View
386
Download
3
Category
Tags:
Preview:
Citation preview
Microbiology of Endodontic Disease
Dr. Ashok Ayer
Assistant Professor
Department of Conservative Dentistry and Endodontics
B. P.Koirala Institute of Health Sciences, Dharan, Nepal
Contents: Introduction
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
Conclusion
References
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.
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.
Mechanisms of MicrobialPathogenicity 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.
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.
Cytoplasmicmembrane Cell wall
Flagellum
LTA
PG
Gram-positive
Exopolysaccharide
(capsule)Fimbriae
ChromosomeRibosomes
Plasmids
Gram-negative
LPS OMP
OM
CM
CM
LPtn
PG
Scanning electron micrograph showing a bacterial biofilm covering dentinin a deep carious lesion. Note the presence of different bacterialmorphotypes (×3500). (From Torabinejad M, Walton RE: Endodontics:principles and practice, ed 4, Saunders/Elsevier, St. Louis, 2009
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 theendodontic microbiota differs consistently between individuals.
From the perspective of the single-pathogenconcept:
◦ Apical periodontitis can be considered as having nospecific microbial etiology.
However, based on the community as-a-pathogen concept:
◦ it is possible to infer that some communities aremore related to certain forms of the disease
Biofilm:◦ a sessile multicellular microbial community
characterized by cells that are firmly attached to asurface and enmeshed in a self-produced matrix ofextracellular polymeric substance (EPS), usuallypolysaccharide
Biofilm infections account for an estimated65% to 80% of bacterial infections that affecthumans in the developed world
Community members form,
◦ distinct populations or microcolonies separated byopen water channels that traverse the biofilmmatrix and create primitive circulatory systems.
vital nutrients and communication moleculescan diffuse, and wastes can be washed outthrough these channels.
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.
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)
Increased Metabolic Diversity and Efficiency
Take part in a number of nutritionalinterrelationships, and food webs.
Products of the metabolism of one speciesmay become the main source of nutrients forother species.
Byproducts of the degradation of complexnutrients are trapped in the biofilm matrixand shared with other community members
Protection From Competing Microorganisms, HostDefenses, Antimicrobial Agents, and EnvironmentalStress
Beta-lactamases, catalase, and proteinases
Retained in the biofilm matrix
protect other bacteria against antimicrobials and host defenses
Genetic Exchanges
Horizontal gene transfer in the community.
Conjugation, transformation, and transduction.
Dissemination of virulence and antibiotic-resistance genes.
A diverse range of virulence traits arerequired for these particular stages of thedisease process,
Require the concerted action of bacteria in a community
Bacterial species that individually have lowvirulence and are unable to cause disease
can do so when in association with others as part of a mixed consortium
(pathogenic synergism)
The antibiotic concentration required to killbacteria 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 ofAntimicrobial Agents
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
cell-cell communication that regulate geneexpression in a cell density–dependent manner
Quorum sensing
Production, release, and subsequent detection of diffusible signaling molecules
Autoinducers
Two predominant types of autoinducers:
◦ N-acyl-l-homoserine lactones (AHLs)
◦ Posttranslationally modified peptides
Used by gram negative and gram-positive bacteria, respectively
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.
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
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
The outer cellular membrane protein profile
Gel electrophoresis, fluorescence under ultraviolet light.
Susceptibility tests to selected antibiotics canbe needed for identification of some species.
Marketed packaged kits that test forpreformed enzymes have also been used forrapid identification of several species
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.
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
Molecular
Biology
Method
There are an estimated 10 billion bacterial cellsin the oral cavity,
Over 50% to 60% of the oral microbiota stillremains to be cultivated and fully characterized
More than 400 different microbialspecies/phylotypes have been found in infectedroot canals
Endodontic infections develop in a previouslysterile place that does not contain a normalmicrobiota.
Culture and molecular studies reveal onlyprevalence of species.
Mixed community conspicuously dominated byanaerobic bacteria.
The number of bacterial cells may vary from 103 –108 per root Canal
Molecular studies have disclosed a mean of 10 to20 species/phylotypes per infected canal.
Canals of teeth with sinus tracts exhibit a meannumber of 17 species
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
Prevalence of bacteria detected in primary infections of teeth with chronic apicalperiodontitis. Data from the authors’ studies using a taxon-specific nested-polymerasechain reaction protocol.
Prevalence of bacteria detected in primary infections of teeth with acute apicalperiodontitis. Data from the authors’ studies using a taxon-specific nested-polymerasechain reaction protocol
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
Geographic Influence:
• Patients residing in different geographic locations and
suggested that significant differences in the prevalence of
some important species can actually exist.
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,
Main pulp canal space and walls
Accessory canals and apical delta
Dentinal tubules
Cementum surface
Extraradicular colonizations
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
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)
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.
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.
The root canal infection is a dynamic process, anddifferent 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.
Ecological conditions in different areas of the root canal. A gradient of
oxygen tension and nutrients (type and availability) is formed.
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.
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.
Other Microorganisms in Endodontic Infections
Fungi:
Candida species,
Archaea:
Viruses:
Noninflamed vital pulps of Patients infected with the humanimmunodeficiency virus.
Human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV)
have been detected in apical periodontitis lesions.
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.
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.
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
• GRAM POSITIVE BACTERIA CAN BE MORE RESISTANT TO ANTIMICROBIAL
TREATMENTMEASURES:
• ABILITY TO ADAPT TO THE HARSH ENVIRONMENTAL CONDITIONS IN INSTRUMENTEDANDMEDICATED ROOT CANALS.
• BACTERIA PERSISTING IN THE ROOT CANAL AFTER CHEMOMECHANICAL
PROCEDURES OR INTRACANAL MEDICATION WILL NOT ALWAYS MAINTAIN AN
INFECTIOUS PROCESS
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 periradicularinflammation.
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.
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
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
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
• E. faecalis can enter a so-called viable but non-cultivable(VBNC) state:
In VBNC state, bacteria lose the ability to grow in culture media
Maintain viability and pathogenicity
Can resume division when optimal environmental conditions are restored
Prevalence of microorganisms detected in root canal–treated teeth with posttreatmentdisease. Data from the authors’ studies using a taxon-specific polymerase chainreaction assay
Prevalence of Enterococcus faecalis in samples from root canal–treated
teeth with apical periodontitis.
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
CONCLUSION:
Microbes seeking to establish in the root canal must leave thenutritionally 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.
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.
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 microorganismsassociated with failed endodontic treatment. Oral Surg Oral Med Oral Pathol OralRadiol Endod 97:85, 2004.
5. Sakamoto M, Siqueira JF, Jr, Rôças IN, Benno Y: Molecular analysis of the root canalmicrobiota associated with endodontic treatment failures. Oral Microbiol Immunol23:275–281, 2008.
6. Zoletti GO, Siqueira JF, Jr, Santos KR: Identification of Enterococcus faecalis in root-filledteeth with or without periradicular lesions by culture-dependent and –independentapproaches. J Endod 32:722, 2006.
7. Rôças IN, Hulsmann M, Siqueira JF, Jr: Microorganisms in root canal-treated teeth froma German population. J Endod 34:926, 2008.
8. Rôças IN, Siqueira JF, Jr, Aboim MC, Rosado AS: Denaturing gradient gelelectrophoresis analysis of bacterial communities associated with failed endodontictreatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 98:741, 2004.
9. Kaufman B, Spangberg L, Barry J, Fouad AF: Enterococcus spp. in endodonticallytreated 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 asthe Cause of Recurrent Disease and Late Endodontic Treatment Failure: A CaseReport. JOE — Volume 38, Number 2, February 2012
Recommended