MICROBIOLOGY

OF
ENDODONTICS
 TEXTBOOKS:
• Ingle’s Endodontics. 6th ed, 2008. J.I.Ingle & LK Bakland.
Chapter 7
• Endodontics. Principles and Practice. 4th ed,2009,
M.Torabinejad & R.Walton.Chapter 3.
• Cohen’s Pathways of the pulp. 10th ed, 2011. Chapter 15.
• Treatment of endodontic infection. Jose F.Siqueira Jr 2011.
Quintessence Publishing. Chapter 1.
• Medical microbiology and immunology for dentistry. Nejat
Düzgünes, 2016.
• Seltzer and Bender’s Dental Pulp. 2nd Ed. 2012
 Learning objectives:
• Define terms associated with endodontic microbiology
• Understand the significance of bacteria in pulpal and PR
diseases
• Describe portals of entry of microorganisms (mo) to the
pulp and periradicular (PR) tissues
• Recognize the different types of endo infections and the
main microbial spp involved in each one
• Understand the bacterial diversity within infected RC
• Describe the reactions of pulp and PR tissues to bacteria
• Discuss the rationale for debridement of root canal system
 Terminology
Colonization: the phenomenon of establishment of bacteria or other
organisms in a living host
occurs if: - appropriate physical or biochemical conditions
are available for growth
- inhibitory factors are inadequate to destroy m.o
Normal oral flora: result of permanent colonization of m.o in a symbiotic
relationship with the hostproduce beneficial results
Opportunistic pathogens : gain access to normally sterile area of
the body and produce disease such as dental pulp or PR tissue
Infection : m.o damage the host & produce clinical signs and
symptoms body defense & immunologic response
Pulpal&PR pathoses: opportunistic pathogens infect the pulp cavity & PR
tissues
• Pathogenicity : the capacity of mo to produce disease
within a particular host
• Virulence : the degree or pathogenicity in a host under
defined circumstances
• Anachoresis : a process by which mo are transported in the
blood to an area of inflammation where they establish an
infection
• Stage in development of an endodontic infection: microbial
invasion, colonization, multiplication and pathogenic
activity
• History perspective of endodontic microbiology :
- 17th century Leeuwenhoek, first observed the presence of
bacteria in the root canal of a decayed tooth that were stuffed
with a soft matter and alive.
- 1894 WD Miller, reported the association between bacteria
and apical periodontitis, and identified 3 basic morphology of
bacteria known today.
- 1939 EW Fish put the basis for successful endodontic
therapy; removal of the nidus of infection would lead to
resolution of the infection.
-1965 Kakehashi et al; no lesions in germ-free
(gnotobiotic) rats
Dental pulps induced with bacteria pulpal and PR
lesions developed
- 1976 Sunqvist : the role of aerobe and anaerobe mo in
the PR pathoses
- 1981 Möller et al: strong evidence of microbial
induction of apical periodontitis. Necrotic pulp tissue per
se is not able to induce and maintain apical periodontitis
lesions.
 Portal of entry of mo to the pulp
• Exposed dentinal tubules
- dental caries
- missing enamel or cementum
- fracture of the tooth/cracks
- microleakage under or around restoration
- restorative procedures
- external or internal root resorption
• Direct pulp exposure
• Periodontal diseaseperio pocket reaches the AF
• Anachoresisblood/lymphatic route mediated by
cytokines
 Types of endodontic infection
• According to anatomic location:
- Intraradicular infection:
Primary infection
Secondary infectionbacterial invasion after
barrier is breached
Persistent infectionafter disinfection and RCT procedures
- Extraradicular infection
• According to the occurrence :
- acute infectionstart rapidly, short duration
- chronic infectioncontinue for a relatively long
time
Primary intraradicular infection
• Initially invade and colonize
the necrotic pulp tissue
• Referred as initial infection
or virgin infection
• Mixed infection ~ 10-30
bacterial species
• Predominant bacteria:
anaerob bacteria and some
facultative anaerob
• Mostly are the participants in early stages of pulp invasion or
late comers after pulp necrosis cause inflammation
• The most prevalent named bacterial species obligate
anaerob Gram + (Parvimonas, Actinomyces,
Peptostreptococcus, Propionibacterium), facultative Gram +
(streptococcus), obligate anaerob Gram – (Fusobacterium,
Porphyromonas, Prevotella, Tanarella etc)
• About 40%-55% of the endodontic microbiota is still
uncultivated
 Secondary intraradicular infection
- Not present in the primary infection but introduced into
the RCS after professional intervention or due to perio
lesion
- Can be during treatment, between appointment or after
root canal filling (loss or leakage of temporary/permanent
restorative materials, fracture, recurrent decay)
- Species oral or non oral mo, depends on the cause of
infection
- Main cause remnants of dental plaque, calculus or
caries on the tooth crown, leaking rubber dam,
contamination of endo instruments, irrigants, intracanal
medication.
 Persistent intraradicular infection
• MO that can resist intracanal antimicrobial proced
• Also termed recurrent infection
• MO involved remnants of 1° or 2° infection
• Composed of fewer species than 1° infection, mostly
gram + facultative or anaerob, fungi↑
• Responsible for several clinical problems: persistent
exudation & symptoms, inter-appointment flare ups,
failure of endo treatment PA lesion
• Most studies- higher occurrence of Gram +
bacteria in both post-instrumentation and post-
medication samples, including Streptococcus
spp,Staphylococcus spp, Lactobacillus, E faecalis
spp, Actinomyces spp, (see Ingle, p.241)
• E faecalis is the most frequent species in root-
canal treated teeth
• Influence the outcome of endo treatment
Extraradicular infection
• Invasion and proliferation in the inflamed PR tissue
almost as a sequel of intraradicular infection
• Can be dependent on the intraradicular infection
acute apical abscess managed by root canal
therapy
can be independent on the intraradicular infection
apical actinomycosis treated by endodontic
surgery
• Establishment of an extraradicular infection :
- a result of direct advance of some bacterial
species that overcome host defenses concentrated
near the apical foramen
- due to bacterial persistence in the apical
periodontitis lesion after remission of acute
apical abscesses
- a sequel to apical extrusion of debris during root canal
instrumentation
Bacterial genera represented in endo infections
Gram Negative Bacteria
Anaerobes Facultatives
Rods:- Dialister* - Capnocytophaga
- Porphyromonas * - Eikenella
- Tannarella * - Haemophilus
- Prevotella*
- Fusobacterium *
- Campylobacter
- Synergistes
- Catonella
- Selenomonas
- Centipeda

Cocci : - Veillonella
- Megasphaera

Spirilla :Treponema *
 Gram Positive Bacteria
• Anaerobes Facultatives
Rods:- Actinomyces -Actinomyces *
- Pseudoramibacter * -Corynebacterium
- Filifactor * -Lactobacillus
- Eubacterium
- Mogibacterium
- Propionibacterium *
- Eggerthela
- Olsenella *
- Bifidobacterium
- Slackia
- Atopobium
- Solobacterium
- Lactobacillus

Cocci :
- Micromonas * - Streptococcus
- Peptostreptococcus * - Enterococcus
- Finegoldia - Granulicatella
- Peptoniphilus
- Anaerococcus
- Streptococcus *
- Gemella
 Gram Positive Bacteria
Anaerobes Facultatives
Cocci :
- Micromonas * - Streptococcus
- Peptostreptococcus * - Enterococcus
- Finegoldia - Granulicatella
- Peptoniphilus
- Anaerococcus
- Streptococcus *
- Gemella

The development of molecular methods have revealed new

suspected endo pathogens and as-yet uncultivated bacteria in
endo infections.
Other MO in endodontic infections
• Fungi, sporadically found in primary infection
• Virusescan’t survive in the root canal with
necrotic pulp (just DNA/RNA)
- found in root canal of non inflamed vital pulps
of HIV patients
- Human cytomegalovirus and Epstein-Barr virus
have been detected in symptomatic apical
periodontitis lesions
• Key ecologic factor that influence the
composition of the MO in the necrotic root
canal :
• Oxygen tension
• Redox potential
• Type and amount of available nutrients
• Bacterial interactions
• geographic influence
• Necrotic pulp :
- fertile environment for bacterial growth and
colonization moist, warm, nutritious, anaerob
- lack of active microcirculation protected from
the host defense

• Patterns of microbial colonization:

- as planktonic(unattached) cells suspended in the fluid
phase of the main root canal
- as aggregates or coaggregates adhered to the root canal
walls forming multilayered biofilms
 Bacterial biofilm
• Biofilm is a mode of bacterial growth where
dynamic communities of interacting sessile
cells are irreversibly attached to a solid
substratum as well as each other and are
embedded in a self-made matrix of extra
cellular polymeric substances.
 Development of biofilm
Occurs in three stages :
1. Adsorption of inorganic and organic molecules to the
solid surface formation of conditioning layer
2. Adhesion of microbial cells to the conditioning layer
affected by pH, temperature, surface energy of the
substrate, nutritional availability, time of contact of
bacteria, bacterial cell surface charge, surface
hydrophobicity
3. Development of biofilm and biofilm expansion
forming microcolony final structure of biofilm
 Endodontic biofilm
Can be categorized as :
• Intracanal biofilms formed on the root canal dentin of
endo infected teeth
• Extraradicular biofilms= root surface biofilms formed
on the root (cementum) surface of endo infected teeth
• Periapical biofilms found in the periapical region of
endo infected teeth  may or may not dependent on the
root canal. Able to overcome host defense mechanisms
• Biomaterial-centered infections adherence of bacteria on
artificial biomaterial surface root canal obturating
materials, can be intraradicular or extraradicular, depends
on the location of the material
 Bacteria invasion capacity:
• Depends on
- antiphagocytosisavoid local host defense by forming capsules
(Streptoc, Bacteroides, Fusiformis)
Phagocytosis is enhanced by forming specific anticapsule
antibodies
- adapt metabolic to pulp condition, from normal to inflammation/
necrotic
- need enzyme to grow and reproduce. The availability of enzyme
is induced by complex mechanism of bacteria
Enzyme produced by bacteria neutralize immunoglobulin and
their components
 Virulence of Bacteria
• Endotoxinderived from components of bacteria
- LPS (lipopolysaccharide) cell wall of Gram-bacteria
associated with pulpal pain, periapical inflammation etc
- effect in bone resorption, induce periradicular pathosis
- have non-specific antigen that are not well neutralized
by antibodies
- enzymes  spreading factors that neutralize
immunoglobulin and the complement components
collagenase, hyaluronidase etc
-PG (peptidoglycan) & LTA (lipoteichoic acid)components
of Gram + cell wallstrongly boosted in the presence of Gr-
- fimbriae (pili)  exchange DNA during conjugation/synergy
relationships between bacteria resistance to antibiotic,
important for attachment to surfaces and to other bacteria.
Found on the surface of Gram – bacteria
- bacterial capsules resistance factor for bacteria, protect the
bacteria from phagocytosis, dessication
- Extracellular vesicles  formed from outer membrane of gram
(-) bacteria  free endotoxin
- able to neutralize antibodies
- involves in hemagglutination, hemolysis, bacterial
adhesion and proteolytic action on host tissues
• Exotoxin released by bacteria
- Short-chain fatty acids  major by products of
fermentation process by obligate anaerobs
including propionic acid, butiric and isobutiric acids
 stimulate inflammatory response bone resorption
- Polyamines polycationic molecules including :
spremine, spermidine, cadaverine, putrescine.
 contribute to clinical symptoms like pain
(eg.percussion pain) and formation of sinus tract
- Superoxide anions biologically toxic, highly reactive
free radicals produced by few bacteria spp and by the
cells of immune system
 Host response/host resistance :
1. Innate immune response (= non specific) :
Predominant cells  odontoblast cells, PMN, macrophage,
dendrite cells, NK cells
Bacteria  PAMPs recognized by PRRs ( called TLRs)

activation of PRRs triggers numerous host responses including

opsonization, activation of complement, coagulation cascades,
phagocytosis, activation of proinflammatory signaling pathways,
and induction of apoptosis.

Macrophages also serve as APC (antigen presenting cells) by

expressing MHC class II molecules for Adaptive immunity
2. Adaptive Immunity (= acquired, specific)
Main role is T cells and B cells.
- the specifity is regulated at genetic levels in B (BCRs)
and T lymphocytes (TCRs)recognize and bind foreign
or self- antigens)
T receptor is TCRs with subpopulations : Thelper cells,
Treg, Tsup, Tcyto cells distinguished by their cell
surface markers, cytokine profiles or transcriptional
factors. Constitute for cell-mediated immunity
- B cells mainly produce antibodies that constitute the
host humoral immune response
Reaction of pulp to bacteria
• Pulp response to irritants :
- non specific/innate inflammationneutrophil, macrophages,
- complement component
- specific/adaptive immunologic reactionsproduction of
IgG (deep).

Response to caries :
- formation of peritubular dentin decreased permeability
of tubular dentin
- production of irregular secondary dentin

• Exposed pulp by caries :

accumulation of PMN liquefaction necrosis
The dynamics of pulp reaction is related to:
• Virulence of the bacteria
• host response/host resistance
• Amount of pulpal circulation
• Degree of lymph drainage
• The ability to release inflammatory fluids to avoid
the increasing of intrapulpal pressure related to
transcapillary fluid exchange, pulpal blood flow,
hydrostatic pressure, colloid osmotic pressure
 Goal of endodontic treatment

Removal of the reservoir of infection (necrotic tissue,

bacteria, bacterial byproducts) through debridement.
- Debridement of the root canal system:
- cleaning and shaping (biomechanical) followed by
irrigation (chemical adjuncts)
- intracanal medication
- Complete obturation of root canal system
• ANTIMICROBIAL IN ENDODONTICS
 Antimicrobial of endo procedures

• Debridement
- mechanical instrumentation: manual or
engine driven/rotary instrumentation
• Irrigation
• Dressings
 Debridement/Mechanical instrumentation

• Biomechanical instrumentation key role in the cascade of

treatment procedures to eradicate mo in the RCS but not
sufficient to disinfect root canals, either stainless steel or Ni-Ti
instruments.Mechanical
• Manual instrumentation (stainless steel)
- hand files used number of bacteria are significantly reduced.
- Rotary NiTi instrumentation faster.
No difference in the ability to eliminate
bacteria compared to manual instrumentation
• Irrigation
• Irrigation solutions support and facilitate mechanical
removal and chemical eradication of the infection
The ideal irrigant or combination of irrigants kill bacteria,
dissolve necrotic tissue, lubricate the canal, remove the
smear layer and non-irritant.

• Intracanal medication such as Ca(OH)2

 Types of irrigants used
- NaOCl (0.5-6%)
- EDTA 10-17% remove smear layer
- ChlorHexidine digluconate 2%
- MTAD (mixture of tetracycline and
detergent) and Tetra clean
- PAD (Photo Activated Disinfection)
low-powered laser light
 Sodium Hypochlorite (NaOCl)
• Most widely used irrigating solutions
• Ionizes and produce Na+ and OCl- , equilibrium with HOCl
(hypochlorus acidresponsible for bacteria inactivation)
• Effectively dissolves pulpal remnants and organic
components of dentin, but lack of effect on inorganic material
• In vitro studieseffective in killing bacteria and yeasts ˃˂ in
vivo studiesroot canal anatomy, substrate, etc
• No significant difference in antibacterial efficiency in vivo
between concentration of 0,5% and 5%
• Higher concentrationcytotoxicity and caustic effect
 EDTA, citric acid and other acids

• EDTA 17% little antibacterial activity, effective

chelating agent in the RCS
• Contributes to the elimination of bacteria by removing
smear layer (together with NaOCl) and inorganic
component of the dentin
• SEM more debris was removed by irrigation with
EDTA followed by NaOCl
• Citric acid 1-50% remove smear layer
• Removal of smear layerenhances disinfection of the
root canal wall and deeper layers of dentin, and
facilitates penetration of sealers into dentinal tubules.
 Chlorhexidine digluconate(CHX)
• Effective as an antimicrobial agent
• In endodontics as an irrigating solution and as an
intracanal medicament
• Permeates the cell wall or outer membrane and attacks the
bacterial cytoplasmic of the yeast
• Higher concentration coagulates intracell components
• Low toxicity and depends on the pH, ineffective in the
presence of organic matter
• Effective both Gram+, Gram – and yeast
 Chlorhexidine digluconate(CHX)

• Mycobacteria and bacterial spores are resistant to CHX

• No effect on the biofilm structure
• Lack tissue dissolving capacity
• Combined benefits is obtained from combining 2 solutions;
CHX and NaOCl, but not soluble in each other, forms
brownish-orange precipitate (flushing with saline to avoid)
• Presently marketed as a water-based solution, as a gel, and
as a liquid mixture with surface active agents
• More in vivo research is needed to identify the optimal
irrigation regimen for various types of endo treatments
 Antibiotic-containing irrigation solutions

MTAD ( a mixture of tetracycline isomer, acid, and

detergent) and Tetraclean
• Low pH (2.5) and contains citric acid
• Benefit of smear layer removal (after NaoCl irrigation) and
antibacterial activity simpler irrigation
• Contains high concentration of doxycycline
• Better performance of NaOCl/EDTA combination
compared to NaOCl/MTAD irrigation
• Antibacterial effect not based on the antibiotic
component, but combined effect from other gradients
 Techniques of irrigation
• Manual agitation /active technique :
Irrigants placed in the root canal are agitated mechanically
in coronoapical and stirring movements performed with a
syringe and a side-vented needle
• Machine-assisted agitation systems:
- sonically activated irrigationoscillating movements
1.5-6 kHz. Examp. Endo-activator
- ultrasonically activated irrigationosc 20-30 kHz; 2 types:
- simultaneous ultrasonic instrumentation & irrigation
- passive ultrasonic irrigation (PUI)
IRRIGATION NEEDLE
ENDOACTIVATOR
• Machine-assisted agitation systems:
- The plastic rotary F fileendo polymer-based
rotary finishing file remove dentinal wall
debris and agitating the NaOCl without
enlarging the canal
- Pressure-alternation technique EndoVac apical
negative-pressure system
ENDOVAC
Intracanal Medication
 Intracanal Medication

• Mode of action : denaturation of cell proteins

• Mode of application: applied on a cotton pellet or
absorbent paper point and placed in pulp space
• Toxicity : kills bacteria and host cells
• Duration of effectiveness : short term except
calcium hydroxide
Intracanal medicaments usually used :

• Phenolics : moderate antimicrobial with short term effect (24 hours)

only effective in direct contact with microorganisms
incld:,camphorated parachlorphenol, cresol, thymol,
eugenol,creosote,cresatin
• Aldehydes : moderate antimicrobial effect formocresol
• Halides : sodium hypochlorit, iodine potassium iodide
• Steroides: single use or in combination with antibiotics
reduce pain post treatment except flare up/severe pain
• Antibiotics : rarely used as intracanal dressing eg.3Mix-MP
• Combination : variety substances or mixed by dentist for double action
by single application
CALCIUM HYDROXIDE
• Current intracanal dressing of choice
Available in many forms and combinations
• Used as intracanal dressing of necrotic pulp
• Can be combined with water, saline, anaestheticum, glycerin,
methylcellulose or other medicaments (not very effective due to
toxicity from other medicaments)
• Combined with water/glycerin paste, placed in root canal with
plugger or lentulo counter clockwise
• Antibacterial action: pH 12.5, block intracanal bacterial growth and
change biological nature of lipopolisaccharide bacteria
WHEN TO CULTURE

• Reasons of culturing bacteria of root canal:

- to determine bacteriologic status of root canal before
obturation
- to assess the effectiveness of debridement procedure
- to isolate flora microbes in order to evaluate the
sensitivity and resistance of antibiotics in persistent
infection
ENDODONTIC PHARMACOLOGY
 Pain Management
Management of painchallenge for endodontist.
Effective pain management  integration of
pharmacological, procedural and behavioral skills.
Major drug classes available to the practitioner such
as: non-narcotic analgesics NSAIDs (non-
steroidal anti-inflammatory analgesics),
Acetaminophen (parasetamol) and steroids
glucocorticoids .
Evidence-based approaches provide information for
pain control
• NSAIDs  inhibition of cyclooxygenase (COX)
• NSAIDs primary class of analgesics for treating acute
pain, surgical or non-surgical procedures.
• Ibuprofen 600 mg:1 hour before local anesthetic injection
with irreversible pulpitis increase depth of anesthesia
• Acetaminophen 650 mg: NSAIDs – contra indicated
patients
• Acetaminophen/NSAID combination very effective for
pain control following surgical or endo procedures  by
blocking inflammatory mediators that sensitize or activate
pulpal nociceptors
• Steroids (dexamethasone, methylprednisolone) used as:
- pulp capping agent
- intracanal medicament, alone or in combination with
antibiotics/antihistaminesmore consisten reductions in
postoperative pain or flare ups when used in vital teeth
- systemicdecrease pain and inflammation
- Double-blind, prospective controlled study IM injection or tablets,
showed significantly reduced pain compared to placebo
- Prescribed for patients with moderate/severe pretreatment pain
- Reduce the inflammatory response by suppressingvasodilation,
neutrophil migration, inhibiting the formation of arachidonic acid
• Opioid analgesics (codein, morphine) additional
pain control is needed
• Opioid+NSAIDs additional analgesia
• Opioid+acetaminophencontrol pain and
patient’s discomfort
• Limited use due to adverse side effects include
nausea, vomiting, dizziness, respiratory depression
and dependence
When to prescribe antibiotic for endodontic infections
• In conjunction with the appropriate endodontic
procedure, when there is :
- systemic involvement, persistent infection or a
spreading infection
signs and symptoms : fever (>38°C), malaise,
cellulitis, unexplained trismus, progressive diffuse
swelling
- Compromise patients
Antibiotic  as an adjunct to debridement and drainage
 Mechanisms of antibiotic action

• Inhibition of cell wall synthesis. e.g. penicillin

• Alteration of cell membranes. e.g. polymyxin
• Inhibition of protein synthesis. e.g. tetracycline
and aminoglycosides
• Inhibition of nucleic acid synthesis. e.g.quinolone,
rifampin
• Antimetabolic activity.e.g. sulfanilamide
 Selection of antibiotic regimen
• Penicillin narrow spectrum of microbial activity
antibiotic of choice for Endo Infec, low toxic, effective,
10% allergy rate.
Oral loading dose 1000 mg, followed by 500 mg/ 6 hours,
6-10 days
• Amoxicillin  broader spectrum than penicillin, for more
resistant organisms,rapid absorption, more sustained serum
level
Oral loading dose 1000 mg, followed by 500 mg/ 8 hours,
6-10 days
Combination with clavulanate not recommended unless
B-lactamase producing bacteria are the cause of infection
.
Clarithromycin and Azithromycin  macrolides
- mild infection and allergic to penicillin
- Clarithromycin dosage : 250-500 mg/12 hours, 6-10 days
(with or without meals)
- Azithromycin : takes 1 hour before meals or 2 hours after
meals
. loading dose 500 mg/1st day, followed by 250 mg/daily
. Block the metabolism of a number of drugswarfarin &
anisindione, lead to serious bleeding in anticoagulated
patients
- Metronidazole :
excellent activity against strict anaerobes, but not
facultative bacteria, can be combined with penicillin, with
loading dose 500 mg, followed by 250-500 mg/6 hours.
During therapy, no alcohol consumption
- Clindamycin
recommended for serious infection and allergy to penicillin,
effective for both facultative and strict anaerobes
loading dose : 300 mg, followed by 150-300 mg/8 hours, 6-
10 days
- Cefixime (3rd generation of cephalosporin) 200 mg/2x
daily
 Bacterial resistance to antibiotics
• Can be intrinsic resistance (result of constitutive
production of enzyme) or acquired resistance (result
of mutation)
• The mechanisms are as follows :
• Impaired permeability of antibiotics ( some of Gram –
bacteria)
• Alteration of the molecular targets of antibiotics
(change of PenBindProteinsemergence of MRSA)
• Enzymatic inactivation of antibiotics (extended
spectrum ß-lactamases-ESBL)
 THANK YOU FOR
YOUR ATTENTION