
pathogen to function in producing disease
should be demonstrated in an animal model
system.

• The two periodontal pathogens that have most thoroughly fulfilled
Socransky's criteria are Actinobacillus actinomycetemcomitans in the form
of periodontal disease known as Localized Juvenile periodontitis (LJP), and
Porphyromonas gingivalis in the form of periodontal disease known as adult
periodontitis.

PERIODONTAL HEALTH
Certain bacterial species have been proposed to be beneficial to the
host, including S. sanguis, Veilonella parvula, and C.
ochraceus(Carranza 10th)
Bacteria associated with periodontal diseases are often found in the
subgingival microflora at healthy sites, although they are normally
present in small proportions(Rose & Maeley, 6th)
Nonmotile nature.
DENTAL PAQUE

GINGIVITIS
104 to 106 bacteria.
Gram-negative bacteria.
Compared with healthy sites, noticeable increase also occur in the
numbers of motile bacteria, including cultivable and uncultivable
treponemas (spirochetes).
Pregnancy associated gingivitis is accompanied by dramatic
increases in levels of P. intermedia.
DENTAL PAQUE

CHRONIC PERIODONTITIS
C. rectus, P. gingivalis, P. intermedia, F. nucleatum and T. forsythia
were found to be elevated in the active sites(Carranza,10th )
Sites with chronic periodontitis will be populated with greater
proportions of gram-negative organisms and motile bacteria.
Certain gram-negative bacteria with pronounced virulence properties
have been strongly implicated as etiologic agents e.g. P. gingivalis and
Tannerella forsythus.

LOCALIZED AGGRESSIVE PERIODONTITIS
 Gram -ve, and anaerobic rods.
The most numerous isolates are several species from the genera
Eubacterium, A. naeslundii, F. nucleatum, C. rectus, and
Veillonella parvula.
However, some populations of patients with LAP do not harbor
Aa, and in still others P. gingivalis may be etiologically more
important.

GENERALIZED AGGRESSIVE
PERIODONTITIS
The sub-gingival flora in patients with
generalized aggressive periodontitis resembles
that in other forms of periodontitis.
The predominant subgingival bacteria in patients
with generalized aggressive periodontitis are P.
gingivalis, T. forsythis A.
actinomycetemcomitans, and Campylobacter
species.

NECROTIZING ULCERATIVE
GINGIVITIS/PERIODONTITIS
More than 50% of the isolated species were strict
anaerobes with P. gingivalis and F. nucleatum
accounting for 7-8% and 3.4%, respectively.
DENTAL PAQUE

Localization of oral flora related to caries
Pit & fissure caries
• A wide variety of microbes may be able
to initiate pit and fissure caries of which
strep. mutans and lactobacillus sp. are
very significant.
• Actinomyces sp. (viscosus, naeslundii or
israelli) may be significant.
• S. sanguis and other strep. (salivarius)
are not very significant.

Smooth surface caries
A limited number of organisms have
proved able to colonize smooth
surfaces in large enough numbers to
cause decay in test animals.
S.mutans is very significant in this
respect. S. salivarius is probably not
significant.

Root caries
• In rodents, gram positive
filamentous rods, including
Actinomyces sp. (viscosus,
naeslundii) have been significantly
associated.
• Strains of Nocardia and S.sanguis
may at times cause root caries, but
S. mutans is still significant.
• S.salivarius, however, is usually not
significant.

• The predominant organism is
lactobacillus which accounts for
about 1/3 (Fairbourn et al, 1980).
• Other frequently isolated gram
positive anaerobic rods and
filaments are Arachnia,
Bifidobacterium, Eubacterium,
Propionibacterium. Actinomyces,
Rothia and Bacillus also occur in
the forefront of deep dentinal
lesions.
• The incidence of gram positive
facultative cocci is low.
Deep dentinal caries

Microflora Of Nursing Caries Lesions
• Streptococcus mutans forms a significantly greater
proportion of the lesion flora while Streptococcus oralis,
Streptococcus sanguis and Streptococcus gordonii forms
a significantly greater proportion of the plaque flora
from sound tooth surfaces.
• The proportions of Actinomyces naeslundii and
Actinomyces odontolyticus are usually significantly
greater in the plaque samples than in the lesion samples.

To understand oral diseases (their etiology, progression,
diagnosis, treatment) it helps to have some understanding
the classic pathogenic potential of various microbes.
To understand the ecology of the oral microbiota and how
it can be affected by various factors helps in treatment
planning.
CONCLUSION

REFERENCES
Oral Microbiology
Marsh, Martin
Oral Microbiology & Immunology
Nisengard & Newman
A Text book of Microbiology
Ananthnarayan
Cariology
Newbrun

Microbiology of Oral Cavity: Resident Oral Flora & Streptococci

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