Endodontic microbiology / rotary endodontics courses

Endodontic Microbiology
CONTENTS
INTRODUCTION
HISTORY
FOCAL INFECTION: NEW AGE OR ANCIENT
HISTORY
ACQUISITION OF NORMAL FLORA
MICROBIAL ECOSYSTEMS IN THE ORAL CAVITY
BIOLOGY OF MICROBES IN THE ROOT CANAL
SYSTEM
MICROBIAL INTERACTIONS
BACTERIAL PATHOGENICITY AND VIRULENCE
IN ENDODONTIC INFECTIONS
PATHWAYS OF PULPAL AND PERIAPICAL
INFECTIONS
CLASSIFICATION OF BACTERIA
TAXONOMY OF ROOT CANAL FLORA
1

MICROORGANISMS ASSOCIATED WITH
PULPITIS
MICROORGANISMS ASSOCIATED WITH
NECROTIC PULPS
MICROORGNAISM ASSOCIATED WITH
PERIAPICAL LESIONS
MICROORGANISM ASSOCIATED WITH
ENDOPERIO LESIONS
MICROORGANISMS ASSOCIATED WITH FAILED
ROOT CANAL THERAPY.
CULTURE MEDIA
 Definition
 Classification
 Contents of culture media
 Methods of anaerobic culture
GRAMS STAIN
ENTEROCOCCUS FAECALIS
2

 Characteristics and strains
 Prevalence in root canal infections
 Methods to detect E. Faecalis:
 Survival and virulence factors
 Methods to eradicate E. Faecalis
FUNGI IN ENDODONTIC INFECTIONS
 Morphologic characteristics of fungi
 Mechanisms of fungal pathogenicity
 Fungi in primary endodontic infections :
 Fungi in persistent or secondary endodontic
infections
 Dentin colonization by fungi :
 Susceptibility to antimicrobial endodontic
medicaments
BIOFILMS IN ENDODONTIC INFECTIONS
 Definition of biofilm
3

 Biofilm formation
 Phenotype of biofilm bacteria
 Biofilm structures in endodontic infections
 Anti-microbial agents and biofilms
HERPES VIRUS IN PERIAPICAL PATHOSIS
EXTRARADICULAR INFECTIONS
CLINICAL BACTERIOLOGICAL TECHNIQUES
 Culturing
 Phase contrast microscopy
 Immunological techniques
MOLECULAR BASED METHODS
POLYMERASE CHAIN REACTION
 Introduction
 Principle
 Steps in PCR
4

 Variations in PCR
 Limitations of PCR
DNA – DNA HYBRIDISATION
FLORESCENCE IN SITU HYBRIDISATION (FISH)
ANTIBIOTICS FOR ENDODONTIC INFECTIONS
CONCLUSION
REFERENCES
5

INTRODUCTION
Microorganisms cause virtually all pathosis of the
pulp & periapical tissues.Bacteria play the primary
aetiological role in the development of necrotic pulps,
periapical pathosis & post treatment disease following
root canal treatment. (Kakehashi et al 1965).
Ever since the demonstration of bacteria in necrotic
pulpal tissue, about 100 years ago, the effect of oral
micro flora in the pathogenesis of pulpal and periapical
lesions has been increasingly evident.
Knowledge of the microorganisms associated with
the endodontic disease is necessary to develop a basic
understanding of the disease process and a sound
rationale for effective management of patients with
endodontic infections.
To effectively treat endodontic infections ,clinicians
must recognize the cause & effect of microbial invasion
of the dental pulp space & surrounding periapical
tissues.One of the crucial factors for the success of
treatment is eradication of microorganisms & their by
6

products from the root canal system. (Gomes et al
1996).
Molecular methods have contributed significantly to
the knowledge about the microbial species involved.
Undoubtedly, a great deal of additional research is
needed to define the specific role played by suspected
endodontic pathogens in the etiology of each form of
periradicular disease and to determine the best
therapeutic measures for the pathogen's eradication.
7

HISTORY
In 1546, Girolamo fracastorius, an Italian physician
was given the credit for being the first to recognize the
existence of tiny living organisms.
In 1667, Antony van Leeuwenhoek, observed the
soft matter of the root canals of grossly decayed tooth
and concluded that these living creatures were similar to
the once he was studying which he called
“Animalcules”. He has given the description of various
types of bacteria. He also invented the simple
microscope.
In 1857, Louis Pasteur (Father of microbiology),
established that fermentation was the result of microbial
activity. Different types of fermentation were associated
with different kind of microorganisms.
In 1876, Robert Koch (Father of Bacteriology), he
introduced staining techniques and also methods of
obtaining bacteria in pure culture using solid media. He
suggested criteria before blaming the organism
responsible for disease. Koch’ postulates
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1. Organism should be present in the pathological
lesion and its demonstration from this lesion.
2. The organism must be able to culture from the
lesion.
3. The cultured organisms must be able to produce
same lesion when injected into animals.
4. Again these organisms should be cultured from
animal lesion.
5. Antibodies against these organisms should be
demonstrable.
In 1854, Joseph Lister (Father of antiseptic
surgery) used carbolic acid spray on wound during
operation.
It took almost 200 years to know the correlation
between microorganism, pulpal and periapical disease,
which was observed by W.D. Miller in 1890, who has
called the (Father of oral microbiology). Miller had
worked in Robert Koch’s had developed methods for
staining bacteria in smears and introduced the solid
9

media techniques which made possible to obtain pure
cultures from mixed infections.
Miller observed that teeth with open pulp
chamber had bacteria in the pulp chamber this was
different from those present in root canal. He also
revealed that only a few strains of bacteria were
cultivable, when strict anaerobic techniques were
introduced it was found that the obligatory anaerobic
bacteria dominated in the infected root canal growth
percentage of bacteria in cultures of (90% of the flora).
In 1936, Fish and Mc Lean demonstrated that the
pulp and periapical tissues of vital healthy teeth are
invariably free of microorganisms when examined
histologically.
Naidorf compiled a list of generalizations
regarding organisms isolated from root canals, as
follows.
1. Mixed infections are more common than single
organim isolates.
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2. The wide variety of organisms found in root
canal partially related to the principal interests
and culture techniques.
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.
8. Treating the obvious source of infection i.e.
RCT.
In 1957, Mac Donald found G +ve organism in
root canal and predominant were streptococci etc.
11

In 1976, a dissertation was published by sandquist,
on the potential role of anaerobic bacteria in endodontic
infections, he evaluated about 32 cases, he used an
anaerobic glove box and mobile anaerobic laboratory to
collect and process the microbial samples.
In 1980, Griffee was the 1st
to compare the
symptoms associated with endodontic infections and
specific bacteria. They found symptoms such as pain,
foul odour, sinus tract, sensitivity to percussion and
localized swelling statistically greater in cases where
Porphyromonas and Prevotella species were present.
This was confirmed by Yoshida in 1987, who
found Peptococcus along with porphyromonas and
prevotella from clinically symptomatic teeth.
In 1996, Odell siqueira et al had shown a definite
relationship between the spread of symptomatic
endodontic infection and enzyme production by
anaerobic bacteria such as Eubacterium, Prevotella,
Peptococcus and Porphyromonas. These enzymes are
collagenase, chondroitinase and hyaluronidase.
12

In 1999, Baumgartner observed that the
predominant anaerobe in endodontic infection was
Prevotella nigrescens, which was separated from
Prevotella intermedia by the DNA studies
Theory of Focal infection
A focus of infection is a confined area that:
(1) contains pathogenic microorganisms
(2) can occur anywhere in the body and
(3) usually causes no clinical manifestations.
A focal infection is a localized or generalized
infection caused by the dissemination of microorganisms
or toxic products from a focus of infection.
These concepts have led to the Focal Theory of
Infection (or Theory of Focal Infection) that postulates a
myriad of diseases caused by microorganisms (bacteria,
fungi, viruses) that arise endogenously from a focus of
infection.
Foci of infection have historically been
postulated to arise from the tonsils, adenoids, sinuses and
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oral cavity with less common foci from the prostate,
appendix, gall bladder and kidney . Oral foci have
traditionally been ascribed to pyorrhea alveolaris
(periodontitis), alveolar abscesses and cellulitis, pulpless
teeth, apical periodontitis, general oral sepsis and
endodontically treated teeth with viridans group
streptococci (VGS) being the principal metastatic
microbial culprits
Ancient history
The first ‘report’ of focal infection has been
ascribed to Hippocrates who attributed the cure of a
case of arthritis to a tooth extraction . In the early 1800s,
Benjamin Rush, an American physician and signer of the
Declaration of Independence, also related arthritis cure
to tooth extraction . With the advent of the germ theory
of disease in England in the 1850s and the United States
in the early 1880s (spurred by Koch’s demonstration of
Mycobacterium tuberculosis as the cause of
tuberculosis), the newly emerging field of microbiology
became, as is common with new discoveries,associated
with wildly excessive claims for causation and cure .
14

The Autointoxication Theory became immensely
popular with the claim that bacterial stasis in the colon
caused systemic disease and colonic purging became a
treatment for gastric cancer, peptic ulcer, neuritis,
headache, endocarditis, stupidity, mentalapathy and
arthritis among other disorders . Stillpracticed today, its
major effect may be to reduce colonization resistance in
the colon against foreignpathogens by eliminating the
local protective flora.
In 1890, the dentist and physician, WD Miller,
published his treatise: The Micro-Organisms of the
Human Mouth: The Local and General Diseases Which
are Caused By Them and a year later in Dental Cosmos
first used the term: ‘focal infection’ . Miller did not
mandate removal of teeth as a focus of infection and also
suggested ‘treating and filling root canals’.
In 1900, the English physician, William
Hunter, reported in the British Medical Journal on
‘Sepsis as a Cause of Disease’ listing poor oral health
and the expanding use of ‘conservative dentistry’ (the
preservation of the dentition by dental treatment) as a
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cause of the multitude of diseases attributed to focal
infectionHunter’s remarks to the medical students at
McGill University in Montreal in 1911 ignited the fires
of focal infection: ‘No man has more reason than I to
admire the sheer ingenuity and mechanical skill
constantly displayed by the dental surgeon. And no one
has had more reason to appreciate the ghastly tragedies
of oral sepsis which his misplaced ingenuity so often
carries in its train.
The era of focal infection in medicine truly began
in 1912 when the physician, Frank Billings , formally
and independently introduced the concept of focal
infection to American physicians via case reports
ascribing distant infections to various pathogens but
going a further step to claim cures of these afflictions by
tonsillectomies and dental extractions that removed
various foci of infections. Billings was the first to
describe microorganisms cultured from septic arthritis
patients that when injected into rabbits also caused
arthritis .
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EC Rosenow was a pupil of Billings and
developed the theories of ‘elective localization’
and‘transmutation’, whereby microorganisms could
possess affinities for certain body organs and then could
alter their biological characteristics (VGS could
‘transmute’ intopneumococci or beta-hemolytic
streptococci). This theory was useful in explaining why
other researchers could not duplicate Rosenow’s results:
the original bacterium injected by Rosenow had
‘transmuted’ to the different bacteria found by other
individuals . As many prominent physicians (Charles
Mayo and Russell Cecil among others) joined Hunter,
Billings and Rosenow in advocating the focal infection
theory of disease and its remedy by surgery millions of
tonsils, adenoids and teeth were removed in an ‘orgy of
extractions’ as described by Grossman.
Endodontics came under particular scrutiny as
many physicians and dentists recommended extraction of
all endodontically treated teeth (the ‘100 percenters’)
with others recommending removal of all non-vital or
‘suspicious’ teeth and yet others suggesting that all teeth
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be removed (diseased or not) for the sake of prevention
as well as treatment (‘therapeutic edentulation’ or ‘the
clean-sweep’)
In the 1920s, Dr Weston Price published a
series of rabbit experiments and case reports
ofremarkable improvements in various medical
conditions after dental extractions and asserted that
‘practically all’ infected non-vital teeth should be
removed rather that endodontically treated to prevent or
cure focal infections.
In a later detailed review of the literature on focal
infection and particularly the studies of Rosenow,
Grossman noted that Rosenow: ‘used massive doses of
bacterial inocula of up to 10mL involume which were
then injected intravenously with the organisms being
particularly virulent’. Grossman further remarked that
Rosenow’s technique: ‘so devastates the laboratory
animal that lesions are sometimes produced in almost
every tissue and organ of the body’.
18

In the 1920s, the theory of focal infection was
widely taught as the cause of a wide range of illnesses
with infected teeth as the principal source . All pulpless
teeth were a probable focus of infection and the
extraction of healthy teeth was justified to prevent focal
infection . Endodontic education was eliminated in most
United States dental schools . C Edmund Kells , the
founder of dental radiology, was one of the few
dissenting voices describing the indiscriminate
extraction of teeth as ‘the crime of the age’ and
recommending that dentists refuse to operate on
physicians’ instructions to needlessly remove teeth.
In 1935, Cecil and Angevine published an
analysis of 200 cases of rheumatoid arthritis that
documented no benefit from tonsillectomy or dental
extractions, but rather occasional exacerbations of the
arthritis and concluded that: ‘focal infection is a splendid
example of a plausible medical theory which is in danger
of being converted by its enthusiastic supporters into the
status of an accepted fact,’ and that ‘the time has arrived
for a complete reevaluation of the focal infection theory.’
19

In 1939, Vaizey and Clark- Kennedy
demonstrated that patients made edentulous for ‘medical
reasons’ developed subsequent arthritis and dyspepsia
and that edentulism actually caused indigestion rather
than cured it.
In 1940, Reimann and Havens published the
most influential critique of focal infection theory and
observed that:
(1) the theory of focal infection had not been proved
(2) its infectious agents were unknown
(3) large groups of people whose tonsils are present are
no worse than those whose tonsils have been removed
(4) patients whose teeth and tonsils are removed often
continue to suffer from the original disease for which
they were removed
(5) any beneficial effects can seldom be ascribed to
surgical procedures alone
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(6)beneficial effects that occasionally occur after
surgical measures are often outweighed by harmful
effects or no effects at all and
(7) many suggested foci of infection heal after recovery
from systemic disease or whengeneral health is
improved with hygiene and dietary measures
The new age
Viridans group of streptococci (VGS) have been
isolated from infections in virtually every body organ
and many disease processes: pneumonia, pleural
empyema, mediastinitis, pericarditis, endocarditis, septic
thrombophlebitis, conjunctivitis, otitis media, meningitis,
osteomyelitis, cellulitis, sinusitis, brain abscess,
prosthetic joint infections, cholangeitis and liver, lung
and splenic abscesses .
These infections are similar to those that would
occur in the oral cavity with the same microorganisms.
VGS are classic purulence-producing microorganisms.
Such infections are not unexpected as VGS are
ubiquitous in the body (skin, conjunctiva, oral cavity,
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pharynx, gastrointestinal and genitourinary tracts),
possess adhesins that allow attachment to virtually any
body surface and are classically opportunistic bacteria
that initiate infections only when host tissues are
damaged, altered or diseased. The question is not
whether these bacteria produce metastatic disease, but
rather how often and what can be done, if anything, to
prevent them.
In an analysis of 281 747 consecutive blood
cultures over an 8-year period at the Mayo Clinic of
which 20 456 were microorganism-positive, 2.8% were
VGS and 4.4% were obligate anaerobes, some of which
are found in the oral cavity: Fusobacteriun nucleatum,
Prevotella intermedia and Veillonella . The prevalence of
oral obligate anaerobes was very low: Prevotella (0.1%),
Fusobacterium nucleatum (0.2%), Peptostreptococcus
(0.2%), Veillonella (0.1%) and Abiotrophia (0.4%) (47).
From these data, it is reasonable to conclude that oral
microorganisms constitute only a very limited presence
in bacteremic cultures.
22

It is apparent from well-performed studies on the
incidence and prevalence of metastatic infections with
oral microorganisms that such bacteria are rarely a cause
of systemic disease. Obligate oral anaerobes do not
appear to survive well in other body locations and VGS
are not primary pathogens but rather opportunistic
bacteria that usually require altered biologic tissue to
produce their suppurative effects.
Endodontics and focal infection
Numerous studies have attempted to determine the
significance of various microbial pathogens in pulpal
and periapical infections . Efforts have been hampered
by small sample sizes, lack of randomization or use of
consecutive cases, varied case definitions and lack of
documentation regarding the presence/absence of dental
caries and periodontal disease, different expertise in
culturing techniques, varied health status of patients and
potential microbial contamination during sampling
procedures.
23

The precise risk of bacteremia associated with
endodontic lesions and therapy is subject to some
controversy. Apparently no study exists that delineates
the incidence/magnitude of spontaneous bacteremias
from infected root canals with chronic periradicular
lesions nor any with acute periodontal abscesses. Such
bacteremias may occur during the management of
infected root canals and a good understanding of their
incidence/magnitude would be of importance.
Bender et al determined a 0–15% incidence of
bacteremia with none if the instrumentation remained
within the canal and 15% if it extended beyond the apex.
Baumgartner et al found a 3.3% incidence with non-
surgical endodontics and a 83–100% incidence with
surgical endodontics.
Al- Karaawi et al determined that the
‘cumulative’ bacteremias with a rubber dam clamp in
children was175 times greater than a tooth extraction,
while a matrix band was only four times greater
whichconflicted with another study by the same group
24

that the incidence of bacteremia using a rubber dam/
wedge/matrix band model was 9–32%.
Endodontic infections can spread to other tissues.
An abscess or cellulites may develop if bacteria invade
periapical tissues.this type of infection spreads directly
from one anatomic space to an adjacent space. This is
not an example of theory of focal infection. Endodontics
has survived the theory of focal infection,because of
recognition by the scientific community that is possible
without endangering systemic health.
25

ACQUISITION OF NORMAL FLORA:
An infant’s mouth at birth is microbiologically
sterile. Within a few hours of birth, streptococci
(especially S. Salivarius) establish themselves in the
mouth. These come from the mother or the environment.
The streptococci alter the oral environment in such a way
that it makes it more hospitable for other
microorganisms.
During infancy and early childhood other species of
streptococci, staphylococci, Neisseria and Veillonella
occupy their habitats. Lactobacilli, Actinomyces and
Fusobacteriumalso colonize in most children. With the
eruption of teeth and the availability of a solid surface,
the microbial flora undergoes dramatic changes.
S.mutans, S.sanguis and A.viscosus establish
themselves on dental hard tissues. Simultaneously,
significant increase in the number of obligate anaerobes
(strict O2 avoiding organisms) takes place. Due to the
eruption of permanent teeth, gingival sulcus depth also
increases. With adolescence spirochaetes and bacteroides
26

tend to appear in the mouth. Adulthood brings in
complex and varied organisms, which vary among
different people, at different times and at different sites.
The loss of normal teeth brings about another drastic
change in microbial flora, which reverts back to the pre-
eruptive days. Interestingly, use of artificial dentures yet
again invites those bacteria, which are fond of hard
dental tissue. The number of fungi can also increase.
ORAL FLORA:
Bacteria are by for the most predominant type of
microorganisms present in the human oral cavity. These
bacteria can be aerobic/ anaerobic depending on O2
requirement bacteria , Gram-positive / Gram negative
according to the gram stain and bacilli / cocci /
treponemes / mycoplasmas according to shape.
Bacteria:
Gram +ve bacteria
Cocci: Of all the bacteria present in the oral cavity
Streptococci constitute the single largest group. A large
number of its species are encountered in the mouth
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including S. faecalis S, Sanguis, S, mitis, S.mutans,
S.milleri and S.salivarius. The other cocci include
peptostreptococcus, micrococcus and staphylococcus.
The last one is a transient member of the oral flora.
S.mutans has been shown to be the most important
organism in the initiation of caries.
Bacilli: Lactobacilli are widely present in the human
body and frequently found in the oral cavity. 27
recognized species of lactobacilli are known with
important ones being L. acidophilus, L.salivarius and
L.casei They are known to be involved in the
progression of caries.
Other G + ve bacilli include actinomyces, Arachnia,
Eubacterium, clostridium etc.
Gram -ve bacteria:
Cocci: Veillonella are the most numerous G -ve bacteria
accounting for 10% of cultivable salivary and tongue
flora. Neisseria and moraxella are also seen.
Bacilli: Bacteroides, Fusobacterium, leptotrichia are the
significant gram -ve bacilli Bacteroid species and F.
28

nucleatum are most commonly occurring bacilli in
gingival sulcus area.
Treponemes: Spirochaetes such as T.denticola, T.orale,
T.vincenti are common inhabitants of gingival crevices
and are often associated with periodontitis
Mycoplasma: These pleiomorphic organisms are
regularly seen in plaque, calculus, periodontal pockets
etc. M. salivarium is the most predominant species while
others like M. pneumonione, M.orale, M. hominess
have also been isolated.
Fungi: Candida albicans is the most common fungus
isolated from the oral cavity & is detected in high
numbers in gingival flora, periodontal abscess, infected
root canals etc. Other fungi isolated are Pencillium,
Aspergillus, Hemispora.
Protozoa: Are present in periodontal diseases Eg:
Entamoeba
Virus: EBV, mumps virus, measles and influenza virus
can be observed during the active stage of the disease.
29

MICROBIAL ECOSYSTEMS IN THE ORAL CAVITY
The mouth provides a congenial environment for
the organisms to grow and survive. Variation in the
different ecological niches causes differences in the
number (quantitative) and in the type (qualitative) of the
organisms. These variations are due to the complex
interactions between the microbe and the host. The
establishment, survival and persistence of the flora in the
oral cavity are determined by:
Microbial Factors Host Factors
Adherence Anatomical
Production of anti-
microbial agents
Saliva
Sensitivity to anti
microbial agents
Crevicular fluid
Metabolic capability Diet
Nutritional requirement Oral hygiene
Oral and systemic disease immune defenses
Microbial Factors:
Adherence: It is a very important property for a bacteria
to become a part of the oral flora. If it cannot be retained
and subsequently multiply, it will be eliminated from the
oral cavity by the washing action of the saliva.
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Adherence occurs reversibly initially via weak, non-
specific electrostatic attraction forces and later becomes
permanent involving specific bridging between host and
normal flora. These 2 phases occur continuously. The
factors influencing adherence are
Microbial factors
1. Pilli and fimbriae
2. Cell wall components
3. Extra cellular polymers
Host factors
a) Acquired Pellicle
b) Salivary factors like IgA lectins
c) Minerals
d) Anatomic factors
e) Crevicular fluid
31

Once adherence occurs via pilli or fimbriae,
aggregation of homotypic and heterotypic organisms
occur via extra cellular polymers, IgA, lectins etc.
Antimicrobial Agents: Certain chemicals and
biologically active substances are produced by one
microorganisms against another. For eg: Bacteriocins are
produced by certain organisms against other species.
Certain metabolic end products like H2O2 can be
antagonistic to unrelated species. Thus, the vulnerability
of an organism to sucuumb to these antimicrobial factors
can determine its survival in the oral cavity.
Metabolic Capability: Organisms, which have the
metabolic machinery to utilize the available nutrients and
neutralize the deleterious chemicals produced in the oral
cavity, have the capacity to survive and propagate
Nutritional Requirements: Organisms use microbial
sources such as intra cellular storage granules or extra
cellular metabolic end products and host sources such as
directory sucrose, salivary proteins and minerals and
crevicular proteins for their growth.
32

HOST FACTORS
Aquired Pellicle: Salivary glycoproteins form a layer on
the tooth surface within minutes of cleaning the tooth.
The organism attach first to this pellicle and not directly
to the enamel.
Salivary Factors: saliva with its composition of 99%
water and 1% solids influences the oral ecology in a
number of ways:
1. Mechanical washing: prevents the overgrowth
of microbes. Swallowing of saliva causes
ingestion of aggregates of microbes.
2. Pellicle formation: salivary Glyco proteins serve
as nutrition and also promotes / inhibits bacterial
adhesion.
3. Inhibitory Action: - Lysozyme, lactoferrin,
lactoperoxididase kills exogeneous bacteria
4. Buffering capacity: to maintain pH at 6.7
prevents overgrowth of microbes which need a
high or a low pH for growth.
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5. IgA usually inhibits adherence and also alters
microbial metabolism.
Anatomical Factors: Untreated malocclusion, irregular
teeth and failing or poorly contoured restorations can
serve as stagnation sites for microbes to proliferate. Pits
and fissures serve as natural stagnation areas.
Crevicular Fluid: Is similar to serum in composition. It
contains albumin, immunoglobulins (IgG,IgA,IgM) and
transferin which aids is the host defense. Apart from
electrolytes, it also contains enzymes such as proteinase,
collagenase etc which helps to combat microbes.
MISCELLANEOUS FACTORS:
There are many other factors which significantly
influence the ecology the oral cavity
a) A low redox potential supports the growth of
anaerobic bacteria in the depth of the dental
plaque.
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b) Administration of antibiotics/ local antiseptics
depresses the number of some species while
allowing some other species to proliferate.
c) Smoking, tobacco chewing, poor oral hygiene,
carbohydrate rich diet, systemic diseases
conditions can influence the oral micro flora in
an unpredictable manner.
ANAEROBIOSIS:
The transit of air through the mouth seems to
preclude the possibility of any anaerobic organisms
thriving in it. However, culturing procedures revealed
that 50-70% of organisms cultured from the mouth were
anaerobes. The factors determining the oral environment
include:
a) Oxygen tension: is the measure of the amount of
oxygen in a gas. For air it is 21%. On the tongue it is 12-
14%, in the buccal mucosa it is 1% and in a periodontal
pocket it is 1-2%. This indicates that the dento-gingival
surface is mainly anaerobic especially where sub-
gingival plaques form. The supra-gingival plaque on the
35

labial, lingual and occlusal surface exhibited an O2
tension of 1-20%, thus producing an ideal niche for
facultative anaerobes and microaerophillic species.
b) Oxidation- reduction potential (Eh) = is the
tendency for a medium or compound to oxidize or
reduce a molecule by the removal or addition of
electrons. Microorganisms that need a +ve Eh are termed
aerobes and those needing a –ve Eh are anaerobes. A low
Eh would be expected in a microbial community which
performs a fermentative metabolism as fermentation
results in formation of reduced end products. Thus, Eh
eventually becomes negative in a site where microbes
accumulate. Portions of plaque keeps changing from
aerobic to anaerobic environment with corresponding
drop in Eh. This change coincides with a shift in the
flora from a facultative microaerophillic to anaerobic.
c) Super-oxide radical and presence of super-oxide
dismutase : The super oxide radical is the most lethal
form of O2 in biologic system as it causes both alteration
is cell membrane and inactivation of enzymes. Aerobes
contain the enzyme super-oxide dismutase which can
36

destroy this super-oxide radical while anaerobes lack it.
Hence, aerobes predominate on tongue and supra-
gingival plaque while anaerobes are in sulcus areas.
ORAL HABITATS:
The oral habitat can either be a non-tooth habitat
or a tooth habitat
The non-tooth habitat includes:
a) Oral Mucosa (lip, cheeks, palate)
b) Dorsum of the tongue
c) Saliva and tonsillar areas
The tooth habitats include:
a) Root Surface
b) Sub- gingival areas
c) Pit and fissure areas
d) Smooth surfaces – gingival to proximal contact
and gingival 1
/3 of facial and lingual surface of
clinical crown.
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The oral mucosa: harbors organisms that can overcome
abrasive forces of food, tongue and teeth for retention.
Due to the washing effects of saliva, these organisms
should be able to reproduce in great numbers to ensure
survival by reattachment.
The dorsum of the tongue: with its surface papilla
provides additional shelter to organisms. Streptococcus
salivarius and Micrococcus mucilaginous are commonly
found here and rarely on teeth.
Saliva: has a wide variety of microbes as most of the
microbes which get detached from the tissue / tooth
surface will appear in saliva. All species of streptococcus
especially S. oralis and S. salivarius are found in the
saliva.
The tooth surface: is unique as it is not protected by
surface shedding mechanisms, which occur in other
tissues. It is stable and gets covered by pellicle, which is
ideal for streptococcal attachment. Tooth habitats
favorable for harboring pathogenic plaque include:
38

Pits and Fissures: provides excellent shelters for
organisms especially S.Sanguis and other streptococci.
S. mutans can also be isolated at these sites even in the
absence of caries. Obligate anaerobes and gram –ve
species are infrequently isolated or are absent.
Smooth surface: The proximal area immediately gingival
to the contact area is protected physically and is
relatively free from the effects of mastication, tongue
movements or salivary flushing. The composition of the
microflora varies and is complex but is predominantly
actinomyces and streptococci. Tooth topography (such
as a rough surface due to defective/ poor restoration), the
size and shape of gingival papilla (apically migrated
papilla) and oral hygiene can predispose the tooth to
caries or periodontal disease.
Root surface: The proximal root surface near the CEJ is
usually unaffected by flossing due to roughness / fluting.
This favors formation of mature, isolated cariogenic
plaque. Mainly, gram -ve obligate anaerobes and
actinomyces are present here.
39

Sub-gingival areas: The initial occupants of the sub-
gingival area are an extension of the community from
the adjacent tooth surface. Metabolites released from the
plaque induce a strong inflammatory response in the
sulcus leading to vascular changes and release of Ig,
PMNL etc. This leads to a variation in the local
environment by removal of some species and
introduction of newer ones. Thus, progressive changes
from the cocci in the supra-gingival plaque to
filamentous bacteria and spirochetes in the sub-gingival
plaque is seen. Pathogenic Bacteroid melaninogenicus
can exploit this habitat and cause destruction of the
gingival epithelium.
ECOLOGICAL DETERMINANTS:
Factors determining the presence of different
organisms in different anatomic sites:
Nutrient Availability: Micro organisms like B
melaninogenicus, B. gingivalis and capnocytophaga
need hemin, certain bacteroids need vit. K, estradiol and
progesterone while T. denticola need spermin as
40

nutrients. As these nutrients are present in serum and not
is saliva / food; these organisms take refuge in the
gingival crevice where the crevicular fluid sustains them.
Thus, an organism’s nutritional requirement decides on
which niche they will occupy.
Inhibitory factors: Specific antibodies, lyzozymes,
lactoperoxidase and lactoferrin, which are present in
saliva will inhibit bacteria from growing.
Microbial acid production which reduces pH, their
reduced end products with reduces Eh, production of
H2O2 which oxidizes enzymes and presence of
bacteriocins can prevent the growth of certain species
while promoting the growth for another.
Thus, interplay of these ecological determinants
will decide which organism will reside where.
41

BENEFITS OF THE ORAL MICROBIOTA
Production of vitamins and co-factors oral bacteria
like the intestinal flora produce certain vitamins- the vit
K, biotin and riboflavin- as well as co-factors required
for our normal systemic functioning.
Production of digestive enzymes Small quantities of
amylase, lipase and protease are produced by the oral
microflora.
Prevention of colonization of exogenous organisms to
establish themselves and produce disease. This may
cause sensitivity to antimicrobial substances produced by
the oral structures.
DENTAL PLAQUE:
It can be defined as a tenacious microbial deposit
which forms on hard surfaces within the mouth and
consists of microbial cells and their products along with
host compounds mainly derived from saliva or crevicular
exudates.
42

Composition of Plaque:
It mainly consists of proliferating micro
organisms along with a scattering of epithelial cells,
leukocytes and macrophages in an adherent intercellular
matrix.
Bacteria may be up 70-80% of this material.
1mm3
of plaque weighing 1mg contains more than 108
bacteria. 200-400 different species of micro organisms
can be present in an extremely complex arrangement at
one site alone. Other than bacteria, mycoplasma,
protozoa and fungi can also be present. The material
among the bacteria is termed “inter microbial matrix”
and accounts for 25% of the plaque volume. It consists
of microbial substances, salivary material and gingival
exudates. The organic portion aids in adherence and co-
aggregation. These include poly saccharides, glyco
proteins. The inorganic components include calcium, P,
K, Mg and Na which increase on calculus formation.
43

Types of plaque:
According to site
a) Supra gingival:
1. Fissure
2. Approximal
3. Smooth Surface
b) Sub Gingival
c) Denture related
Formation of plaque:
Stage I Acquired pellicle formation:
It involves the adsorption of salivary proteins to
apatite surfaces via electrostatic ionic interactions
Stage 2: Transport and Adherence of pioneering
organisms:
The transition between pellicle to plaque is rapid.
The first constituents are cocci with small numbers of
epithelial cells and PMNL’s which initially adhere via
44

electrostatic interactions these long range interactions
facilitate a reversible adhesion. A little later specific
short range interactions occur between adhesins on
microbial surface and receptors in the pellicle and results
in irreversible adhesions. S. mutans and S. Sanguis
produce glycans in the presence of sucrose which aids in
adherence to pellicle and also promotes adherence to
other micro organisms Within 8-10 hours, about 10,000
cells/ mm2
are deposited. The pioneer ent. An extra-
cellular matrix develops consisting of polysaccharides,
salivaryorganisms multiply to produce micro colonies
which with time become conflu glycoproteins.
Stage 3: Co-Aggregation (Within 1-3 days):
The metabolic products of the pioneering
organisms alter the immediate environment such as
creating conditions with a low redox potential suitable
for anaerobes. Other organisms become incorporated
into the plaque with a resulting gradual increase in
microbial complexity, biomass and thickness
Stage 4: Multiplication
45

The proliferation of the attached micro-
organisms and further aggregation produces a confluent
growth and a biofilm.
Stage 5: Seeding:
Detachment of cells form this bio film into the
saliva results in colonization of fresh sites.
Growth And Accumulation Of Supra-Gingival
Plaque:
The total plaque mass that develops is mainly
determined by the multiplication of the attached bacteria
as well as cohesion of bacterial cells. Thus, growth is by:
a) Adhesion
b) Cohesion
Factors which determine the ultimate composition
and Pathogenicity of plaque are:
Bacterial factors
Extra-cellular products: eg.glucans produced by
S.mutans are sticky and help in coaggregation
46

Bacterial interactions: are important for bacteria that
cannot attach directly to the tooth.Eg. Violonella which
of incapable of direct attachment accumulates on
A.viscosis.
Plaque ecology: formation and growth of plaque is an
orderly sequence of ‘replacement community’ with each
community modifying the local environment of that site.
This process of mutual change of community and its
environment is called ‘ecological succession’.
Host factors:
Oral cleansing mechanism such as salivary flow,
movements of tongue and cheek control the plaque
formation rate.
Saliva influences
a) The plaque pH by its buffering action and acid
neutralization.
b) Inhibition of adherence by coating the surface
receptors
47

c) Inhibition of adherence via promotion of
bacterial agglutination
Immune response: Main sources of immune
components in oral cavity are:
a) IgA and antibodies in saliva which compete with
bacterial adherence and influences their
metabolism, growth and accumulation.
b) Crevicular fluid which contains antibodies,
leukocytes, complement factors etc.
Structure of plaque:
1) Bacterial cells near to the enamel surface tend to
have a reduced cytoplasm:cell wall ratio,
indicating metabolic inactivity.
2) In some areas (esp. outer surface) cocci attach
and grow on the surface of filamentous
microorganisms giving a ‘corn-cob’ appearance.
3) There is a tendency for filamentous bacteria to
orient themselves at right angles to the enamel
surface producing a ‘palisade effect’.
48

4) Bacteria containing glycogen-like storage
granules intra-cellularly are seen indicating that
these bacteria are relying on themselves for
nutrition.
Sub gingival plaque: Supra-gingival plaque can induce
inflammatory changes in the gingiva, which leads to
edematous enlargement of the gingiva. This leads to an
increase in the capacity of the sub-gingival area for
bacterial colonization. Since this area is protected from
normal cleaning mechanisms, has increased crevicular
fluid and has desquamated epithelial cells, it favors a
new ecological environment favoring anaerobic bacteria.
Thus, Gram +ve filaments, Gram –ve cocci, rods and
spirochetes predominate here. This flora is associated
with root caries periodontitis, gingivitis etc.
49

Bacterial Biofilms
Biofilm is a mode of microbial growth where
dynamic communities of interacting sessile cells are
irreversibly attached to a solid substratum, as well as
each other, and are embedded in a self-made matrix of
extracellular polymeric substances (EPS).
A microbial biofIlm is considered a community that
meets the following four basic criteria: The
microorganisms living in the community
(1) must possess the abilities to self-organize
(autopoiesis)
(2) Resist environmental perturbations (homeostasis)
(3) Must be more effective in association than in
isolation (synergy)
(4) Respond to environmental changes as a unit rather
than single individuals (communality).
Dental plaque is the typical example of a biofilrn.
50

Bacteriological studies were conducted on free-
floating bacterial cells (planktonic state), ignoring the
importance of the sessile bacterial cells (biofilm state).
BiofIlms can be formed wherever there is a flow
of fluid, microorganisms, and a solid surface. It is one of
the basic survival strategies employed by bacteria in all
natural and industrial ecosystems in response to
starvation.
The sessile bacterial cells in a biofilrn state differ
greatly from their planktonic counterparts. Inside a
biofIlm, the bacterial cells exhibit altered phenotypic
properties and are protected from antimicrobials,
environmental stresses,nbacteriophages, and phagocytic
amoebae.
Biofilms are responsible for most of the chronic
infections and almost all recalcitrant infections in human
beings, as bacteria in a biofilm are resistant to both
antibiotic therapy and host defense mechanisms.
However, common biofilms found in the oral cavity and
gastrointestinal tract are protective in nature. These
51

biofilms featuring a large number and diverse array of
commensal bacteria hinders the adherence of pathogenic
microorganisms.
ULTRASTRUCTURE OF BIOFILM
The basic structural unit of a biofilm is the micro
colonies or cell clusters formed by the surface adherent
bacterial cells. Microcolonies are discrete units of
densely packed bacterial cell (single or multispecies)
aggregates.
There is a spatial distribution of bacterial cells
(microcolony) of different physiological and metabolic
states within a biofilm. A glycocalyx matrix made up of
EPS surrounds the micro colonies and anchors the
bacterial cell to the substrate.
Eighty-five percent by volume of the biofilm
structure is made up of matrix material, while 15% is
made up of cells.
A fresh biofilm matrix is made of biopolymers
such as polysaccharides, proteins, nucleic acids, and
salts.671,6 The structure and composition of a matured
52

biofilm is known to modify according to the
environmental conditions (growth conditions, nutritional
availability, nature of fluid movements, physicochemical
properties of the substrate, etc).
Representation of the structure of a mature
biofilm.
A viable, fully hydrated biofilm appears as
"tower-" or "mushroom" –shaped structures adherent
to a substrate. The overall shape of a biofilm structure is
determined by the shear forces generated by the flushing
of fluid media.
53

Biofilms formed in high-shear environments
have shown that the microcolonies are deformed by
these forces to produce tadpole-shaped oscillation in the
bulk fluid.
Advanced microscopy of living biofilms have
revealed that single-species biofilms growing in the
laboratories to complex multispecies biofilms growing in
the natural ecosystems have similar basic community
structure, with some subtle variations.
The water channels, are regarded as a primitive
circulatory system in a biofilm, intersect the structure of
biofilm to establish connections between the
microcolonies. Presence of water channels facilitates
efficient exchange of materials between bacterial cells
and bulk fluid, which in turn helps to coordinate
functions in a biofilm community.
The structural feature of a biofilm has the highest
impact in chronic bacterial infection is the tendency of
rnicrocolonies to detach from the biofilm community.
54

There- are two main types of detachment
process: erosion (the continual detachment of single cells
and small portions of the biofilm) and sloughing (the
rapid, massive loss of biofilm). Detachment has been
understood to play an important role in shaping the
morphological characteristics and structure of mature
biofilm. It is also considered as an active dispersive
mechanism (seeding dispersal).
Bacterial colonization and biofIlm formation can
alter the physicochemical properties of many substrates.
The EPS and the metabolic activities of bacteria within a
biofIlm determine the physicochemical characteristics of
the substrate.
Bacterial cell surfaces are typically anionic due to
the presence of carboxylate or phosphate moieties in
capsular or cell wall polymers.
Therefore, a colonized substrate will acquire an
anionic character, regardless of its original
physicochemical properties. Under a favorable
55

environment, metal ions including Ca2+, Mg2+, and
Fe3+ will readily bind to and precipitate within anionic
biofilms, inducing biofilm- mediated mineralization.
CHARACTERISTICS OF BIOFILM
Bacteria in a biofIlm state show distinct capacity
to survive tough growth and environmental conditions.
This unique capacity of bacteria in a biofilm state
is due to the following features:
a) BiofIlm structure protects the residing bacteria
from environmental threats
b) Structure of biofIlm permits trapping of nutrients
and metabolic cooperativity between resident
cells of same species and/or different species
c) Biofilm structures display organized internal
compartmentalization, which allows bacterial
species with different growth requirements to
survive in each compartment
56

d) Bacterial cells in a biofIlm community may
communicate and exchange genetic materials to
acquire new traits.
Protection of biofilm bacteria from environmental
threats
Bacteria residing in a biofilm community
experience certain degree of protection and homeostasis.
Many bacteria are capable of producing polysaccharides,
either as cell surface structures (eg., capsule) or as
extracellular excretions (eg., EPS).
EPS covers biofIlm communities and creates a
micro niche favorable for the long-term survival and
functioning of the bacterial communities.
EPS protects the biofilm bacteria from a variety
of environmental stresses, such as UV radiation, pH
shifts, osmotic shock, and desiccation. EPS can sequester
metals, cations, and toxins.
Metallic cations such as magnesium and calcium
minimize electrostatic repulsion between negatively
57

charged biopolymers, increasing the cohesiveness of the
EPS matrix.
Diffusion is the predominant transport process
with in cell aggregates. The diffusion distance in a
planktonic cell is on the order of magnitude of the
dimension of an individual cell, while the diffusion
distance in a biofilm is on the order of the dimension of
the multicellular aggregate.
A biofilm that is 10 cells thick will exhibit a
diffusion time 100 times longer than that of a single cell.
Nutrient trapping and establishment of metabolic
cooperativity in a biofilm
An important characteristic of biofilms growing
in a nutrient-deprived ecosystem is its ability to
concentrate trace elements and nutrients by physical
trapping or by electrostatic interaction.
The water channel connects the outer fluid
medium with the. interior of the biofilm, ensuring
nutrient availability to microbial communities deep
inside the biofilm structure.
58

The complex architecture of a biofilm provides
the opportunity for metabolic co-operation, and niches
are formed within these spatially well-organized
systems.
Bacterial microcolonies in a biofilm structure are
exposed to distinct environmental signals. For example,
cells located near the center of a microcolony are more
likely to experience low oxygen tensions compared to
cells located near the surface. Moreover, due to the juxta
positioning of different microorganisms, cross feeding
and metabolic co operativity between different species of
microorganisms are seen in a biofilm.
Studies have reported the production of essential
growth factors such as hemin by W. recta to support the
growth of fastidious organisms such as P. gingivalis in a
biofilm. In addition, each bacterial species residing in a
biofIlm possess different array of lytic enzymes, and a
biofilm as a unit is equipped with a wide spectrum of
enzymes that can degrade complex organic materials.
For instance, bacterial species possessing proteolytic
59

enzymes make nutrients available to all other bacteria in
a protein-rich environment.
Organized internal compartmentalization in biofilm
A mature biofilm structure displays gradients in
the distribution of nutrients, pH, oxygen, metabolic
products, and signaling molecules within the biofilm.
Cell-cell communication in a biofilm. .Some bacteria
can produce chemical signals (green) and other bacteria
60

from the same species or from different species or strain
can respond to them (red).
This would create different microniche that can
accommodate diverse bacterial species within a biofilm.
The gradients in nutrients, chemicals, and gases,
observed in a biofilm structure, are influenced by the
type of nutrients and the physiological requirements of
the residing microorganisms.
In a multispecies biofilm involving aerobic and
anaerobic bacteria, oxygen is consumed by the aerobic
and facultative anaerobic species, making the
environment rich in carbon dioxide and other gases.
When the aerobic bacteria residing on the surface
of the biofilm consumes all available oxygen, the interior
of the biofilm can be absolutely anaerobic that it can
even support the growth of obligatory anaerobes.Despite
the fact that oral cavity is abundant in oxygen, anaerobic
microbes are found to dominate oral biofilms because of
the possible redox gradient formed within the biofilm
structure.
61

Bacterial cells residing in a biofilm communicate,
exchange genetic materials, and acquire new traits
Bacterial biofilm provides a setting for the
residing bacterial cells to communicate with each other.
Some of these signals, produced by cells, may be
interpreted not just by members of the same species, but
by other microbial species too. Communications
between bacterial cells residing in a biofilm is attained
through signaling molecules, by a process called quorum
sensing. Quorum sensing is mediated by low molecular
weight molecules, which in sufficient concentration can
alter the metabolic activity of neighboring cells, and
coordinate the functions of resident bacterial cells within
a biofilm. Exchange of genetic materials between
bacterial species residing in a biofilm will result in the
evolution of microbial communities with different traits.
62

Planktonic bacteria; The concentration of chemical
signals secreted by the planktonic cells is low.
Close proximity of microbial cells in a biofilm
facilitates genetic exchange between bacteria of
genetically distant genera. Even the possibility of gene
transfer between a commensal organism (Bacillus
subtilis) and oral biofilm bacteria (Streptococcus
species) has been demonstrated.
The horizontal gene transfer is of importance in
human diseases caused by bacterial biofilm as it can
63

result in the generation of antibiotic-resistant
bacterial population.
Biofilm bacteria; Biofilm cells are held together in
dense populations, so the secreted chemical signals
higher concentrations. Signal molecules then re-cross the
cell membranes and trigger changes in genetic activity.
Gene transfer between bacteria residing in a
biofilm is thought to be mediated by bacterial
conjugation. The presence of diverse bacterial species in
a biofilm presents a pool of genetic codes for nutrient
breakdown, antibiotic resistance, and xenobiotic
metabolism.
64

Cell-cell communication can result in the
coordinated behavior of microbial population residing in
a biofilm.
DEVELOPMENTOF BIOFILM
The three major components involved in biofilm
formation are bacterial cells, a solid surface, and a
fluid medium.
65

Diagram showing different factors influencing
initial bacteria-substrate interaction
Stage 1; the first step involved in the development of
biofilm is the adsorption of inorganic and organic
molecules to the solid surface creating what is termed a
conditioning layer
Stage 2: During dental plaque formation, the tooth
surface is conditioned by the saliva pellicle. Once the
conditioning layer is formed, the next step in biofilm
formation is the adhesion of microbial cells to this layer.
Amongst the pioneer organisms, the oralis group
of streptococci is the major population to form a
bacterial monolayer on the salivary pellicle coated tooth
surface.
Factors that affect bacterial attachment to a solid
substrate. These factors include pH, temperature, surface
energy of the substrate, flow rate of the fluid passing
over the surface, nutrient availability, length of time the
bacteria is in contact with the surface, bacterial growth
66

stage, bacterial cell surface charge, and surface
hydrophobicity.
Physicochemical properties such as surface
energy and charge density determine the nature of initial
bacteria-substrate interaction (Phase 1: transport of
microbe to substrate surface).
In addition, the microbial adherence to a
substrate is also mediated by bacterial surface structures
such as fimbriae, pili, flagella, and EPS (glycocalyx).
Molecular-specific interactions between bacterial
surface structures and substrate become active in this
phase (Phase 2: initial non-specific microbial-
substrate adherence phase).
Initially, the bonds between the bacteria and the
substrate may not be strong. However, with time these
bonds gains in strength, making the bacteria-substrate
attachment irreversible. Finally, a specific bacterial
adhesion with a substrate is produced via polysaccharide
adhesin or ligand formation (Phase 3: specific
67

microbial substrate adherence phase). In this phase,
adhesin or ligand on the bacterial cell surface will bind
to receptors on the substrate. Specific bacterial adhesion
is less affected by many environmental factors such as
electrolyte, pH, or temperature.
Stage 3; the bacterial growth and biofilm expansion.
During this stage, the monolayer of microbes attracts
secondary colonizers forming microcolony, and the
collection of micro colonies gives rise to the final
structure of biofilm.
The lateral and vertical growth of indwellers
gives rise to microcolonies similar to towers. A mature
biofilm will be a metabolically active community of
microorganisms where individuals share duties and
benefits.
The bacterial cells in a matured biofilm will
exhibit considerable variation in its genetic and
biochemical constitutions compared to its planktonic
counterparts. Two types of microbial interactions occur
at the cellular level during the formation of biofllm. One
68

is the process of recognition between a suspended cell
and a cell already attached to substratum. This type of
interaction is termed co-adhesion
69

Stages in the development of biofilm
70

In the second type of interaction, genetically
distinct cells in suspension recognize each other and
clump together. This type of interaction is called
coaggregation
Resistance of Microbes in Biofilm to Antimicrobials
The nature of biofilm structure and physiological
characteristics of the resident microorganisms offer an
inherent resistance to antimicrobial agents, such as
antibiotics, disinfectants, or germicides.
71

The resistance to antimicrobial agents has been
found to amplify more than thousand times for microbes
in biofilm, when compared to planktonic cells.
An identical biofilm exposed to vancomycin and
rifampin for 72 hours at concentrations exceeding the
minimum inhibitory concentration (MIC) and minimum
bactericidal concentration (MBC) for the microorganism.
ENDODONTIC BIOFILMS
Endodontic microbiota is established to be less
diverse compared to the oral microbiota. This transition
in the microbial population is more conspicuous with the
progressionof infection. Progression of infection alters
the nutritional and environmental status within the root
canal.
The root canal enviroment apparently becomes
more anaerobic and the nutriton level will be depleted.
These changes will offer a tough ecological niche
for the surviving microorganisms. Furthermore, clinical
investigations have shown that the complete disinfection
of root canal is very difficult to achieve. Microbes are
72

found to persist in the anatomical complexities such as
isthmuses and deltas and in the apical portion of root
canal system. Often, bacterial activities may not be
confined to intracanal spaces, but also access regions
beyond the apical foramen.
These anatomical and geometrical complexities
in the root canal systems shelter the bacteria from
cleaning and shaping procedures.
Additionally, biofilm mode of bacterial growth
offers other advantages such as
a) resistance to antimicrobial agents
b) increase in the local concentration of nutrients
c) opportunity for genetic material exchange
d) ability to communicate between bacterial
populations of same and/or different species
e) produce growth factors across species
boundaries.
Endodontic bacterial biofilms can be categorized as
73

1. Intra canal biofilms
2. Extra radicular biofilms
3. Periapical biofilms
4. Biomaterial centered infections.
INTRACANAL MICROBIAL BIOFILMS
lntracanal microbial biofilms are microbial
biofilms formed on the root canal dentine of an
endodontically infected tooth.
A detailed description on the intracanal bacterial
biofilm was documented by Nair in 1987.
74

It was suggested that the intracanal micro biota in
an endodontically infected teeth existed as both loose
collection and biofilm structures, made up of cocci, rods,
and filamentous bacteria.
Monolayer and/or multilayered bacterial biofilms
were found to adhere to the dentinal wall of the root
canal. The extracellular matrix material of bacterial
origin was also found interspersed with the cell
aggregates in the biofilm
Studies have established the ability of E. faecalis
to resist starvation and develop biofilms under different
environmental and nutrient conditions (aerobic,
anaerobic, nutrient-rich, and nutrient-deprived
conditions).
E. faecalis under nutrient-rich environment (aerobic and
anaerobic) produced typical biofilrn structures with
characteristic surface aggregates of bacterial cells and
water channels. Viable bacterial cells were present on the
surface of the biofilm. Under nutrient-deprived
75

environment (aerobic and anaerobic), irregular growth of
adherent cell clumps were observed.
76
Nutrient- deprived condition after 4 weeks,
Nutrient- deprived condition after 1 week,

Laser scanning confocal microscopy displayed
many dead bacterial cells and pockets of viable bacterial
cells in this biofilm structure.
In vitro experiments have revealed distinct stages
in the development of E. faecalis biofIlm on root canal
dentine.
77
Nutrient-rich condition after 4 weeks.
Nutrient- rich condition after 1 week

In stage 1, E. faecalis cells adhered and formed
microcolonies on the root canal dentine surface. In stage
2, they induced bacterial-mediated dissolution of the
mineral fraction from the dentine substrate. This
localized increase in the calcium and phosphate ions will
promote mineralization (or calcification) of the
E.faecalis biofilm in stage 3.
Extraradicular Microbial Biofilms
Extraradicular microbial biofilms also termed
root surface biofilms are microbial biofilms formed on
the root (cementum) surface adjacent to the root apex of
endodontically infected teeth.
The extraradicular biofilm structures were
dominated by cocci and short rods, with cocci attached
to the tooth substrate. Filamentous and fibrillar forms
were also observed in the biofilm.
A smooth, structureless biofilm structure
consisting of extracellular matrix material with
embedded bacterial cells was noticed to coat the apex of
the root tip adjacent to the apical foramen. There was no
78

obvious difference in the biofilm structures formed on
the apical root surface of teeth with and without sinus
tracts.
Bacterial biofilms in the areas of the root
surfaces between fibers and cells and in crypts and holes.
The biofilm contained varying degrees of extracellular
matrix materials (glycocalyx).
The root surface biofilms were mostly
multispecies in nature associated with periapical
inflammation and delayed periapical healing in
orthograde treatment .
79

PERIAPICAL MICROBIALBIOFILMS
Periapical microbial biofilms are isolated
biofilms found in the periapical region of an
endodontically infected teeth. Periapical biofilms mayor
may not be dependent on the root canal. The micro biota
in the majority of teeth associated with apical
periodontitis is restricted to the root canal, as most of the
microbial species that infect the root canal are
opportunistic pathogens that do not have the ability to
survive host defense mechanism in the periapical tissues.
80

Members of the genus Actinomyces and the
species P. propionicum have been demonstrated in
asymptomatic periapical lesions refractory to endodontic
treatment. These microorganisms have the ability to
overcome host defense mechanisms, thrive in the
inflamed periapical tissue, and subsequently induce a
periapical infection.
Clinical investigation detected Actinomyces in 72
of 129 (55.8%) clinical samples. Of those, 41 of 51
(80.4%) were from infected root canals, 22 of 48
(45.8%) were from abscesses, and 9 of 30 (30%) were
associated with cellulites.
ENDODONTIC MICROBIOLOGY
APICAL PERIODONTITIS is caused by a
microbial infection in the root canal. [Miller, 1894;
Kakehashi et al, 1965; Sundquist; Bergenholtz;
Moller; Fabricius and others].
In majority of the cases, the causative agents are
bacteria, although yeasts are also occasionally reported.
NON – MICROBIAL CAUSES like:
81

a) Severe occlusal interference
b) Chemical irritation caused by materials used in
endodontic therapy.
c) allergic reactions to these materials may also be
responsible for inflammatory apical reactions
with accompanying bone loss.
HOWEVER,SCIENTIFIC EVIDENCE IS LACKING
Bacterial Pathogenicity and Virulence in
Endodontic Infections
Bacterial pathogenicity and virulence depends on:
(i) Ability to cause the disease
(ii) Ability to cause one or more of the following
symptoms – pain, tenderness to percussion, swelling
and / or open sinus tract, or
(iii) The ability of bacteria to have a negative impact on
the long term prognosis of the endodontic treatment of
apical periodontitis.
82

(i) ABILITY TO CAUSE DISEASE
The pathogenicity of bacteria in apical
periodontitis is based on ANTIGENIC STRUCTURES
or OTHER PROPERTIES with a potential to induce a
cascade of immunological reactions in the periapical
tissues, resulting in bone resorption at the tooth apex
around the root canal opening.
[Endodontics and Dental Traumatology 1990, 6, 89-
96]
(ii) ABILITY TO CAUSE ACUTE SYMPTOMS
In endodontics, the number of species regarded
virulent (ability to cause acute symptoms) is
considerably lower than the number of pathogenic
species. Presence of only a limited number of species in
the root canal flora seems to be closely related to the
occurrence of symptoms.
[JOE, 1982; JOE, 1998 ; JADA, 1989; Oral
Microbiology and Immunology, 1992]
83

Most of the bacteria regarded as virulent in apical
periodontitis are Gram –ve anaerobic rods.
[Oral Surgery, oral Medicine, Oral Pathology –
1980, 50, 457-61 ; JOE – 1989 (Sundquist et al);
Endodotnics and Traumatology – 1989 (Haapasalo)]
Microbial factors responsible for acute symptoms
may be surface components (CAPSULE) and proteolytic
enzymes that are capable of degrading host defense
proteins (immunoglobulins, complement proteins, etc.).
[Journ Periodon Res (1987) – Sundquist et al. Journ
Med Microbiol (1985) – Sundquist et al]
(iii) ABILITY OF BACTERIA TO HAVE A
NEGATIVE IMPACT ON PROGNOSIS OF THE
THERAPY
Success in RCT depends on success in
controlling the infection. If bacteria are resistant to
therapy or the host defense mechanisms and surrive
within the root canal system or in the periapical area,
healing of the periapical lesions is compromised. For
example, the role of E. faecalis in failed RCT.
84

Microbial factors responsible for long term failures
are probably related to
(a) In vivo resistance to phagocytosis
(b) The ability to survive in changing ecological
conditions within the root canal system with only
limited supply of nutrients.
BACTERIAL VIRULENCE FACTORS :
Microbes have numerous virulence factors:
(i) CAPSULE – protects the microbes from phagocytosis
(ii) FIMBRIAE – participate in aggregation of bacteria
or attachment to tissues
(iii) PILLI – extend from one bacterium to another
during conjugation and exchange DNA for virulence
factors.
(iv) LIPOPOLYSACHARIDES – (ENDOTOXIN) –
weak antigens that bind to host antibodies and decrease
host response; capable of activation of complement
system and bone resorption.
85

Conc. of endotoxin in symptomatic teeth is
higher than asymptomatic teeth.
(v) ENZYMES – neutralise immunoglobulins and
complement system components.
(vi) EXTRACELLULAR VESICLES – Carry same
surface antigen as parent bacteria; may contain enzymes
or other toxic agents; involved in haemagglutination
hemolysis, bacterial adhesion and proteolytic action on
host tissue.
(vii) FATTY ACIDS – short chain F.A. like – propionic
acid, butyric acid and isobutyric acid
a) Affect neutrophil chemotaxis, degranulation,
chemiluminescence and phagocytosis.
b) Butyric acid exerts greatest inhibition of T cell
blastogeneisis and stimulates the production of
IL-1 (bone resorption).
(viii) POLYAMINES – Spermine, spermidine,
cadaverine, putresceine
86

a) biologically active compounds involved in
regulation of growth, regeneration of tissues and
modulation of inflammation.
b) Teeth that are painful to percussion or have
spontaneous pain have been shown to have a
higher conc. of total polyamines in necrotic pulp.
[Oral Microbiol Immunol – 1991, 6 : 17]
(ix) MICROBIAL COAGGREGATION AND SELF –
AGGREGATION
a) The combination of different species of bacteria
was found to be more virulent in mice than the
organisms in pure culture. [Baumgarther et al]
b) The additive and synergistic relationships
between organisms in polymicrobial infections
may increase the overall pathogenicity.
c) Efficacy of various irrigants and intra canal
medicaments is related to the nature of the
organisms in a biofilm and to contact time. [Int
Endodon J – 34 : 300, 2001]
87

BACTERIAL COMPONENTS
 Endotoxin (LPS)
 Lipoteichoic Acid  COMPLEMENT
ACTIVATION
 Peptidoglycan ↓
↓ ↓ Chemotaxis
of PMNs
B-CELL STIMULATION PERSISTENCE IN
AND STIMULATION OF
MACROPHAGES
↓ ↓
Lymphokines (OAF) Interleukin 1
a. Stimulation of T-
lymphocytes
↓ b. Stimulation of
fibroblasts
c. Production of
prostaglandins
BONE RESORPTION
↓
↓
PERIAPICAL GRANULOMA
PERIAPICAL CYST
88

Role of bacteria and their components in the
pathogeneisis of pulpal and periapical inflammatory
lesions. [JOE 1988; 14 ; 363-71]
CLASSIFICATION OF BACTERIA
Need for classification?
(i) Helps to improve our understanding of the
etiology and pathogeneisis of odontogenic
infections
(ii) In cases of persistent infections, detailed
information of the species involved is
conducive to efficient and successful therapy.
CLASSIFICATION SCHEME FOR BACTERIA IN
ENDODONTIC INFECTIONS
Gram-ve
cocci
Gram-ve rods Gram +ve
cocci
Gram +ve
rods
Anaerobic
bacteria
Veillonella Prevotella
Porphyromonas
Bacteroides
Peptotrepto-
Coccus
Eubacterium
Bifidobact
Clostridium
89

Fusobacterium
Campylobacter
Selemonas
Treponema
Propioniba
Lactobacillus
Actinomyces
Gram-
cocci
Gram-rods Gram + cocci Gram + rods
Facultative and aerobic bacteria
Neisseria Actinobacillus
Hemophilus
Eikenella
Caphocytophaga
Enterobacter
Klebsiella
Escherichia
Citrobacter
Pseudomonas
Xantomonas
Proteus
Streptococcus
Gemella
Enterococcus
Staphylococcus
Micrococcus
Propionibact
Lactobacillus
Actinomyces
Bacillus
Cornebact
Development Of Classification Of Dark Pigmenting
Anaerobic Rods (Formerly Black Pigmented
Bacteroides) In Humans
90

The “Bacteroides melanogenicus” and “other
bacteroides strains” have been BIOCHEMICALLY
DIFFERENTIATED into two major genera,
porphyromonas (assacharolytic) and prevotella
(saccharolytic)
Porphyromonas
asaccharolyticus
BACTEROIDES
ASSACHAROLYTICS
Porphyromonas
gingivalis
Porphyromonas
endodontalis
BACTEROIDES
MELANOGENICUS
BACTEROIDES
INTERMEDIUS
Prevotella
intermedia
Prevotella
nigrescens
Prevotella
corporis
BACTEROIDES
MELANOGENICUS
Prevotella
melaninogenica
Prevotella
denticola
Prevotella
loescheii
91

DISTRIBUTION OF CANDIDATE ENDODONTIC
PATHOGENS ACCORDING TO THEIR
RESPECTIVE PHYLA :
According to a study by Siqueira (2003), the
bacteria as yet identified by molecular and cultural
analyses fall into 6 bacterial groups or phyla
Actinobacteria, proteobacteria, fusobacteria
Bacteroidetes, Firmicutes and spirochetes.
Species from primary endodontic infections are
basically from grp 4, 5, 6
MICROBIOLOGY OF THE DENTINO-PULPAL
COMPLEX
Microbiology of Dentin and Pulp under Caries
Caries is regarded the main source of bacteria in
infections of the pulp and periapical area.
According to a study by Engstrom (1964):
93% - Gram +ve rods
92

32% - Gram +ve cocci
11.6% - Gram –ve cocci
5% - Gram –ve rods
LACTOBACILLUS was the most common group
followed by PLEOMORPHIC RODS & FILAMENTS.
a) Microorganisms in the front line are etiologically
significant in development of pulpitis.
b) These bacteria are the first to invade the pulp and
root canal.
c) However, microbial products, organic acids,
other metabolites and various enzymes can be
found ahead of the front line and may cause both
reversible and irreversible pulpitis. [Bergenhaltz
et al 1986].
BIOLOGY OF MICROBES IN THE ROOT CANAL
SYSTEM
HOST – PARASITE RELATION
93

Regulated by several factors. In case of an open
communication to the root canal – whole saliva and
plaque flora. Whereas in an invasion via carious lesion –
predominantly streptococci, gram +ve facultative and
anaerobic rods.
Various possibilities for entrance (dentinal
tubules, fractures, lateral canals, hematogenous route)
regulate the number of infecting bacterial species.
Fate of bacteria entering the pulp is determined by
a) Environmental factors in RCs.
b) Host related factors
c) Microbial interactions
Due to lack of Collateral Circulation,
inflammatory reactions of pulp are irreversible and the
pulp gradually becomes necrotic. The necrotic tissue
and inflammatory exudate exert selective pressure to
give an ecological advantage to those bacteria which are
favored by this specific environment.
94

Proteolytic Bacteria – Prevotella, Porphyromonas,
Peptostreptococcus and Fusobacterium may use tissue
and serum proteins as nutrients.
May also evade host defense factors by
destruction of immunoglobulins and complement factors.
Prevotella and Porphyromonas produce a
POLYSACHARIDE CAPSULE which makes them
resistant to phagocytosis and intracellular destruction by
neutrophils.
Thus, these bacteria not only surrive the host’s
defenses, they start to multiply and their growth is
continuously regulated by availability of nutrients.
Also, because of the limited availability of O2
which can penetrate the root canal, the facultative
microorganisms consume the available o2 and favors
anaerobic microorganisms.
Thus, flora in the necrotic root canal changes
with time to a predominantly proteolytic anaerobic flora
capable of evading host defense mechanism.
MICROBIAL INTERACTIONS
95

No bacteria can be regarded as Indigenous as the
pulp and canal are sterile from the start. Competition
between different species starts once they enter the root
canal.
a) Competition for nutrients and space
b) Antagonistic forces – production of toxic
metabolites – H2O2, NH3, S – Compounds, acids,
bacteriocins. (lactobacilli and streptococci) which
inhibits the access of exogenous bacteria to oral
cavity and resp. tract.
c) Symbiotic relations – (1) Consumption of O2 by
facultative bacteria favours anaerobic species (2)
Proteolytic degradation of immunoglobulins and
serum proteins not only destroys host defense
factors but also releases peptides and amino acids
which are nutrients to other species.
Thus, positive and negative microbial
interactions may therefore significantly regulate the
flora by selecting bacterial species that are present
together those that avoid each other.
96

A/c to Sundquist, 1992 – by odds, ratio certain
bacteria were more likely to be found together in the root
canal flora.
High Odds Ratio (>0. 5)
P. intermedia – P.
anaerobius
P. intermedia – P. micros
P. endodontalis – F.
nucleatum
P. anaerobius – E.
alactolyticum
P. anaerobius – E. lentum
P. micros – E. lentum
Low Odds Ratio (< 0.5)
propionicus
endodontalis
E. alactolyticum –
Actinomyces
F. nucleatum – S.
anginosus
F. nucleatum – S. mitis
F. nucleatum –
Actinomyces
P. anaerobius – V. Parula
P. micros – A. israelli
P. micros – P. Propionicus
97

Morphologic Localisation Of Bacteria
(A) Infection in the Main Canal and Lateral Canals
The living conditions of infected microbial flora
in the root canal system depend on:
(i) Redox Potential (amount of O2 present) : After the
development of pulp necrosis, the redox potential is
quite low which contributes to dominance of anaerobic
bacteria.
(ii) Availability Of Nutrients : Possible sources of
nutrients are:
a) Necrotic pulp tissue
b) Diffusion of inflammatory exudates and body
fluids (via apical foramen, lateral canals, patent
dentinal tubules).
c) Diffusion of oral fluids (via caries lesion,
dentinal tubules, leakage by a filling)
98

(iii) Host Defenses
Limited possibilities to function due to absence
of vascular system.
All these factors result in microorganisms in
apical periodontitis being located in main canal.
a) Usually infection does not extend beyond the
apex and bacteria cannot be detected outside the
root. [JOE, 1992].
b) The apical flora is often delineated from the
periradicular tissues by a dense accumulation of
polymorphonuclear leukocytes at and near apical
foramen. [Nair et al JOE – 1987].
c) Histologic studies of apical periodontitis have
shown bacterial penetration in lateral canals also.
(B) Dentin Canal Invasion
Bacteria from the main canal can also spread into
surrounding dentin by invading the dentinal tubules. [Int
Endod Journ 1994; 27, 218 – 212]
99

Frequency and quantity of bacterial penetration
into dentin canals. Comparative ability of different
bacterial species to invade dentin canals in vivo or even
in vitro . Gram +ve species (LACTOBACILLI and
STREPTOCOCCI) can invade dentin tubules more
easily than Gram –ve species. [Endodon Dent
Traumatol 1990; 6, 142-149]
Mechanism of invasion not known.
Does not depend on motility of microorganisms
as most are non-motile and occurs at random; canal
packed with bacteria is often surrounded by empty
canals. Bacteria in dentin canals are seen as sporadic,
dense accumulations rather than a continuously growing
row of cells. There is also a possibility of bacteria
breaking out from dentin canal into surrounding dentin
leading to destruction. [JOE 1987; 13, 29-39]
(C) Bacteria in periapical Tissues
(i) Until recently, it was accepted that bacteria in
apical periodontitis are located within the dental
root canal system (Main canal, lateral canal,
100

dentin canals). [JOE 1990; 16; 534-538. JOE
1992; 18; 216-227]
(ii) Periapical Actinomyces is regarded as an
exception where Actinomyces spp. is present in
the periapical area. [JOE – 1986, 12, 76-79;
JOE – 1988, 14, 147-149; JOE – 1980, 6, 602-
606]
(iii) Presence of a sinus tract also indicates a
permanent presence of bacteria in extraradicular
area but most fistula close after routine
endodontic treatment.
(iv)However, recent studies have shown that in cases
of persistent apical periodontitis after RCT,
bacteria can survive outside the root canal system
on the root surface or in periapical tissues.
[Haapasalo et al 1987, Transtadt et al 1987,
Waltan et al 1992].
101

(D) Bacteremia in Connection With Apical
Periodontitis.
(i) Bacteremia from Apical periodontitis occurs
through tissue and lymphatic vessel causing
swelling & tenderness of lymph nodes (which is
seldom)
(ii) Other cause for bacteremia in apical periodontitis
is the trauma which occurs often.
(iii) Transient in nature
(iv)Number of bacteria/ml blood in endodontic
bacteremia is lower than in bacteremia during
tooth extraction or scaling. [Heimdahl et al.
1990]
Species recovered from blood were [Debelian et al
1992, 1995]
(i) Fusobacterium nucleatum
(ii) Prevotella intermedia
(iii) Peptostreptococcus prevotii
102

(iv) Propionibacterium acnes
(v) Actinomyces israelli
(vi) Streptococcus intermedius
(vii) Streptococcus sanguis
(viii) Saccharomyces cerevisae
After 10 min only Gram +ve were found.
i. Actinomyces israelli
ii. Propionibacterium acnes
General Description of Microbial Flora
Apical periodontitis is a polymicrobial infection
dominated by obligately anaerobic bacteria. [Haapasalo
1986, 1993]. No. of species per case is 2-8, and never
more than 20 species in one canal. Many studies have
shown that species isolated in A.P. are to a great extent
same as those in infected periodontal pocket.
Microscopic studies have shown the presence of
SPIROCHETES in infected root canal [Dahle et al
1995, 1996].
103

Flora of OPEN CANALS shows wide variation
and corresponds to oral flora while flora of CLOSED
CANALS shows less variation
Teeth with periapical radiolucent areas have
increase incidence of infected root canals.[Int Endod J
1982; 15 ; 79-86]
Pathways of pulpal and periapical infections (or)
routes of microorganism’s ingress:
Microorganism’s can reach the dental pulp by
any of the six routes:
1) Through the open cavity
2) Through the dentinal tubules
3) Through the gingival sulcus or PDL
4) Through blood stream and anachoresis.
5) Through a broken occlusal seal / faulty
restoration of a tooth.
6) Through extension of periapical infection from
adjacent infected teeth.
104

Through the open cavity:
Enamel and dentin provide excess protection to
the pulp against inflammation when they are intact.
However, once they are damaged by caries or trauma,
the underlying pulp is invaded. As the irritant approach
the pulp, new protective layer of reparative dentin may
be laid down to avert exposure. sometimes due to the
rapid deposition of the reparative dentin the
microorganisms are prevented from entering the pulp,
but this rarely happens. When a healthy vital pulp is
exposed, as a result of trauma, the penetration of the
microorganisms into the tissues is relatively slow,
bacterial penetration is less than 2mm after 2 weeks. If
the pulp is necrotic, dead tracts of empty dentinal tubule
are rapidly penetrated.
Through the dentinal tubule:
As the diameter of dentinal tubule range from 1
to 4mm and majority of bacteria are less than 1mm, they
can easily pass through the dentinal tubule and reach the
105

pulp. The pressure of impression materials, temporary
restorative materials, acid and cements may drive
microorganisms from the surface of the preparation
through the dentinal tubules into the pulp. This effect
must be kept in mind when preparing cavities and
grinding dentin for full crowns. Experiments have
demonstrated that the pulp can be infected through
prepared and exposed dentin up to a thickness of 0.2mm.
If the exposure is because of caries, removal of
caries helps in protecting the pulp, if not treated bacteria
in the dentinal tubule reaches the pulp tissues and the
inflammatory response enlarges. Pulp infected through
the dentinal tubule reveals by a few bacterial strains,
mostly facultative anaerobic bacteria such as lactobacilli,
Viridans streptococci for which the dentinal tubules are
selective. It might lead to pulpities without direct
exposure. Removal of cementum during periodontal
therapy exposes dentinal tubules to the oral flora.
Through the gingival sulcus or PDL
Microorganisms and other irritants from the PDL
may reach the pulp through the portals at the apex of the
106

root or through the lateral accessory or furcation canals.
Langerland et. al found that pulpal necrosis occurred
when the apical foramen was involved. If the periodontal
disease destroys the protecting bone and soft tissue to a
sufficient degree, the canal may be exposed to the
microorganisms present in the gingival sulcus through
the lateral foramina. In this way pulp exposure occur
without caries or trauma with heavy ingress of irritants.
Through blood stream:
Anachoresis may be defined as and transportation
of microbes through the blood to an area of
inflammation where they establish an infection. It has
been demonstrated in animals but is not believed to
contribute to significant disease in humans. It does seem
possible that anachoresis may be the mechanism by
which some traumatized teeth may become infected.
Anachoresis is defined as the localization of
transient bacteria in the blood into an inflamed area, such
as such as traumatized (or) inflamed pulp.
107

Through broken occlusal. Seal (on faulty restoration
of a tooth previously treated by endodontic therapy.
Torabinajed et al have proven that salivary
contamination from the occlusal aspect can reach the
periapical area in less than 6 weeks in canals obturated
with Gutta-percha and sealer. If there is a delay in
restorative procedure following endodontic therapy and
if the temporary seal is broken, if the tooth structure
fractures before final restoration (or) if the final
restoration is inadequate or become inadequate due to
subsequent decay, bacteria may gain access to the peri
apical tissue and result in infection.
Through extension of a periapical infection from
adjacent infected teeth.
There is considerable question whether or not
bacteria from a periapical area will enter an adjacent,
non-infected tooth. Large periapical radiolucencies may
appear to encompass the roots of multiple teeth, yet be
caused by pulp necrosis of only one tooth. This occurs
with greatest frequency in the mandibular anterior teeth,
108

on treating the causative tooth endodontically, the entire
radiolucency heals. Despite of the presence of
granulomas the nerves and blood vessels can safely
penetrate and course through the lesion. If pulpitis or
trauma severely affects a tooth, its neighboring tooth
does have an infected periapical area, which should also
be treated endodontically.
RECENT TAXONOMIC CHANGES
Using, DNA studies, the black-pigmented
bacteria previously in the genus Bacteroides have now
been placed in the geneses porphyromonas and
prevotella. Porphyromonas being asacchorolytic.
Prevotella being sacchorolytic, which even includes the
non-pigmented species.
Pathogenicity – Ability of the microorganisms to
produce disease.
Virulence – Ability of a particular strain to cause
disease.
Microorganisms associated with pulpitis:
109

In general streptococci predominate than other
microorganisms such as staphylococci, diptheroids,
Fusiform bacteria, filamentous forms. Some studies have
shown gram-ve anaerobic bacteria to be the most
predominant which are predominant in endodontic
infections.
Endodontic disease will persist until the source of
irritation is removed by debridement. Therefore, chemo
mechanical debridement using a suitable irritant should
be done to eliminate the microorganism.
NaOcl is one of the most widely used irrigating
solutions because of its excellent antimicrobial activity
against G +ve, G-ve, aerobes, facultative anaerobes &
few obligate anaerobes and also because of the tissue
dissolving properly. Naocl is an effective antimicrobial
irrigating solution but has toxic effects on the periapical
tissues at higher concentrations. Naocl shows best results
when the contact time is at least 10 min.
Chlorhexidine has been widely used in the
treatment of periodontal disease and has been suggested
110

to be used as irrigating solution or intra canal
medicaments during inter appointments. It is active
against a wide range of microorganisms, which include
gram +Ve, gram-Ve, bacterial spores, lipophilic virus,
yeasts and dermatophytes. It is bacteriostatic at low
concentration and bactericidal at high concentration.
Symptomatic pulpitis usually does not require
course of antibiotics. Removal of irritant gives relief
from pain.
Microorganisms associated with necrotic pulps
The microorganisms isolated from necrotic
pulps include obligate, facultative anaerobes, and a
fungus has been isolated from few cases, most frequent
are prevotella, fusobacterium, lactobacillus, streptococci,
clostridium and peptostreptococcus.
In acute endodontic lesions, large numbers of
obligate anaerobes are present which include prevotella,
porphyromonas, vieillonella, Actinomyces
porphyromonas gingivalis and porphyromonas
endodontalis were commonly isolated from teeth with
111

acute symptoms of pain, swelling, open sinus tract and
tenderness to percussion. In addition prevotella denticola
was isolated from asymptomatic teeth.
The microbial evaluation of root canals with
pulpal necrosis suggested the presence of polymicrobial
infections, mainly involving obligate anaerobes and
shows that the infection may persist even after treatment.
Naocl is the irrigant that is most widely used;
even chlorhexidine gluconate can be used because of its
wide antibacterial spectrum. Naocl used alternatively
with chlorhexidine has shown better antimicrobial
activity than using the either alone. If the endodontic
treatment is completed in a visit then there is no need of
an intra canal medicament but if it is a multi visit
treatment the chlorhexidine can be used as an intra canal
medicament.
Antibiotic coverage is usually not needed unless
it is associated with systemic signs and symptoms such
as fever and malaise.
112

Leonardo et al 1999 evaluated the
antimicrobial activity of 2% chlorhexidine gluconate as a
root canal irrigant in teeth with pulpal necrosis and radio
graphically visible chronic periapical lesions and
concluded that it is equally effective in both the
conditions.
Ahmad et al 2003 studied the effect of 2%
chlorhexidine rinse and 1% Naocl irrigant. They used
1% Naocl during BMP and then flushed the canals with
2% chlorhexidine and then dried the canals and used Ca
(OH) 2 as an intra canal medicament microorganisms
were isolated from only 1 of the 12 teeth, where as in the
control group 7 of the 12 teeth has microorganisms.
Microorgnaism associated with teeth with periapical
lesion
It was believed that bacteria usually confined to
the root canal system of an infected tooth except when
associated with an abscess or cellulites they include
several strains of bacteria from each infection.
113

Tronstad et al studies 6 patients who had
asymptomatic periapical lesions and isolated
propionibacterium acnes, porphyromonas gingivalis and
porphyromonar endodontalis. Anaerobic infection often
produces pain, swelling and a febrile state. A purulent
foul swelling discharge indicates the presence of
anaerobic metabolites such as ammonia, indole, urea and
aminoacids.
Black pigmented bacteria are mainly isolated
from symptomatic teeth. Most of them associate with
acute periapical abscess. P. Gingivalis, p.endodontalis
isolated from symptomatic teeth. Where as P. intermedia
from both symptomatic and asymptomatic teeth.
T. Socranski was the most common spirochaete
isolated from teeth with endodontic abscess or cellulites,
usually teeth with periradicular lesions cannot be treated
in a single visit especially those with weeping canals.
Naocl is the choice of irrigant
An intracanal medicament is needed for
114

1) To eliminate any remaining bacteria after canal
instrumentation
2) To reduce inflammation of periapical tissues and
pulp remnants.
3) To help to dry persistently wet canals.
Ca(OH)2 is the current intracanal medicament of
choice. Ca(OH)2 has some efficacy in the dissolution of
pulp tissue and may increase the ability of Naocl to
dissolve the remaining organic tissue at subsequent
appointments.
Studies were done on the efficacy of Ca(OH)2
using different vehicles. Ca(OH)2 + CMCP + glycerine,
Ca(OH)2 + saline, Ca(OH)2 + H2O, Ca(OH)2 +
polyethylene glycol. Ca(OH)2 pastes with oily vehicles
showed larger zones of inhibition compared to those
with aqueous solutions.
Andreas et al studies the additive antimicrobial
activity of Ca(OH)2 and chlorhexidine on common
endodontic pathogen such as E. Faecalis, Fusobacterium
nucleatum, peptostreptococcus micros, porphyromonas
115

gingivalis and streptococcus intermedia and found that
G-Ve bacteria were significantly killed by Ca(OH)2. For
the inactivation of peptostreptococcus micros and
streptococcus inter media a combination of Ca(OH)2 +
Zno+ chloroxidine killed the bacteria faster than
Ca(OH)2 alone. For enterococcus the combination led to
decrease in the number of viable bacteria but did not
completely eliminate them.
Camphorated paramonochlorophenol has also
been used as an intracanal medicament but it uses its
effectiveness in the 1st
24hrs and eliminate only about
67% of bacteria, whereas as Ca(OH)2 takes almost 24hrs
to become active and eliminate about 97% of bacteria by
one month.
Antibiotic regimen
An antibiotic regimen should be prescribed in
conjunction with appropriate endodontic therapy when
there are systemic signs and symptoms or a progressive /
persistent spread of infection. The presence of fever,
malaise, cellulites, unexplained trismus and progressive
116

swelling are all signs and symptoms of systemic
involvement and the spread of infection.
Pencillin VK remains the initial antibiotic of
choice because it is effective against many facultative
and strictly anaerobes it is not effective against B-
lactamase producing bacteria, which include
staphylococcus, and black pigmented bacteria like
porphyromonas, prevotella and fusobacterium species.
Metronidezole is very effective against an G-ve
bacilli, including those that produce lactamase. But is not
effective against aerobic organisms and has only
marginal coverage for some of the G+ ve anaerobic
organisms (Actinomyces, peptostreptococcus).
Therefore pencillin in combination with
metronidazole is effective against both G+ve, G-ve,
aerobic and anaerobic organisms including those that
produce B-lactamase. Amoxicllin: An oral dose of
1000mg should be followed by 500mg every 6 hrs for 7
to 10 days. Metrodizole :- 400mg loading dose should be
followed by 250mg to every 6hrs.
117

Approximately 10% populations are allergy to
pencillin. In such patients erythromycin may be choice
of antibiotics even erythromycin should be given in
combination with metronidazole as many anaerobes are
developing resistance to erythromycin. Antibacterial
spectrum of erythromycin includes gram+ ve facultative
and anaerobic bacteria. They include Eubacterium,
Propionibacterium, Bifido bacterium. Lactobacillus,
peptostreptococcus, actinomyces israeili. An oral loading
dose of 1000mg should be followed by 500mg at 6hrs
interval for 7 to 10 days.
Clarithromycin or azithromycin
They have limited spectrum than clindamycin but
are advantageous over erythromycin. In addition to the
organisms susceptible to erythromycin they are also to
be effective against some of the anaerobic species, which
are associated with endodontic infections.
They produce less G.I.T problems than
erythromycin. Clarithromycin may be given without
118

meals in a dose of 250mg or 500mg every 6 to 12hrs for
7 to 10 days.
Azithromycin should be taken 1hr before meals
or 2hrs after meals with a loading dose of 500mg on the
1st
day followed by 250mg daily.
Clindamycin – These agents are effective against aerobic
gram+ve and have broader coverage anaerobes including
B- lactamase producing bacteria. Should be prescribed
with a 300mg loading dose and then 150mg every 6hrs
for 7-10days.
In addition it has good ability penetrate bone and
joints is also useful when B- lactamase antibiotics have
been ineffective or contraindicated.
Amoxicillin when combined with clavulanic acid
makes active against most aerobic and anaerobic B-
lactomase, producing bacteria. It does not inhibit gram-
ve aerobic bacteria such as Citrobacter, Enterobacter,
Serratia, Pseudomonas and Escherichia Coli. Severe
infections 4 tabs 6hourly.
119

Baumgaether in 2002 studied the antibiotic
susceptibility of bacteria associated with abscess and
found amoxicillin + clavulanic acid to the most effective.
Metronidazole had the greatest amount of bacterial
resistance however if not used in combination with
pencillin or amoxicillin susceptibility of the
microorganisms increased to 93 to 98%.
Microorganisma associated With Endo-perio Lesions
Microorganisms isolated from non-vital, infected
teeth that had intact pulp chambers no apparent
communication with the oral environment where the
infection might have occurred through the gingival
crevice include Actinomycosis species, Bacteroides
species, Fusobacterium species, Camphylobacter
sputorum, Eubacterium species, peptococcus species and
several unidentified anaerobic facultative
microorganisms.
Porphyromonas endodontalis seems to be very
rare in oral infections than those of endodontic origin. In
general abscess of periodontal origin. In general abscess
120

of periodontal origin contains 30 to 58% sprirocheates.
Where as abscesses of endodontic origin contains less
than 10% Spricheates, studies have also shown the
presence of CMV and Epstein barr virus in teeth having
intact crowns and calcified necrotic pups.
Naocl, chlorhexidine gluconate, ciprofloxacin,
metronidazole and minocycline have been used as
intracanal disinfection against strict anaerobes and the
combination of then was found to completely eliminate
the bacteria.
In teeth associated with endodontic-perio
relation, the cleaning and shaping of the root canal in
combination with irrigation alone cannot render the root
canal system free of cultivable bacteria, but an additional
inter appointment medication with hydroxide dressing
should be given.
Calt in 1999 demonstrated the use of EDTA and
NaOcl to maximally remove Ca(OH)2 dressing from the
root canal system.
121

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