Microbiology of Endodontic Infection.Mechanisms of MicrobialPathogenicity and Virulence Factors Biofilm and Community-Based Microbial Pathogenesis Biofilm and Bacterial Interactions Biofilm Community Lifestyle Quorum Sensing—Bacterial Intercommunication Methods for Microbial Identification Diversity of the Endodontic Microbiota Primary Intraradicular Infection Spatial Distribution of the Microbiota Microbial Ecology and the Root Canal Ecosystem Secondary/Persistent Infectionsand Treatment Failure

1. Microbiology of Endodontic Disease
Dr. Ashok Ayer
Assistant Professor
Department of Conservative Dentistry and Endodontics
B. P.Koirala Institute of Health Sciences, Dharan, Nepal

2. Contents:
 Introduction
 Mechanisms of Microbial
Pathogenicity and Virulence Factors
 Biofilm and Community-Based Microbial Pathogenesis
 Biofilm and Bacterial Interactions
 Biofilm Community Lifestyle
 Quorum Sensing—Bacterial Intercommunication
 Methods for Microbial Identification
 Diversity of the Endodontic Microbiota
 Primary Intraradicular Infection
 Spatial Distribution of the Microbiota
 Microbial Ecology and the Root Canal Ecosystem
 Secondary/Persistent Infections
and Treatment Failure
 Conclusion
 References

3. Introduction:
 Microorganisms cause virtually all pathoses of the
pulp and periapical tissues.
 Once bacterial invasion of pulp tissues has taken
place, both non-specific inflammation and specific
immunologic response of the host have a
profound effect on the progress of the disease.

4.  Endodontic infection develops in root
canals devoid of host defenses,
 pulp necrosis (as a sequel to caries, trauma,
periodontal disease,or iatrogenic operative
procedures) or
 pulp removal for treatment.
 Biofilm-induced oral diseases.

5. Mechanisms of Microbial
Pathogenicity and Virulence Factors
Pathogenicity :
The ability of a microorganism to cause disease.
Virulence:
Degree of pathogenicity of a microorganism.
• Microbial products,
• structural components of bacteria
 Bacterial strategies that contribute to pathogenicity
include the ability to coaggregate and form bifilms.

6. Bacterial cell and its structural components that can act as virulence factors.
Detailed scheme of the bacterial cell walls from gram-positive and gram-
negative bacteria. CM, Cytoplasmic membrane; LPS, lipopolysaccharide
(endotoxin); LPtns, lipoproteins; LTA, lipoteichoic acid; OM, outer membrane;
OMP, outer membrane protein; PG, peptidoglycan.
Cytoplasmic
membrane Cell wall
Flagellum
LTA
PG
Gram-positive
Exopolysaccha
ride
(capsule)
Fimbriae
Chromoso
meRibosomes
Plasmids
Gram-negative
LPS OMP
OM
CM
CM
LPtn
PG

7. Scanning electron micrograph showing a bacterial biofilm covering dentin
in a deep carious lesion. Note the presence of different bacterial
morphotypes (×3500). (From Torabinejad M, Walton RE: Endodontics:
principles and practice, ed 4, Saunders/Elsevier, St. Louis, 2009

8. Biofilm and Community-Based Microbial
Pathogenesis
Single Individual Cell
Population
Community
Ecosystem
(a functional selfsupporting system that includes the microbial community
and its environment)
• Each population occupies a functional role (niche) within the community
• Community profiling studies revealed that bacterial composition of the
endodontic microbiota differs consistently between individuals.

9.  From the perspective of the single-pathogen
concept:
◦ Apical periodontitis can be considered as having no
specific microbial etiology.
 However, based on the community as-a-
pathogen concept:
◦ it is possible to infer that some communities are
more related to certain forms of the disease

11.  Biofilm:
◦ a sessile multicellular microbial community
characterized by cells that are firmly attached to a
surface and enmeshed in a self-produced matrix of
extracellular polymeric substance (EPS), usually
polysaccharide
 Biofilm infections account for an estimated
65% to 80% of bacterial infections that affect
humans in the developed world

12.  Community members form,
◦ distinct populations or microcolonies separated by
open water channels that traverse the biofilm
matrix and create primitive circulatory systems.
 vital nutrients and communication molecules
can diffuse, and wastes can be washed out
through these channels.

13. During the early stages of biofilm formation,
bacteria bind to many host proteins and
coaggregate with other bacteria.
changes in growth rate, gene expression, and
protein production.

14. A Broader Habitat Range for Growth
 The metabolism of early colonizers alters the
local environment,
setting the stage for attachment and growth of
latecomers
(including more fastidious species)

15. Increased Metabolic Diversity and Efficiency
 Take part in a number of nutritional
interrelationships, and food webs.
 Products of the metabolism of one species
may become the main source of nutrients for
other species.
 Byproducts of the degradation of complex
nutrients are trapped in the biofilm matrix
and shared with other community members

16.  Protection From Competing Microorganisms, Host
Defenses, Antimicrobial Agents, and Environmental
Stress
Beta-lactamases, catalase, and proteinases
Retained in the biofilm matrix
protect other bacteria against antimicrobials and
host defenses

17. Genetic Exchanges
 Horizontal gene transfer in the community.
 Conjugation, transformation, and transduction.
 Dissemination of virulence and antibiotic-
resistance genes.

18.  A diverse range of virulence traits are
required for these particular stages of the
disease process,
Require the concerted action of bacteria in a
community
 Bacterial species that individually have low
virulence and are unable to cause disease
can do so when in association with others as part of a
mixed consortium
(pathogenic synergism)

19.  The antibiotic concentration required to kill
bacteria in the biofilm:
About 100 to 1000 times higher than that needed to
kill the same species in planktonic state
 Biofilm Structure May Restrict Penetration of
Antimicrobial Agents

20.  Altered Growth Rate of Biofilm Bacteria :
Bacteria grow slowly under conditions of low
availability of nutrients in an established biofilm
much less susceptible than faster-dividing cells.
Most antibiotics require at least some degree of
cellular activity to be effective.
 Presence of “Persister” Bacteria

21.  cell-cell communication that regulate gene
expression in a cell density–dependent manner
Quorum sensing
Production, release, and subsequent detection
of diffusible signaling molecules
Autoinducers

22.  Two predominant types of autoinducers:
◦ N-acyl-l-homoserine lactones (AHLs)
◦ Posttranslationally modified peptides
 Used by gram negative and gram-positive
bacteria, respectively

23.  Bacteria can perform specific functions only
when living in groups:
◦ Regulate virulence,
◦ Competence for DNA uptake,
◦ Entry into stationary phase, and
◦ Biofilm formation
 Entry into stationary phase dramatically alters
patterns of gene expression
Allow extended cell survival in the absence of
nutrients.

24.  Endodontic samples are collected and
transported to the laboratory
◦ in a viability-preserving, nonsupportive, anaerobic
medium.
Dispersed by sonication or vortex mixing
Diluted, distributed onto various types of agar
media
cultivated under aerobic or anaerobic
conditions

25.  After a suitable period of incubation:
Individual colonies are subcultivated
Identified on the basis of multiple phenotype-
based aspects
 colony and cellular morphology,
 gram-staining pattern,
 oxygen tolerance,
 comprehensive biochemical characterization,
 metabolic end-product analysis by gas-liquid
chromatography

26.  The outer cellular membrane protein profile
Gel electrophoresis, fluorescence under
ultraviolet light.
 Susceptibility tests to selected antibiotics can
be needed for identification of some species.
 Marketed packaged kits that test for
preformed enzymes have also been used for
rapid identification of several species

27.  Not all microorganisms can be cultivated
under artificial conditions
Nutritional and physiologic needs of most
microorganisms are still unknown
 Limitations of culture
Molecular biology
substantially improved to achieve a more realistic
description of the microbial world without the need
for cultivation.

28.  Molecular approaches for microbial
identification
◦ rely on certain genes that contain revealing
information about the microbial identity.
 16S rRNA gene (or 16S rDNA) has been the
most widely used

29. M
o
l
e
c
u
l
a
r
B
i
o
l
o
g
y
M
e
t
h
o
d

31.  There are an estimated 10 billion bacterial cells
in the oral cavity,
 Over 50% to 60% of the oral microbiota still
remains to be cultivated and fully characterized
 More than 400 different microbial
species/phylotypes have been found in infected
root canals

32.  Endodontic infections develop in a previously
sterile place that does not contain a normal
microbiota.
 Culture and molecular studies reveal only
prevalence of species.

33.  Mixed community conspicuously dominated by
anaerobic bacteria.
 The number of bacterial cells may vary from 103 –
108 per root Canal
 Molecular studies have disclosed a mean of 10 to
20 species/phylotypes per infected canal.
 Canals of teeth with sinus tracts exhibit a mean
number of 17 species

34.  The size of the apical periodontitis lesion has
been shown to be proportional to the number
of bacterial species and cells in the root canal
 The larger the lesion, the higher the bacterial
diversity and density in the canal

35. Prevalence of bacteria detected in primary infections of teeth with chronic apical
periodontitis. Data from the authors’ studies using a taxon-specific nested-polymerase
chain reaction protocol.

36. Prevalence of bacteria detected in primary infections of teeth with acute apical
periodontitis. Data from the authors’ studies using a taxon-specific nested-polymerase
chain reaction protocol

37. Prevalence of bacteria detected in primary infections of teeth with acute apical
abscesses. Data from the authors’ studies using a taxon-specific nested-polymerase
chain reaction protocol

38. Geographic Influence:
• Patients residing in different geographic locations and
suggested that significant differences in the prevalence of
some important species can actually exist.

39. Spatial Distribution of the Microbiota
 Bacterial cells from endodontic biofilms are very
often seen penetrating the dentinal tubules
 Dentinal tubule infection can occur in about 70% to
80% of the teeth with apical periodontitis.
 Bacteria present as planktonic cells in the main root
canal may be easily accessed and eliminated by
instruments and substances used during treatment,

40.  Main pulp canal space and walls
 Accessory canals and apical delta
 Dentinal tubules
 Cementum surface
 Extraradicular colonizations

41.  Those organized in biofilms:
 Attached to the canal walls or
 Located into isthmuses, lateral canals, and
 Dentinal tubules
More difficult to reach and may require special
therapeutic strategies to be eradicated

42.  Whenever dentin is exposed,
Pulp is put at risk of infection
Permeability of normal dentin dictated by its tubular structure
 largest diameter located near the pulp
(mean, 2.5 μm)
 Smallest diameter in the periphery, near the enamel or
cementum.
(mean, 0.9 μm)

43.  The smallest tubule diameter is entirely compatible with the
cell diameter of most oral bacterial species:
 Which usually ranges from 0.2 to 0.7 μm
 Bacterial invasion of dentinal tubules occurs more rapidly in
nonvital teeth than in vital.
Presence of tubular contents (In Vital teeth)
the functional or physiologic diameter of the tubules is only 5% to
10% of the anatomic diameter.

44. Most of the bacteria in the carious process
are non-motile;
Invade dentin by repeated cell division which
pushes cells into tubules
Bacterial cells may also be forced into tubules
by hydrostatic pressures
Developed on dentin during mastication.

45.  The root canal infection is a dynamic process, and
different bacterial species apparently dominate at
different stages.
In the very initial phases of the pulpal infectious process:
facultative bacteria predominate.
After a few days or weeks, oxygen is depleted
loss of blood circulation in the necrotic pulp.
Growth of obligate anaerobic bacteria.

46. Ecological conditions in different areas of the root canal. A gradient of
oxygen tension and nutrients (type and availability) is formed.

47. The main sources of nutrients for bacteria colonizing
the root canal system include:
 The necrotic pulp tissue
 Proteins and glycoproteins from tissue fluids and
exudate that seep into the root canal system via
apical and lateral foramens
 Components of saliva that may coronally penetrate
into the root canal
 Products of the metabolism of other bacteria.

48. Interbacterial nutritional interactions that can take place in
infected root canals where growth of some species can be
dependent upon products of metabolism of other species.

49. Other Microorganisms in Endodontic Infections
 Fungi:
Candida species,
 Archaea:
 Viruses:
 Noninflamed vital pulps of Patients infected with the human
immunodeficiency virus.
 Human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV)
have been detected in apical periodontitis lesions.

50.  Persistent or secondary intraradicular infections are
the major causes of endodontic treatment failure
 Involved microorganisms are remnants of a primary
or secondary infection
 Microorganisms that at some time entered the root
canal system secondary to professional
intervention.

51.  Bacteria at the Root Canal–Filling
Stage
 Diligent antimicrobial treatment may still fail to
completely eliminate bacteria from the infected
root canal system.
Persisting bacteria are either resistant or inaccessible
to treatment procedures.
 When bacteria resist treatment procedures, gram-
positive bacteria are more frequently present.

52.  Gram-positive facultatives or anaerobes often detected in
these samples include:
 Streptococci,
 P. micra, Actinomyces species,
 Propionibacterium species,
 P. alactolyticus,
 lactobacilli,
 E. faecalis, and
 Olsenella uli

53. • GRAM POSITIVE BACTERIA CAN BE MORE RESISTANT TO ANTIMICROBIAL
TREATMENT MEASURES:
• ABILITY TO ADAPT TO THE HARSH ENVIRONMENTAL CONDITIONS IN INSTRUMENTED
AND MEDICATED ROOT CANALS.
• BACTERIA PERSISTING IN THE ROOT CANAL AFTER CHEMOMECHANICAL
PROCEDURES OR INTRACANAL MEDICATION WILL NOT ALWAYS MAINTAIN AN
INFECTIOUS PROCESS

54.  Some apical periodontitis lesions healed even after
bacteria were found in the canal at the filling stage:
 Residual bacteria may die after filling because of toxic
effects of the filling material,
 Access denied to nutrients, or disruption of bacterial
ecology.
 Residual bacteria may be present in quantities and
virulence subcritical to sustaining periradicular
inflammation.

55.  Residual bacteria remain in locations where access to
periradicular tissues is denied
Host resistance to infection is also an important and
probably decisive counteracting factor.

56. MICROBIOTA IN ROOT CANAL–
TREATED TEETH
 Canals apparently well treated harbor one to
five species
 The number of species in canals with inadequate
treatment can reach up to 10 to 30 species
Which is very similar to untreated canals

57.  Several culture and molecular biology studies
have revealed:
E. faecalis is the most frequent species in root
canal– treated teeth
Prevalence values reaching up to 90% of cases
commonly recovered from cases treated in
multiple visits and/or in teeth left open for
drainage

58. The ability of E. Faecalis:
• penetrate dentinal tubules, sometimes to a deep
extent
Enable it to escape the action of endodontic
instruments and irrigants
• Resistant to calcium hydroxide:
• Acidify the cytoplasm

59. • E. faecalis can enter a so-called viable but non-cultivable
(VBNC) state:
InVBNC state, bacteria lose the ability to grow in culture media
Maintain viability and pathogenicity
Can resume division when optimal environmental conditions are
restored

60. Prevalence of microorganisms detected in root canal–treated teeth with posttreatment
disease. Data from the authors’ studies using a taxon-specific polymerase chain
reaction assay

61. Prevalence of Enterococcus faecalis in samples from root canal–treated
teeth with apical periodontitis.

62.  E. faecalis as the main causative agent of endodontic failures
has been questioned by some studies:
I. Even when present, E. faecalis is rarely one of the
most dominant species in retreatment cases
II. E. faecalis has been found not to be more
prevalent in root canal–treated teeth with lesions
when compared to treated teeth with no lesions

63. CONCLUSION:
Microbes seeking to establish in the root canal must leave the
nutritionally rich and diverse environment of the oral cavity.
Breach enamel, invade dentine.
overwhelm the immune response of the pulp and
settle in the remaining necrotic tissue within the root canal.
During that time they have to compete in a limited space with
other microbes for the available nutrition.

65.  The microbiota of root canal– treated teeth with apical
periodontitis is more complex than previously anticipated
by culture studies.
 The bacterial community profiles in treated cases vary
from individual to individual, indicating that distinct
bacterial combinations can play a role in treatment
failure.

66. References:
1. Kenneth M. Hargreaves, Stephen Cohen. Cohen’s Pathways of the Pulp. 10th edition.
Elsevier, Mosby.2011
2. Ingle, Bakland, Baumgartner. Ingle’s Endodontics 6. BC Decker. 2008
3. Sabeti M, Slots J: Herpesviral-bacterial coinfection in periapical pathosis. J Endod 30:69,
2004
4. Siqueira JF, Jr, Rôças IN: Polymerase chain reaction-based analysis of microorganisms
associated with failed endodontic treatment. Oral Surg Oral Med Oral Pathol Oral
Radiol Endod 97:85, 2004.
5. Sakamoto M, Siqueira JF, Jr, Rôças IN, Benno Y: Molecular analysis of the root canal
microbiota associated with endodontic treatment failures. Oral Microbiol Immunol
23:275–281, 2008.
6. Zoletti GO, Siqueira JF, Jr, Santos KR: Identification of Enterococcus faecalis in root-filled
teeth with or without periradicular lesions by culture-dependent and –independent
approaches. J Endod 32:722, 2006.
7. Rôças IN, Hulsmann M, Siqueira JF, Jr: Microorganisms in root canal-treated teeth from
a German population. J Endod 34:926, 2008.
8. Rôças IN, Siqueira JF, Jr, Aboim MC, Rosado AS: Denaturing gradient gel
electrophoresis analysis of bacterial communities associated with failed endodontic
treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 98:741, 2004.
9. Kaufman B, Spangberg L, Barry J, Fouad AF: Enterococcus spp. in endodontically
treated teeth with and without periradicular lesions. J Endod 31:851, 2005.
10. Aruna Kanaparthy, Rosaiah Kanaparthy:Biofilms-The Unforgiving Film in Dentistry
(Clinical Endodontic Biofilms) . Dentistry 2012, 2:7
11. Adalberto R. Vieira, Jose F. Siqueira, Domenico Ricucci. Dentinal Tubule Infection as
the Cause of Recurrent Disease and Late Endodontic Treatment Failure: A Case
Report. JOE — Volume 38, Number 2, February 2012