ORAL

MICROBIOLOGY

ORAL
MICROBIOLOGY
Dentistry 17
Semester 6

 MOUTH MICROBES
The human mouth hosts a panoply of microbes, some
taking up residence on the mouth lining (blue) within
days after birth.
Harmful species form biofilms, like the plaque that
encourages tooth decay, or colonize the crevices
between teeth and gums, causing periodontal disease.
Oral probiotics designed to boost the population of
species that outcompete pathogenic ones could help
prevent or reverse dental disease.

ORAL MICROBIOLOGY
Normal oral flora
The mixture of organisms regularly found at any
anatomical site is referred to as the normal flora.
Normal Flora of the Oral Cavity
The presence of nutrients, epithelial debris, and
secretions makes the mouth a favorable habitat for
a great variety of bacteria
Diverse organisms, including bacteria, fungi,
mycoplasmas, protozoa and viruses.
Mouth>700 bacterial species in mouth.
1 ml saliva = millions of bacteria.
~ 350 cultivable species. What about the rest ????

Normal oral flora

Some Spirochaetes, are also members of oral
cavity (dark field microscope for Spirochaetes).
Candida albicans is the most common fungal
isolates.
Epstein- Barr virus and human herpes virus six
are frequently shed in the saliva of healthy
individuals
Unculturable flora (identified by molecular
techniques)
Oral flora exists in a harmonious relationship
with the host.
Disturbance by any changes to the habitat,
affect the stability (broad-spectrum antibiotic)

Normal oral flora

At birth the oral cavity is sterile, but rapidly
becomes colonized from the environment,
particularly from the mother in the first feeding.
Streptococcus salivarius is dominant and may
make up 98% of the total oral flora until the
appearance of the teeth (6 - 9 months in
humans).
By one year of age , when teeth have erupted,
Streptococcus, Neisseria, Staphylococcus, less
frequently Lactobacillus, Actinomyces, Prevotella
& Fusobacterium.
Tooth surface and gingival tissues provide new
habitats for colonization with the resultant
formation of dental plaque.

Diverse ecological niches in the

oral cavity
The oral cavity contains various different forms
of tissue which have different properties;
The heterogeneity of tissue types in the oral
cavity, such as teeth, tongue and mucosa, means
that a variety of sites are available for
colonization by oral microorganisms;
Each site has unique characteristics and allows
those microorganisms best suited to the
environment to inhabit the site;
The role of microorganisms in a habitat is
referred as an ecological niche and a number of
ecological niches exist in the oral cavity, including
supragingival plaque, subgingival plaque and
tongue coating.

Mucosal Surfaces:
These include the palate, cheek and tongue,
which have cells which are constantly replaced
due to the normal wear-and-tear of the mouth;
The different mucosal surfaces also have
different properties which contribute to the
presence of different types of bacteria:
Areas such as the palate contain keratinized
squamous epithelium and the softer nonkeratinized areas such as the cheeks;
The tongue is a unique surface with papillary
structures, which provides ample crevices for a
wide variety of different strains of bacteria to
reside;
The presence of these crypts allows for
bacteria to be protected from the normal

Pathogenicity of
microflora
Microflora usually is nonpathogenic and form an integral
part of the host;
The presence of friendly resident
microorganisms on oral surfaces
contributes to the bodys
defense against foreign
pathogens, which are generally
transient and can give rise to
harmful infections.

Function of Microflora
Colonization resistance is firstly achieved
through the saturation of oral surfaces
with preexisting resident bacteria, which
reduces the available sites left for the
attachment of exogenous organisms;
Essential nutrients derived from the
saliva and various proteins in the oral
cavity are also more effectively utilized by
the resident microflora, which inhibits
infections via competition for resources;

Resident microflora modified the conditions

of the mouth
It maintains unfavorable conditions
for invading microbes;
Producing substances such as
hydrogen peroxide that actively
inhibits pathogen growth;
As a result, the harmony between the
oral microflora and the bodys natural
defenses acts as an effective
deterrent to infections, and thus
maintains the health of the host.

Variations inMicroflora
Everyone has microflora, but it
varies between people
depending on the condition of
their oral cavity;
Not all individuals would have
the same microflora profile;
Not all bacteria are ubiquitous
in all individuals.

Gingival crevicular fluid:

At the gingival margin, serum flows from
the inside the gingiva into the mouth;
The flow of this fluid removes foreign
microbes which do not adhere to these
surfaces;
For the resident population, the flow of
crevicular fluid provides these organisms
with a source of nutrition;
They have specialised enzymes which can
break down these proteins to provide them
with the nutrients necessary for growth.

Teeth and artificial hard

surfaces:
Microbes can accumulate on teeth;
This generally forms a dense layer of
bioflim known as dental plaque;
Fissures and deep grooves in the
teeth provide ideal conditions for the
formation and build-up of dental
plaque;
Most of the bacteria found in dental
plaque is absent in individuals without
teeth, such as babies.

The role of saliva in the maintenance

of a healthy oral environment
By acting as a buffer, saliva maintains the
pH of the mouth, ensuring the optimal
growth of the resident colonies;
Most of the microbes present in the mouth
utilize the glycoproteins and proteins in
the saliva as their main source of nutrition;
Proteins and glycoproteins of saliva are
responsible for the formation of the
pellicle, a film on the surface of the tooth
enamel where the microorganisms of the
microflora can adhere to.

Salivary flow
It is responsible for the removal of nonendogenous bacteria which is unable to
adhere to specific sites in the mouth;
Saliva acts a collector of these cells and
facilitates their removal;
Areas which receive markedly less saliva
flow, such as deep gingival crevices,
proximal spaces and occlusal fissures, tend
to have significantly higher levels of
bacterial buildup;
Saliva is carrying bacteria - the main source
of microbial transmission between
individuals;
Saliva also circulates bacteria within the

The normal bacterial flora of the

oral cavity
Benefit from the host who provides
nutrients and habitat;
Occupy available colonization sites
which makes it more difficult for other
microorganisms to become established;
Contribute to host nutrition through
the synthesis of vitamins.
Exert microbial antagonism against
exogenous species by production of
inhibitory substances such as fatty
acids, peroxides and bacteriocins.

The oral flora are the usual

cause of various oral diseases
Abscesses, dental caries, gingivitis, and
periodontal disease;
If oral bacteria can gain entrance into deeper
tissues, they may cause abscesses of alveolar
bone, lung, brain, or the extremities;
Such infections usually contain mixtures of
bacteria
If oral streptococci are introduced into
wounds created by dental manipulation or
treatment, they may adhere to heart valves
and initiate subacute bacterial endocarditis.

Analysis of The Oral

Microflora

1- Collect sample
The outcome of microbiological diagnostics may depend on
sampling techniques
1- Aspirate.
2- Paper point sampling
3- Oral rinse.
2- Transport to the laboratory
3- View sample
Gram stain.
Dark field microscope.
4- Treat sample
Disaggregate sample (plaque, glass granules)
5- Culture sample
Sabourauds agar.
Mitis Salivarius Bacitracin for Mutans.(MSB)
Anaerobic media
6- Identify organism
Enzyme profile, sugar fermentation, serology, DNA & API kit for
streptococcus.

Sabouraud agar

 Paper points was introduced successively into the depth of

the periodontal pocket for 10 s to harvest subgingival
plaque.
After removal from the pocket, the apical 3 mm of the point
was cut and immediately placed into a vial containing 3 ml
of pre-reduced anaerobically sterilized Ringers solution.
Sample was dispersed by vortexing at maximal setting for
60s.

 Mitis Salivarius Agar

Mitis salivarius agar is used to differentiate among species of Streptococcus
and the very close genus Enterococcus (formerly in the Streptococcus genus)
that are flora in the mouth. These organisms are very common in the mouth,
and have been associated with dental caries, as well as agents of endocarditis.
The sugars in this medium are sascharose and glucose, as well as the dyes
trypan blue and crystal violet.
Trypan blue is absorbed by the bacterial colonies, causing them to become
blue. The crystal violet, along with the 1% tellurite added to this medium,
inhibit gram - bacteria as well as many other gram + bacteria.

Strep. salivarius uses the sugars to produce sticky, mucoid, gum-drop colonies.
Strep mitis colonies will be small, flat, light blue.
Strep mutans will be undule-shaped colonies, with a granular frosted-glass
appearance (making dextran from sugar)
Enterococcus will produce dark, blue-black colonies.

Normal oral flora

Gram +

Comment

Gram Haemophilus
spp.

Comment

Streptococcus
spp.

Largest
proportion of
oral flora

Commonly
present in saliva,
dental plaque

Staphylococcus
spp.

Coagulase -,
Neisseria spp.
ex.
S.epidermidis

Early colonizer.
Isolate in low
number

Actinomyces
spp.

Dental
plaque

Actinobacillus
spp.

Found in
periodontal
pockets

Lactobacillus
spp.

Small
percentage
of O.F.

Porphyromona
s spp.

Adult
periodontitis

Eubacterium
spp.

Periodontitis,
dental
abscess

Prevotella spp. Adult

periodontitis
Fusobacterium Adult
spp.
periodontitis

Flora of normal, healthy dentate

mouth
% (approx)

85%

Remainder

Bacteria
Streptococci
Veillonella
Gram positive Diptheroids
Gram negative anaerobic rods

5-7%

Neissaeria

2%

Lactobacilli

1%

Staphylococci & Micrococci

Other bacteria, fungi,
protozoa & viruses

Difference between mature supra &

sub-gingival plaque
Characteristic

Supra gingival

Sub gingival

Grams stain

Gram + or -ve

Mainly Gram ve

Morphotypes

Cocci, branching
rods, filaments &
spirochaetes

Mainly rods &

spirochaetes

Energy Metabolism

Facultative, some
anaerobic

Mainly anaerobic

Energy source

Mainly ferment
carbohydrate

Many proteolytic
forms

Motility

Few

Many

Pathology

Caries & gingivitis

Gingivitis &
periodontitis

Oral flora changes with age

Time during a
lifetime

MAJOR COMPONENTS & CHANGES IN ORAL

FLORA

Newborn

Oral cavity sterile. Soon colonised by facultative

and
aerobic organisms; esp S. salivarius

6 months

Flora becomes more complex & includes

anaerobic
orgs eg. Veillonella sp. & Fusobacteria

Tooth eruption

Increase in complexity. S sanguis, S mutans and

A.viscosus appear. New habitats include hard
surfaces and gingival crevice.

Child to adult

Various anaerobes frequently found in c.

Members
of the Bacteroidaceae. Spirochaetes isolated
more frequently

Loss of teeth

Disappearance of S mutan, S sanguis,

spirochaetes
and many anaerobes

Dentures etc

Reappearance of bacteria able to grow on hard

Ecological succession
1 colonizers (Gram+)
Streptococci bind pellicle
proteins from saliva
2 colonizers (Gram-)
Bridge species - F.
nucleatum
Bind other bacteria
3 colonizers (Gram-)
Porphyromonas gingivalis

Primary
enemy of the
teeth
Streptococcus
mutans

ORAL MICROBIOLOGY
Streptococci

are the largest proportion

of the oral flora, play major role in dental
caries, purulent oral infection and infective
endocarditis.
Four main groups:
A- Facultative anaerobe
Mutans
Salivarius
Anginosus
Mitis
B- Obligate anaerobe
Peptostreptococcus

Schachtele et al.,2007 Oral Streptococci: Commensal and opportunistic

pathogens (chapter 16) in Molecular Biology of Streptococci.

Normal oral flora

Gram-positive bacteria
Dental disease is one of the most prevalent and costly
infectious diseases
1- Streptococcus (Viridans Streptococci)
These are a diverse group of species commonly found
orally (including S. mutans) and cause endocarditis after
release into the bloodstream from tooth extraction . They
are also involved in dental caries.
What causes rheumatic fever?
1-mutans group (mutans streptococci):
On tooth surfaces; produce acid rapidly; & can grow in
acid conditions
S. mutans: Most common spp. Isolated and implicated in
human caries. (fissures)
Primary pathogen in enamel caries in children & young

Normal oral flora

2- mitis group:
May act as opportunistic pathogens;
may cause infective endocarditis
S. sanguis: Found in plaque; produces IgA protease

Also known as: Streptococcus.sanguinis

As a key agent to infective endocarditis, S. sanguinis can
attach to the bloodstream and damage heart valves.
Specifically, "fibrin and platelets are deposited at the site of
endothelial cell trauma, forming a sterile vegetation where
bacteria may adhere and colonize" in the presence of bacteria
in the bloodstream. Endocarditis can proceed through
entrance of oral streptococci to the bloodstream during
dental procedures or even during normal daily activities such
as eating
S. oralis: Found on tongue, cheek, plaque, saliva; produces IgA
protease & glucans, initial colonizer
S. mitis: On soft & hard tissues; initial colonizer
(S. Pneumoniae is also in this group)
Most are A haemolytic

Normal oral flora

3- Salivarius group:
S. salivarius
Mucosal surfaces, esp. tongue; initial colonizer.
S. vstibularis
Most are A haemolytic
4- Anginosus group (Group A streptococci):
S. anginosus
Dental plaque & mucosal surfaces.
S. intermedius
An important cause of purulent disease, ex.
dental and brain abscesses.
Most are B haemolytic

Normal oral flora

Anaerobic streptococci
Peptostreptococcus species:
Carious dentine, root canals, advanced
periodontal disease
Obligate anaerobe
Lactobacillus
L. casei, L. fermentum, L. acidophilus:
~ 1% of micro flora; % increases in
advanced caries lesions of the enamel
and root surface (facultative anaerobe)

Normal oral flora

Eubacterium
Pleomorphic rods or filaments.
Caries & periodontal disease
50% of anaerobes of periodontal
pockets
Obligate anaerobe
Propionibacterium
Strict anaerobic bacilli.
Root surface caries & plaque.

Normal oral flora

Actinomyces:
Filamentous bacteria, bacilli.
Facultative anaerobe
A. israelii -> Dental plaque;
associated with dental calculus
formation
A. naeslundii -> Dental plaque

Normal oral flora

Gram-negative bacteria
Haemophilus
Facultative anaerobes (plaque, saliva
& mucosal surfaces)
Acute bacterial epiglottitis,
pneumonia,
sinusitis, meningitis
Veillonella sps
Gram - cocci

Normal oral flora

Actinobacillus
A. actinomycetemcomitans
(coccobacilli)
Subgingival plaque, -> juvenile
periodontitis,-> adult periodontitis
Capnophilic
Fusobacterium
F. nucleatum
Subgingival plaque, adult periodontitis.
Obligate anaerobe

Normal oral flora

Porphyromonas
P. gingivalis, (pleomorphic rods)
Gingival crevice, subgingival plaque -> chronic periodontitis,
dentoalveolar abscess
Obligate anaerobe
Prevotella
P. intermedia, (pleomorphic rods)
Dental plaque -> chronic periodontitis, dentoalveolar abscess
Obligate anaerobe
Treponema
T. denticola, (motile, helical cells):
Found mainly in periodontal pockets
Difficult to culture or stain , visualized by dark-field
microscope.
Obligate anaerobe

Summary
Of all bacteria present in mouth, streptococci
are predominant group.
Various species of Streptococci are
encountered in mouth. Most of these are alphahaemolytic streptococci, collectively named as
viridians streptococci
Lactobacilli are frequently found in mouth.
They are imp bacteria to promote the progress
of carious lesion after the initiation of dental
caries by Strep mutans
Veilonella is the most common among Gramnegative cocci bacteria. Fusobacterium
nucleatum is the most commonly occurring
Gram-negative anaerobic bacillus.

Relationships with the

host
Host defenses in the mouth:
Epithelial cells:
Barrier function;
Innate immunity - sensors (Toll-like receptors);
Inflammatory mediators, antimicrobial peptides;

Salivary antimicrobial factors

Mucosal antibodies (secretory IgA);
Cell-mediated immunity (T-cells);

In most cases, host defenses tolerate

oral bacteria
The predominant relationships are
commensal.

Host defense
mechanisms
Removing the microorganisms through
stimulation of salivary flow;
Specific protection:
SIg A;

Nonspecific protection:
Mucin;
Antimicrobial factors:

Lysozyme;
Lactoferrin;
Salivary peroxidase;
Leukocytes;
Complement.

Removing of the
microorganisms
The majority of microorganisms
in the mouth is removed by
washing action of saliva;
Salivary flow is stimulated by
muscle activity of lips and
tongue.

Specific protection - Secretory

IgA system
SIgA-antibodies reduce microbial
adhesion to enamel epithelium and
through:
Neutralizing enzymes microorganisms;
Neutralize toxins and viruses;
Synergy with other antibacterial agents
such as lysozyme, lactoferrin, peroxidase
and mucin;

Protects the mucosa of penetration of

antigens;
Helps complement activation.

Mucin
Provides a protective coating of
enamel and mucosa;
Catches microorganisms and
antigens like in a trap;
Limits microorganisms penetration
into tissues;
Eliminates microorganisms with
continuous updating of mucin layer
combined with washing action of
saliva flow