Endodontic microbiology / dental implant courses

ENDODONTIC
MICROBIOLOGY
INDIAN DENTAL ACADEMY
Leader in continuing Dental Education
www.indiandentalacademy.com

Contents
 Introduction
 History
 Gram +ve and Gram –ve organisms
 Aerobic and Anaerobic microorganisms
 Microflora of oral cavity
 Portals of root canal infection
 Bacterial pathogenicity
 Bacterial defence
 Host response
 Endodontic microflora

 Microorganisms of persistent infections
 Identification of microorganisms
culture tests
DNA probes
PCR
Immunoassays
Immunofluorescence
 Treatment of endodontic infection

Introduction
 Over three centuries that have passed since
Leeuwenhoek first observed bacteria and
protozoa with his primitive microscope , a
vast amount of knowledge ,has been
accumulated about the “small animals” ,now
known as microorganisms.

Microorganisms cause virtually all pathoses of the pulp
and the periradicular tissues. To effectively treat
endodontic infections, clinicians must recognize the
cause and effect of microbial invasion of the dental
pulp space and the surrounding periradicular tissues.
A thorough understanding of these organisms including
their growth and destructive potential, sensitivity to
pharmacotherapeutic agents and their relationship to
clinical symptoms is necessary to formulate a sound
approach to root canal therapy.

History
1890
W.D .Miller
 Authored a book called : “Microorganisms of
Human Mouth”
 First researcher to identify bacteria in the diseased
pulp
1909
E. C. Rosenow,
“Theory of Focal Infection”
 localized or generalized infection caused by
bacteria traveling through the bloodstream from a
distant focus of infection.

1939
Winfred Fish
 Fish Zones of reaction
 Fish related these bone changes to infections from
the dental pulp and theorized that removal of the
nidus of infection would lead to resolution of the
infection
Schuller
 Anachoresis : Process by which blood borne
bacteria,dyes ,pigments,metallic substances,foreign
proteins are attracted to and fixed in, circumscribed
areas of inflammation

 1941
Anachorectic pulpitis
Robinson and Boiling cites the movement of systemic
bacteria into inflamed pulps, a major concern in dental
related bacterial endocarditis.
 1981
Moller and associates illustrated the importance of
bacteria in the development of pulp and periradicular
disease

 Sundquist
Studied the significance of bacteria and their association
with periradicular disease.
Analysis of aerobic and anaerobic bacteria
Positive cultures for teeth with radiographically exhibited
periradicular disease.
Noted –Bacteriodes melaninogenicus was sessential for
the development of periradicular destruction.

Gram +ve and Gram-ve
 Cell wall
 The cell wall of a bacterium is an essential structure that
protects the delicate cell protoplast from osmotic lysis.
 The cell wall of Bacteria consists of a polymer of
disaccharides cross-linked by short chains of amino
acids (peptides). This molecule is a type of
peptidoglycan called murein.


 In the Gram-positive bacteria,
the cell wall is thick (15-80 nanometers), consisting of
several layers of peptidoglycan complexed with
molecules called teichoic acids .
 In the Gram-negative bacteria,
the cell wall is relatively thin (10 nanometers) and is
composed of a single layer of peptidoglycan surrounded
by a membranous structure called the outer membrane.

 Gram staining by Christian Gram
Gram +ve bacteria retain the primary
stain and resist decolorisation,and appear
violet
Gram –ve bacteria get decolorised and
take up the counter stain and appear red.

Oxygen requirement
 Assimilation of glucose results in terminal
generation of free oxygen radical
superoxide(O2
-
)
 Reduced to oxygen gas and H2O2-
superoxide dismutase
 H2O2 is converted into water and oxygen-
Catalase

Oxygen requirement
Anaerobic bacteria
grow only in the absence of oxygen but vary in their
sensitivity to oxygen. They function at low oxidation-
reduction potentials
lack the enzymes superoxide dismutase and catalase.
Microaerophilic bacteria
can grow in an environment with oxygen but
predominantly derive their energy from anaerobic energy
pathways.

Facultative anaerobes
grow in the presence or absence of
oxygen and usually have the enzymes
superoxide dismutase and catalase.
Obligate aerobes
require oxygen for growth and possess
both superoxide dismutase and catalase.

BACTERIA COMMONLY FOUND IN ORAL
CAVITY
 Staphylococcus epidermidis
 Staphylococcus aureus
 Streptococcus mitis
 Streptococcus salivarius
 Streptococcus mutans
 Enterococcus faecalis
 Streptococcus pneumoniae
 Streptococcus pyogenes
 Neisseria sp
 Neisseria meningitidis
 Veillonellae sp
 Enterobacteriaceae
(Escherichia coli)
 Proteus sp

 Pseudomonas aeruginosa
 Haemophilus influenzae
 Bacteroides sp
 Bifidobacterium bifidum
 Lactobacillus sp
 Clostridium sp
 Clostridium tetani
Corynebacteria
Mycobacteria
 Actinomycetes
 Spirochetes
Mycoplasmas

PULPAL INFECTION
 Endodontic infections are polymicrobial
 The number of CFU is usually between 102
to 108
.
 Majority of the microbes associated with endodontic
infections are ANAEROBIC.
 The anaerobic bacteria that constitute
endodontic infections are,
Obligate Anaerobes 60-63%,
Facultative Anaerobes 34-36%.

 Anaerobic Bacteria causing endodontic infections can be
grouped into,
Gram Positive Rods:
 Actinomyces sp.,
 Lactobacillus,
 Eubacterium sp.
Gram Positive Cocci:
 Peptostreptococcus sp.,
 Streptococcus sp.,
 Staphylococcus sp.

 Gram Negative Rods:
 Bacteroides sp.,
 Fusobacterium sp.,
 Campylobacter,
 Prevotella sp.,
 Porphyromonas sp.

PORTALS OF ROOT CANAL INFECTION
 Openings in the dental hard tissue wall—resulting from
caries,
clinical procedures, or
trauma-induced fractures andcracks
 Gingival sulci or periodontal pockets throughsevered
periodontal blood vessels
 Through exposed dentinal tubules at the cervicalroot
surface, due to gaps in the cemental coating
 Anachoresis

Dental Caries
Invasion of the pulp cavity by bacteria is most often
associated with dental caries. Bacteria invade and multiply
within the dentinal tubules .
Dentinal tubules range in size from 1 to 4 ∝m in diameter,
whereas the majority of bacteria are less than 1 µm in
diameter. If enamel or cementum is missing, microbes
may invade the pulp through the exposed tubules.

Trauma
Following trauma and direct exposure of the pulp,
inflammation, necrosis, and bacterial penetration are
no more than 2 mm into the pulp after 2 weeks. In
contrast, a necrotic pulp is rapidly invaded and colonized.
The “dead tracts” of empty dentinal tubules following
dissolution of the odontoblastic processes may leave
virtual highways for the microbes’ passage to the pulp
cavity.
Microbes may reach the pulp via direct exposure
of the pulp from restorative procedures or trauma
injury and from pathways associated with anomalous tooth
development.

Gingival sulci or Periodontal ligament
 Possible sources-
 Hard tissue communication
Lateral canals in the furcation area and in the apical third of
tooth roots
 Lymphatic and hematogenous routes
 Root planing
 Trope and Tronstad
Theorized that spirochete count from the exudate of
periradicular lesions ,differentiate an abscess of endodontic
origin from one of periodontal origin.
0 – 10 % -endodontial origin
30 -58 % - periodontal origin

Anachoresis
Anachoresis is a process by which microbes may be
transported in the blood or lymph to an area of
inflammation such as a tooth with pulpitis, where they
may establish an infection.
Anachoresis may be the mechanism through which
traumatized teeth with intact crowns become infected.
The process of anachoresis has been especially
associated with bacteremias and infective endocarditis.

Endodontic microflora of a
human tooth with apical
periodontitis (GR). The areas
between the upper two and
the lower two arrowheads in
are magnified in and ,
respectively. Dense bacterial
aggregates (BA) sticking to
the dentinal (D) wall .A
transmission electron
microscopic view (d) of the
pulpo-dentinal interface
shows bacterial condensation
on the surface of the dentinal
wall, forming thick, layered
biofilm.

Well-entrenched biofilm at
the apical foramen of a
tooth affected with apical
periodontitis . The canal
ramifications on the left and
right in (b) are magnified in
(c) and (d), respectively.
Note the strategic location
of the bacterial clusters (BA)
at the apical foramina. The
bacterial mass appears to
be held back by a wall of
neutrophilic granulocytes
(NG).

Pathogenicity/Infection
 Microbial pathogenicity has been defined as the structural and
biochemical mechanisms whereby microorganisms cause disease.
Pathogenicity in bacteria may be associated with unique
structural components of the cells (e.g. capsules, fimbriae, LPS or
other cell wall components) or active secretion of substances that
either damage host tissues or protect the bacteria against host
defenses.
 Infection may imply colonization, multiplication, invasion or
persistence of a pathogen on or within a host,
 Disease is used to describe an infection that causes significant
overt damage to the host.

Colonization is the establishment of microbes in a host
if appropriate biochemical and physical conditions are
available for growth.
Normal oral flora is the result of a permanent microbial
colonization in a symbiotic relationship with the host.
Although the microbes in the normal oral flora participate
in many beneficial relationships, they are opportunistic
pathogens if they gain access to a normally sterile area
of the body such as the dental pulp or periradicular
tissues and produce disease.

 Invasiveness is the ability to invade tissues. This
encompasses mechanisms for adherence and initial
multiplication, ability to bypass or overcome host defense
mechanisms, and the production of extracellular substances
("invasins") which facilitate the actual invasive process.
 Toxigenesis is the ability to produce toxins.Toxic
substances, both soluble and cell-associated, may be
transported by blood and lymph and cause cytotoxic effects at
tissue sites remote from the original point of invasion or
growth

PATHOGENICITY OF ENDODONTIC
FLORA
 Interactionswith other micro-organisms in the root
canal, to develop synergisticallybeneficial partners;
 ability to interfere with andevade host defenses;
 Action of virulence factors

 Interactionswith other micro-organisms
Microbial interactionsthat influence the ecology of
the endodontic flora may be positive(synergistic) or
negative associations, as a result of certainorganisms
influencing the respiratory and nutritional environmentsof
the entire root canal flora
Sundqvist et al., 1979;

 Microbial interference
Certain microbes have the ability to shirk and interfere
withthe host defenses
Bacterial toxins can effectively interfere with the various
mechanisms of host immune system
A. israelii aggregate to form large cohesive colonies that
cannotbe killed by host phagocytes

Virulence factors
 Capsules
-Present external to the outer layer of the cell
wall
-Composed of polysaccharide,rarely polypeptide
-Functions :
 Protects the cell from dessication and from
toxic materials in the environment
 Promotes the concentration of nutrients at the
bacterial cell surface
 Adherence of bacteria to host cells
 Resistance to bactericidal action of
complement and serum antibodies

 Glucan capsule of streptococcus mutans
Practical importance of virulence factor as it
forms the matrix of dental plaque
Adherence of bacteria to this matrix and
subsequent formation of acids from dietary
sucrose leads to initiation of caries

 Fimbriae(pili)
They are small appendages fond on the surface of many
gram-ve bacteria.
Although the terms “pili” and “fimbriae” are used
interchangeably,
Fimbriae- non flagellar hair like appendages
Pili- fimbriae of gram –ve bacteria that function
specifically in the transfer of DNA from one cell to other
during process of conjugation(sex pili)

Functions
 participates in the aggregation of bacteria or
attachment to tissues.
 Pili may extend from one bacterium to another
during conjugation and exchange DNA for
virulence factors, including resistance to
antibiotics.

Enzymes as spreading factors
 Hyaluronidase. is the original spreading factor It
is produced by streptococci. Staphylococci. The
enzyme attacks the interstitial cement ("ground
substance") of connective tissue by
depolymerizing hyaluronic acid.
 Collagenase is produced by Clostridium
histolyticum and Clostridium perfringens. It breaks
down collagen, the framework of muscles, which
facilitates gas gangrene due to these organisms.

Neuraminidase
It degrades neuraminic acid (also called sialic acid), an
intercellular cement of the epithelial cells
Streptokinase and Staphylokinase
produced by streptococci and staphylococci,
respectively. Kinase enzymes convert inactive
plasminogen to plasmin which digests fibrin and
prevents clotting of the blood. The relative absence of
fibrin in spreading bacterial lesions allows more rapid
diffusion of the infectious bacteria

 Enzymes
Porphyromonas and Prevotella species to break down
plasmaproteins—particularly IgG, IgM and the
complement factor C3
is of particularsignificance, since
these molecules are opsonins necessary forboth
humoral and phagocytic host defenses.
In abscesses, neutrophils lyse and release their
enzymes to the surrounding milieu to form purulent
exudates. This enzyme-rich exudate has an adverse
affect on the surrounding tissues.

Endotoxins and Exotoxins
 Exotoxins are proteins formed by Gram +ve bacteria
and are highly potent in minute quantities.
 Endotoxins are polysacharide protein complexes which
form an integral part of the cell wall of Gram –ve
bacteria.
They are less potent than the exotoxins

Lipopolysaccharides (LPS)
Endotoxins consists primarily of Lipopolysaccharide.It
is the structural component of Gram –ve outer
membrane.
It has been shown that the concentration of endotoxin
in the canals of symptomatic teeth is higher than that in
the canals of asymptomatic teeth.

Functions of LPS
 It is a permeability barrier to toxic molecules
 Barrier to lysozyme and many antimicrobial agents
 Impedes destruction of the bacterial cells by serum
components and phagocytic cells.
 Important role as a surface structure in the interaction of
the pathogen with its host.
 LPS may be involved in adherence (colonization), or
resistance to phagocytosis, or antigenic shifts that
determine the course and outcome of an infection.

Extracellular vesicles

 The vesicles are formed from the outer membrane of gram-
negative bacteria and have a trilaminar structure similar to the
parent bacteria. These vesicles may contain same antigens which
neutralizes antibodies against parent organism. The vesicles may
contain
 Enzymes, or
 Toxic agents.
These vesicles are involved in,
 Hemagglutination,
 Hemolysis,
 Bacterial adhesion, and
 Proteolytic action on host tissues.

Fatty acids
 The short-chain fatty acids most commonly produced by
bacteria in infected root canals are
Propionic,
Butyric, and
Isobutyric acids.
Short-chain fatty acids affect neutrophils chemotaxis,
degranulation, chemiluminescence, and phagocytosis.
 Butyric acid has the greatest inhibition of T-cell growth
and stimulates the production of interleukin-1 which is
associated with bone resorption.

Polyamines
 Polyamines are biologically active compounds involved
in regulation of growth, regeneration of tissues, and
modulation of inflammation. Teeth that are painful to
percussion or have spontaneous pain have been shown
to have higher concentration of total polyamines in
necrotic pulps.
 Ammonia,
 Hydrogen sulphide.

Host Response
 CELLULAR ELEMENTS
PMN
Though PMN are essentially protective cells, theycause
severe damage to the host tissues Their cytoplasmic
granules contain several enzymes that, on release,
degrade thestructural elements of tissue cells and
extracellular matrices. Becausethey are short-lived cells,
PMN die in great numbers at acute inflammatory sites.
Therefore,the accumulation and massive death of
neutrophils are a majorcause for tissue breakdown in
acute phases of apical periodontitis.

 Lymphocytes
Among the three major classes of lymphocytes—T-
lymphocytes,B-lymphocytes, and the natural killer (NK)
cells.They are involve din antibody production.
 Macrophages
cytokinesIL-1, TNF- , interferons (IFN), and growth
factors that are of particularimportance periadicular
infection. They also contribute serumcomponents and
metabolites, such as prostaglandins and leukotrienes,
that are important in inflammation.

 Osteoclasts
A major pathological event of peri apicalinfection is the
osteoclastic destruction of bone and dental hard tissues
 the pro-osteoclasts migrate through blood as monocytes
to the periradicular tissues and attach themselves to the
surface of bone. Several daughter cells fuse to form
multinucleated osteoclasts that spread over injured and
exposed bone surfaces.

 Bone resorption takes place beneath the ruffled border,
facing the bone surface
 The bone destruction happens extracellularly at the
osteoclast/bone interface and involves:
(i) demineralization of the bone by solubilizing the mineral
phase in the resorption compartment, as a result of ionic
lowering of pH in the micro-environment; and
(ii) enzymatic dissolution of the organic matrix.
 Root cementum and dentin are also resorbed by fusion
macrophages designated as ‘odontoclasts’

 Epithelial cells
During periapical inflammation, the epithelial cell rests
are believed to be stimulated by cytokines and growth
factors to undergo division and proliferation, a process
commonly described as ‘inflammatory hyperplasia’.
These cells participate in the pathogenesis of radicular
cysts by serving as the source of epithelium.
 However, ciliated epithelial cells are also found in
periapical lesions ,particularly in lesions affecting
maxillary molars. The maxillary sinus-epithelium was
suggested to be a source of those cells

MOLECULAR MEDIATORS
1. Pro-inflammatory & chemotactic cytokines
2. IFN
3.Colony-stimulating factors (CSF)
4.Growth factors
5.Eicosanoids
a-Prostaglandins
rapid bone loss
Apical hard-tissue resorption can be suppressed by parenteral
administration of indomethacin, an inhibitor of cyclo-oxygenase (
Torabinejad et al., 1979).
b-Leukotrienes
6.Antibodies
IgG have been shown to b epresent in plasma cells residing in
the periapical cyst wall and in the cyst fluid

Bacterial Defense Against Phagocytes
 Most successful pathogens, however, possess additional
structural or biochemical features that allow them to
resist the host cellular defense against them, i.e., the
phagocytic and immune responses. If a pathogen
breaches the host's surface defenses, it must then
overcome the host's phagocytic response to succeed in
an infection.

 Bacteria can avoid the attention of phagocytes in a
number of ways.
 remain confined in regions inaccessible to
phagocytes
 avoid provoking an overwhelming inflammatory
response
 inhibit phagocyte chemotaxis
 Some pathogens can cover the surface of the bacterial
cell with a component which is seen as "self" by the host
phagocytes and immune system. Such a strategy hides
the antigenic surface of the bacterial cell.

 Inhibition of Phagocytic Engulfment
 Survival Inside of Phagocytes
 Products of Bacteria that Kill or Damage
Phagocytes

The seriousness of an infection beyond the apex of a tooth
depends on the number and virulence of the organisms,
host resistance, and anatomic structures associated with
the infection
Once the infection has spread beyond the tooth socket, it
may localize or continue to spread through the bone and
soft tissue as a diffuse abscess or cellulitis.

Abscess is a cavity containing pus (purulent exudate)
consisting of bacteria, bacterial by-products,
inflammatory cells,numerous lysed cells, and the
contents of those cells.
Cellulitis is a diffuse, erythematous, mucosal, or cutaneous
infection that may rapidly spread into deep facial spaces
and become life threatening

The periapical inflammatory responses that occur
following bacterial infection of the root canal system result
in the formation of granulomas and cysts with the
resorption of surrounding bone. Interleukin-1 and
prostaglandins have been especially associated with
periapical bone resorption.

Bacteria From The Root Canals Of Teeth With Apical
Rarefactions
Rarefactions
 Fusobacterium nucleatum
 Streptococcus sp
 Bacteroides sp
 Prevotella intermedia
 Peptostreptococcus micros
 Eubacterium alactolyticum
 Peptostreptococcus
anaerobius
 Lactobacillus sp
Fusobacterium sp
Eubacterium lentum
Campylobacter sp
Peptostreptococcus sp
Actinomyces sp
Eubacterium timidum
Capnocytophaga
ochracea
Eubacterium brachy
Selenomonas sputigena
Veillonella parvula

 Porphyromonas endodontalis
 Prevotella buccae
 Prevotella oralis
 Proprionibacterium propionicum
 Prevotella denticola
 Prevotella loescheii
 Eubacterium nodatum
It was shown that Black pigmented Bacteriodes were the
bacteria most reactive with IgG produced by explant
cultures of periapical lesions

 Recent Taxonomic Changes for Previous
Bacteroides Species
Porphyromonas—black-pigmented (asaccharolytic
Bacteroides species)
Porphyromonas asaccharolyticas
Porphyromonas gingivalis
Porphyromonas endodontalis

 Prevotella—black-pigmented (saccharolytic Bacteroides
species)
Prevotella melaninogenica
Prevotella denticola
Prevotella loescheii
Prevotella intermedia
Prevotella nigrescens
Prevotella corporis
Prevotella tannerae

 Prevotella—nonpigmented (saccharolytic Bacteroides
species)
Prevotella buccae
Prevotella bivia
Prevotella oralis
Prevotella oris
Prevotella oulorum
Prevotella ruminicola

 Bacteroides, a group of
Gram-negative,
anaerobic, non-
sporeforming bacteria

Microorganisms isolated from periapical lesions
with refractory apical periodontitis
 Actinomyces sp-A.israelli
 Bacillus
 Bacteriodes
 Candida sp-C.albicans
 Clostridium sp
 E.faecalis
 Hemofillus sp
 Streptococcus sp
 Staphylococcus sp
 Veillonella sp

A microbial biofilm at the
root-tip of a failed root
canal. The mixed bacterial
flora consists of numerous
dividing cocci, rods
,filaments (FI), and
spirochetes (S).Rods often
reveal a Gram-negative cell
wall

Axial sections through the
surgically removed apical portion
of the root with a therapy-
resistant apical periodontitis.
visible cluster of bacteria (BA ) in
the root canal.
emerging and gradually
widening profiles of an
accessory root canal (AC) that is
clogged with bacteria (BA )

Actinomyces sp
 Species of Actinomyces have been associated with
endodontic treatment that failed to heal
 A. viscosus
 A. israelii
 A. naeslundii were detected in clinical samples from
infected root canals, and abscesses,and few were
associated with cellulitis.

Actinomyces-
infected periapical
pocket cyst
typical ‘ray-fungus’
type of
actinomycotic
colony

Fungi in endodontic infections
Species
 C.albicans –dentinophilic organism
C.glabrata
 C.guillermondii
 C.inconspicua
 Geotrichium candidum
Dentin colonisation by fungi
Invasion of fungi into dentinal tubules protect
it from intracanal procedures
Availability of Ca ions help in growth and
adhesion

Fungi as a potential cause of
endodontic failures
axial section of a root-filled
(RF) tooth with a persisting
apical periodontitis lesion
showing microbial clusters
and budding forms

 Susceptibility to antimicrobial endodontic medicaments
Nystatin
Sodium carylate
 Sen et al,
EDTA –most effective –reduces adhesion
decreases metabolic activity
Nystatin,ketaconazole,1.5%chorhexidine gluconate
 Resistant to Ca(OH)2
It can survive wide range of pH
 Combination with CPMC/glycerin
Chlorhexidine gluconate and zinc oxide
Chlorhexidine iodine
Iodine potassium iodide
OOO 2004

Enterococcus faecalis
 Invasion into dentinal tubules and remain viable
within the tubule.
 Adherence to collagen
 Regulates internal pH with an efficient proton pump
 Withstands high pH upto 11.5,resisting effects of
Ca(OH)2
 E. faecalis can survive prolonged starvation.
 It can grow as a mono-infection in treated canals in
the absence of synergistic support from other
bacteria
JOE 2001

Enterococcus
faecalis

Viruses in Periapical pathosis
 Herpes Virus-Human cytomegalo virus
Epstein Barr virus
 FIV –Feline immunodeficiency virus
antiviral medications for Rx in periapical infection.
root canal irrigants- NaOcl and iodine
JOE 2004
IEJ 2004,IEJ 2001

ISOLATION AND DETECTION OF MICROBES

Clinically identification of bacteria provides a more
specific targeting of the microbes.
Identification of microbe through laboratory support is
necessary especially in,
Patients who are immunosuppressed
Patients with progressive/ persistent infection
High risk of developing an infection (e.g., history of
infective endocarditis)

CULTURE TESTS:

Clinical reasons for culturing root canals
1.To determine the bacteriologic status of the root canal
system before obturation and assess the efficacy of
debridement procedure.
2.To isolate microbial flora for antibiotic sensitivity and
resistance profiles in cases of persistent infections.

When culturing is done a proper media must be used that
will support the growth of aerobic and anaerobic
organisms.
Common endodontic culture media
 Thioglycollate
 Trypticase soy broth(with 1% agar)
 Dextrose broth
 Brain heart infusion broth
 Serum dextrose broth


SPECIMEN COLLECTION:
Specimen collection in involved root canal or periradicular region
should be under strict aseptic conditions without any error.
Fundamental consideration in specimen collection:
1. Specimen collection should be from the actual infection site with
minimal contamination,
2. Optimal timing:
Culturing should be done before and after cleaning and shaping
of the root canal system.
3. Sufficient quantity: adequate quantity of specimen is required to grow
the microbes.
4. Appropriate collection devices, specimen containers and culture
media

Steps in Specimen Collection:
Specimen collection from root canal:
1) The tooth must be isolated with a rubber dam to
obtain aseptic sample
2) The surface of the tooth and surrounding field must
be disinfected with Sodium hypochlorite or other
disinfectants
3) Access to the canal is made with sterile burs and
instruments,
4) If there is drainage the sample is collected using
sterile needle and syringe or sterile paper points,

 When samples are taken for anaerobic
bacteria,it is important that the canal orifice be
free from atmospheric oxygen by the proper use
of nitrogen gas flow over the canal orifice before
the samples are taken.The anaerobic
environment is thus maintained.

 Specimen collection from surgical site or mucosal
swelling:
1) A surgical soap scrub is first done on the collection
site,
2) Apply 70% ethyl alcohol and tincture iodine to
disinfect the surface layer of mucosal site,
3) Removal of iodine with alcohol,
4) Sample from mucosal swelling is best acquired by
needle aspiration with a 16 to 20 gauge needle,
5) The aspirate is then injected into an anaerobic
transport media.

CULTURE REVERSAL
Grossman found that 2٪ of the cultures were negative
after 48 hours of incubation, but they turned positive
when incubated for 10 days.
It is advisable to allow more than 48 hours between
taking the culture and filling the root canal, preferable 96
or more hours, and it is recommended that the culture
tube be re-examined immediately before obturating a
canal to make certain that no evidence of growth is
present.

False-positive culture:
1. Failure in sterilization of the operating field, and
instruments
2. Rubber dam leaks
3. Use of unsterile paper points and cotton tube plugs;
4. Air or hand contamination during collection or
transport

 A false-negative culture can occur with the following,
1. Incomplete penetration of a paper point ;
2. Use of a paper point that is too narrow;
3. Undetectable microbes ( hidden in dentinal tubules,
accessory or lateral canals, or cementum lacunae);
4. Inadequate amount of specimen

6. Presence of antimicrobial materials in the canal;
7. Insufficient incubation time;
8. Failure to consider culture reversal (negative culture
at the obturation visit becoming positive after the
obturation visit); and
9. Use of a single culture medium that fails to allow for
growth of obligate anaerobic microbes and other hard-to-
culture species.

DNA PROBES
The advances in the field of immunology and
molecular biology have affected the field of diagnosis
and detection of microorganisms based upon unique
sequences of DNA or RNA. With the development of
gene cloning technique almost any nucleic acid
sequence can be prepared in large quantities for use as
a probe.

APPLICATION OF NUCLEIC ACID PROBES
• Detection of organisms difficult to culture
• Detection of organisms which do not have
diagnostic antigens
• Differentiation of a virulent strains from
pathogenic ones
• Identification of antibiotic resistance genes
• Detection of latent virus infection
• Rapid confirmation of cultured organisms

 DNA probes provide reliable results in a short
time (usually less than one day) on a large
number of specimens.
 The identification and production of a nucleotide
sequence is highly sophisticated and
expensive.

POLYMERASE CHAIN REACTION

The development of the polymerase chain reaction
(PCR) in 1983 was major methodological break-through
in molecular biology.

PCR is an in vitro method for producing large
amounts of specific DNA fragment of defined length and
sequence from small amounts of complex template.
By exponentially amplifying a target sequence, PCR
significantly enhances the probability of detecting rare
sequences in a heterologous mixture of DNA.

Applications:
1. The PCR amplification permits the detection of as
few as 100 microbes per 100 gm sample,
2. PCR Is useful for measuring gene expression by
viable microorganisms as well as detecting specific
populations based upon diagnostic gene sequence.
3. Also useful for cloning genes.

LIGASE CHAIN REACION (LCR)
The LCR, or ligase amplification reaction, was first
described in 1989 and modified in 1991. The principal
advantage of is its ability to detect single base-pair
mismatches between target DNAs.
LCR based probe amplification is used detection of
M. tuberculosis,
Borrelia burgdorferi and
N. gonorrhoeae.

 Immunoassays demonstrating antigen antibody
reactions –EIA
RIA
 Immunofluoresence method detecting a specific antigen
of the pathogen using fluorescent antibody

Treatment of endodontic infection
The goal of clinical treatment is to completely disrupt
and destroy the bacteria involved in the endodontic
infection. Endodontic disease will persist until the
source of the irritation is removed.

Root Canal Debridement
and antisepsis
Root canal débridement includes the removal of the
microorganisms and their substrates required for
growth.
Cleaning and shaping of the root canal system remove a
great deal of the irritants, but total débridement is
impeded because of the complex root canal systems with
accessory canals, fins, cul-desacs, and communications
between the main canals.
Irrigants and intra canal medicaments support mechanical
instrumentation of root canal system for complete
asepsis.

TREATMENT OF ENDODONTIC
ABSCESSES/CELLULITIS
Vast majority of infections of endodontic origin can be
effectively managed without the use of antibiotics.
Systemically administered antibiotics are not a substitute
for proper endodontic treatment. Chemomechanical
débridement of the infected root canal system with
drainage through the root canal or by incision and
drainage of soft tissue will help in normal healing
process.

Because of the lack of circulation, systemically
administered antibiotics are not effective against a
reservoir of microorganisms within an infected root canal
system.
A minimum inhibitory concentration of an antibiotic may
not reach a space filled with pus because of poor
circulation.
Incision for drainage will allow drainage of the purulent
material and improve circulation
to the area.
.

 Indications for Adjunctive Antibiotics
(Antimicrobial Therapy)
 Systemic involvement
Fever > 100°F
Malaise
Lymphadenopathy
Trismus
 Progressive infections
Increasing swelling
Cellulitis
Osteomyelitis
 Persistent infections

Selection of antimicrobial agent
 Empirical selection of an antibiotic (antimicrobial
agent) must be based on one’s knowledge of which
bacteria are most commonly associated with
endodontic infections and their antibiotic
susceptibility.
The antibiotic should generally be continued
for 2 to 3 days following resolution of the major
clinical signs and symptoms of the infection.

Antibiotic senstivity test
Pathogenic bacteria exhibit great strain variations in
susceptibility to antibiotics. It is, therefore, essential to
determine the susceptibility of isolates to antibiotics that
are likely to be used in the treatment.
Diffusion tests
Stokes disc diffusion method;
Kirby – Bauer disc diffusion method.
Dilution tests
Broth dilution method ;
Agar dilution method.

Diffusion method
The ‘disc diffusion’ method uses filter paper discs charged with
appropriate concentration of the drugs. The test bacterium is
inoculated on the medium and antibiotic discs are applied.
Sensitivity to the drug is determined from the inhibition of bacterial
growth around the disc
.
Dilution tests
Serial dilutions of the drug in broth are taken in tubes and a
standardized suspension of the test bacterium is inoculated.
An organism of known sensitivity should also be titrated and
Incubated.
The minimum inhibitory concentration (MIC) is read by noting the
lowest concentration of the drug at which there is no visible growth

 Antibiotics For Medically Compromised Patients
Standard general prophylaxis
Amoxicillin
Adults: 2.0g
Children: 50mg/kg orally 1hr. before procedure
Unable to take oral medications
Ampicillin
Adults: 2.0g IM or IV
Children: 50mg/kg orally 30 min. before procedure

Allergic to penicillin
 Clindamycin or
Adults: 600mg
Cephalexin or
Adults: 2.0g
Cefadroxil or
Adults: 2.0g
Azithromycin
Adults: 500mg

 Allergic to penicillin unable to take oral medications
Clindamycin or
Adults: 600mg
Children: 20mg/kg IV 30 min. before procedure
Cefazolin
Adults: 1.0g
Children: 25mg/kg IM or IV 30 min. before procedure

Conclusion
Knowledge of the microorganisms
associated with endodontic disease is necessary
to develop a basic understanding of the disease
process and a sound rationale for effective management
of endodontic infections.

Thank You

 Edwardson’s study on the bacterial composition of dental caries
 Predominantly Gram + ve
Actinomyces
Bifidobacterium
Arachina
Eubacterium

 Even as the irritants approach the pulp, new protective layers
of reparative dentin may be laid down to avert exposure which
rarely can prevent microorganism entry without intervention by
some type of caries excavation


Traumatic injuries or operative procedures
also may remove the protective dentin barrier and allow
access to pulp. To prevent contamination, only materials
with good sealing ability should be used following
operative procedures when any possibility of pulp
exposure is present.

 Traumatic injuries
 Predominantly gram +ve organisms
 Mode of entry is through communication of the fracture with gingival
sulcus.
 Or from the canal space itself

Through The Dentinal Tubules

Dentinal tubules range from 1 to 4 µm,
whereas the majority of bacteria are less than 1µm in
diameter. These invaders may enter the tubules from
salivary contamination during operative procedures or
thorough adjacent carious lesions.

 The microorganisms able to penetrate after cavity preparation are
usually low in number and virulence and rarely cause clinical
symptoms of pulpitis. The pressure of impression materials,
temporary restorative materials, acids, and cements may drive
microorganisms from the surface of a preparation the defense cells
of the pulp can frequently remove these invaders and retain a
healthful environment.


Even though irritation of this dimension may not cause
clinical symptoms, protection of the pulp is available.
 Tubule sealants such as varnishes, sedative bases, or sedative
cements should be used over exposed dentin in proximity to the
pulp immediately after the completion of cavity or crown preparation.


However, when a deep carious lesion brings high numbers of
microorganisms to tubules in proximity to the pulp, it has been
shown that bacteria will penetrate to the pulp well in advance of the
carious process.
 The pulpitis that may result occurs without direct pulp exposure.

Through The Ginigival Sulcus Or Periodontal Ligament.
 Microorganisms and other irritants from the periodontal ligament may reach
the pulp through the vessels in the apical foramen or any auxiliary foramina
present. Also, in some teeth auxiliary canals may be present some distance
from the apex of the root, toward the crown of the tooth. If periodontal
disease destroys the protecting bone and soft tissues to a sufficient degree,
the canal may be exposed to microorganisms present in the gingival sulcus.



 Predominantly in periodontal disease
Strptococcus
Peptostreptococcus
Eubacterium
Bacteriodes
Fusobacterium

Through The Bloodstream:
 ANACHORESIS may be defined as the transportation of microbes through
the blood or lymph to an area of inflammation, such as tooth with pulpitis.
 ANACHORESIS may be mechanism by which some traumatized teeth may
become infected. ANACHORESIS could not be demonstrated in
instrumented but unfilled canals.

Through A Broken Occlusal Seal Or Faulty Restoration
Of Tooth Previously Treated By Endodontic Therapy:
 Controlled studies by Torabine Jad et al have proved that salivary
contamination from the occlusal aspect reach the periapical area in less
than six weeks in canals obturated with gutta-percha and sealer. If there is a
delay in restorative procedures following endodontic therapy and the
temporary seal is broken, if the tooth structure fractures before final
restoration, or if the final restoration is inadequate or becomes inadequate
due to subsequent decay, bacteria may gain access to the periapical tissues
and result in infection.

 This problem is often compounded by hydrophilic composite cores placed
under cast restorations that begin to leak with time, absorb contaminants,
and serve as a reservoir for bacteria. Unused post space further
complicates the situation in a leaking restorative system acting as an
incubator for anaerobic bacteria, inviting them to travel to the periradicular
tissues through the apex. Microbes traveling around only 4 to 5 millimeters
of gutta-percha and sealer-or through the much shorter route of
contamination, are causing an increasing number of endodontic failures
over extended periods of time.

Through Extension Of Periapical Infection From Adjacent
Infected Teeth
 There is considerable question whether or not bacteria from a periapical
area will enter an adjacent, noninfected tooth. Large periapical radiolucency
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 lower anterior teeth. Only the
causative tooth is treated endodontically, and the entire radiolucency heals.
Despite the presence of the granulomatous tissue and multiple colonies of
microorganisms, the nerves and blood vessels can safely penetrate and
course through the lesion.


If a pulpitis or trauma severely affects a tooth and if its
neighbor has an infected periapical area, the microorganisms may easily
reach the newer problem by the interlacing blood and lymph systems, by
physical extension, or by pressure.
 In a process similar to the anachoretic effect, the injured pulp is invaded,
and the proximity of the source of microorganisms may yield a high number
of organisms.

Microorganisms found in root canals
 Mostly gram negative anaerobic microorganisms

Significance of microorganisms in
endodontic therapy
 Hobson’s equation
Number of MO x Virulence of MO
Resistance of host
 All the factors,except for the number of microorganisms,are
qualitative in nature.
 Host resistance depends on many factors and can even vary
significantly from host to host.
 Disease severity can be quantitatively compared on factors suuh as
pain,swelling and tissue destruction.
Severity
of
disease
process

Rarefactions
 Percentage of incidence
Eubacterium lentum 31 Fusobacterium nucleatum 48
Streptococcus sp. 40 Bacteroides sp. 35
Prevotella intermedi 34 Peptostreptococcus micros 34
Lactobacillus sp. 32 Fusobacterium sp. 29
Actinomyces sp. 15 Eubacterium timidium 11
Eubacterium brachy 9 Campylobacter sp. 25
Veillonella parvula 9 Prevotella buccae 8
Prevotella oralis 8 Peptostreptococcus sp. 15
Prevotella loescheii 6 Eubacterium alactolyticum 34
Prevotella denticola 6
Peptostreptococcus anaerobius 31
Porphyromonas endodontalis 9
Propionibacterium propionicum 8
Eubacterium nodatum 6

 The presence of microorganisms does not ensure
endodontic failure nor does the absence of
microroganisms guarantee success.However,the
presence of microorganisms,particularly those of certain
types,provides an additional source of irritation that the
body must overcome to gain optimum
results.Therefore ,the control of microorganisms and
possible substrate must be an objective in every
endodontic case .

Bacterial culturing
 Common endodontic culture media
 Thioglycollate
 Trypticase soy broth(with 1% agar)
 Dextrose broth
 Brain heart infusion broth
 Serum dextrose broth
When samples are taken ,it is important that the canal orifice
be free from atmospheric oxygen by the proper use of
nitrogen gas flow over the canal orifice before the samples
are taken.The anaerobic environment is thus maintained.

 Carbon di oxide
 Some bacteria use atmospheric CO2 as a principle
source of carbon for biosynthetic reactions.
 CO 2 is required for certain macromolecular synthetic
pathways,such as fatty acid biosynthesis.

 Because apical periodontitis is essentially a disease of root canal
infection, the logical treatment has been to eliminate infection from
the root canal and exclude further infection of the canal. Since the
essential role of root canal microbes in both primary and post-
treatment apical periodontitis has been well-recognized, the major
thrust of treatment procedures should be with the clinical
management of problems associated with the control and
elimination of infection. In recent years, there has been a trend to
focus on the purely mechanical aspects of treating the disease.
While those are important, a clear understanding of the etio-
pathogenic factors involved is necessary for the therapeutic
application of intelligent solutions to solve the problem.

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