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