Microbiology aspect in endodontics

1. Microbiolo
gy aspect
in
endodontic
Thalerngsak Samaksamarn
Department of restorative dentistry
Faculty of dentistry.
KKU

2. Terminology
 Colonization : the establishment of bacteria or
orther microorganism in a living host.
 Infection : damage the host and produce clinical
signs and symptom
 Pathogenicity : The capacity of organisms to
produce disease within a particular host
 Virulence : the degree of pathogenicity in a host
under defind circumstance

3. State of disease Spread of infection
No.&Virulence of bacteria Associated anatomical structure
Abscess
formation
PA Pathology
Host defense
Status of host defensive system

4. Entries of bacteria
to the pulp system

5. •Caries
•Periodontal disease
•Trauma and restoration leakage
•Anachoreasis

6. Dental caries
Facultative gram positive
bacteria
Propionibacterium,
Eubacteruim, Arachnia,
Lactobacilli, Bifidobacterium ,
Actinomyces
Some of gram negative
bacterial such as Bacteroides
Hoshino et al 1985

7. DENTAL CARIES
 the most common pathway to the root canal
system for microbes. When the tooth is
intact, enamel and dentin protect it against
invasion of the pulp space.
 As caries approaches the pulp, reparative
dentin is laid down to avert exposure, but this
rarely can prevent microbial entry without
caries excavation

8. Dentinal tubules
 1 to 4 μm in diameter,
 bacteria are less than 1 μm in diameter.
 the protective cementum layer is missing or if it has been
lost through trauma, the dentinal tubules may be
exposed and may serve as a pathway for microbial
invasion of the pulp space.
 Bacterial movement is restricted by
:outflow of dentinal fluid,
: odontoblastic processes,
: mineralized crystals
: macromolecules, including immunoglobulins in the
tubules.

10. •Accessory canal
•Furcation canal
•Lateral canal
•Apical foramen
•Exposed dentinal tubule
Anerobic bacteria  Gram negative rod,
spirochete

11. Inflammation and local necrosis have been
demonstrated in pulp adjacent to entry pathway.
Immune system via circulation
Whole pulp tissue change
due to periodontal disease

12. Necrotic
pulp
Periodontal lesion involve apical foramen
Guldenor et al 1985

13. Pulp exposure due to trauma give access to
oral bacteria, this will cause bacterial invasion
to the pulp  inflammation
Pulp necrosis
Kakehashi et al 1965

14. Healthy exposed vital pulp, the penetration of
tissue by bacteria is relative slow ≤2 mm./wk
Cvek et al 1978

15. Laboratory experiments indicate that bacteria
can enter through even minor crack in enamel
or dentine.
Love et al
1996
Dentinal tubules exposed by tooth fracture
during cavity preparation or marginal leakage
are the potential pathway.
Bender & Seltzer 1959
Smulson & Sieraski 1989

16. Anachoresis
“Blood borne bacteria is
preferentially localized in
areas of inflammation.”
Burke & Knigton 1960
Gier & Mitchell 1968
Intravenous bacterial injection into bloodstream could
be demonstrated pulp that were inflamed due to
deep cavity preparation and chemical irritation.

17. Polymicrobia
l
Microorganism
Endodontics failure
Pulpal and periapical
disease
In 1890 W.D. Miller, the father of oral microbiology, was the first
investigator to associate the presence of bacteria with pulpal disease.
A classic study published in 1965 by Kakehashi et al

18. Polymicrobial
 the number of microorganisms detected in
endodontic infections increased to a range of
three to 12 organisms per infected root canal
associated with an apical lesion
 The number of colony forming units (CFU) is
usually 102 to 108
 A positive correlation exists between the number
of bacteria in an infected root canal and the size
of periradicular radiolucencies

19.  Strict anaerobes: function at low oxidation-reduction
potential and grow only in the absence of oxygen, but
they vary in their sensitivity to oxygen.
 Obligate anaerobic bacteria lack the enzymes superoxide
dismutase and catalase. Some species of bacteria are
microaerophilic; they can grow in the presence of oxygen,
but they derive most of their energy from anaerobic
energy pathways.
 Facultative anaerobic bacteria :can grow in the presence
or absence of oxygen.
 Obligate aerobic bacteria : have both superoxide
dismutase and catalase and require oxygen for growth

20.  Intact teeth with necrotic pulp
: strict anaerobes more than 90% of the bacteria
( Sundqvist 1989)
 The apical 5 mm of carious exposed teeth
: 67% of the bacteria were strict anaerobes
( Baumgaetner 1999)
 Gram-negative bacteria, especially species of
Porphyromonas and Prevotella that are dark
(black) pigmented, have been associated with
endodontic infections.

22. Type of microorganisms found in
endodontic Infections
ตาราง 2
Pisano and

23. Type of microorganisms found in
endodontic Infections
Pisano and

25. Failed Endodontic Treatment
 complete periapical healing occurred in
: 94% of roots with negative culture
: 68% of cases with positive culture ( Sjogren 1997 )
 Enterococcus faecalis has been the predominant
microbe in canals undergoing retreatment
 E. faecalis was found in 77% of cases, confirming
that this microbe is the most prevalent species in fai
led endodontic treatment

26. Sundqvist 1998

27. Bacterial virulence factors
Newman, M. B., H. H.

28. Bacterial virulence factors
 Bacteroides มี polysaccharide capsule
เป็น virulence factor ที่สำำคัญ
(Samaranayake 2002)
 Porphyromonas endodontalis มี
capsule ที่ทำำให้ต้ำนทำนต่อ
phagocytosis และยังมี proteolytic
capability ที่สำมำรถทำำลำย
immunoglobulin G และ E และ
complement factors C3 และ C5 ได้
นอกจำกนี้ยงมี toxic metabolic
ั
products เช่น butyrate และ
propionate ที่สำมำรถพบได้ในบริเวณที่มี

29. Type of microorganisms found in
endodontic Infections
 Aerobes
 Streptococcus sp.: S.mitis, S.salivarius
 Anaerobic streptococci :
Peptostreptococcus
 Enterococci: E. faecalis
 Bacteroides sp.: Porphyromonas, Prevotella
 Provotella nigrescens
 Actinomyces
 Fungi: Candida albicans
 Fusobacteria
 Spirochetes
 Biofilm

30. Aerobes
 Beta hemolytic และ non-
hemolytic streptococci ก่อโรค
ได้น้อย
 peptidoglycans กระตุ้น
lymphokine เช่น osteoclast-
activating factor, prostaglandin
 lipoteichoic acid  กระตุ้น
complementbone resorption
 S.mitis from root canal

31. Anaerobes: Enterococci
 เป็น facultative bacteria, frequency
in Endodontic failure (Ingle and
K.Bakland 2002)
 Common in GI tract
 streptococcus faecalis กำำจัดออกจำก
คลองรำกฟันได้ยำก(resistant to
antibiotic)
 ต้ำนทำนต่อ benzylpenicillin,
ampicillin, clindamycin,

32. Anaerobes: Enterococci
 sensitive ต่อ erythromycin และ
vancomycin (Dahlen,
Samuelsson et al. 2000)
 Molander and Dahlen 2003 :Tx
with calcium hydroxide+
erythromycin / tetracycline

33. Anaerobes: Bacteroides
 เป็น strictly anaerobes, short chain,
gram negative rods and
coccobacilli, common in dental
plaque, non motile, no spore
 มี polysaccharide capsule เป็น
virulence factor
 serious anaerobic infection เช่น
sepsis, abscess

34. Anaerobes: Bacteroides
 Polymicrobial infection
(facultative anaerobes  reduced
oxygen Bacteriodes growth)
 Sensitive to metronidazole และ
clindamycin
 Resistance to penicillin (beta-
lactamase)

35. Anaerobes: Bacteroides
 2 genera คือ Porphyromonas และ
Prevotella
 Porphyromonas เป็น asacharolytic
bacteria, short chain, gram negative
rods, non motile, no spore
 P.gingivalis common in subgingival
sulcus  periodontal infection
 P.endodontalis (first from root canal
infection) common in dental root canal,
periodontal pocket, dental plaque
(Samaranayake

36. Anaerobes: Bacteroides
 Prevotella เป็น saccharolytic
bacteria, gram negative rods, non
motile
 Prevotella melaninogenica
(Bacteroides melaninogenica)=
black pigment (melanin) พบได้ใน
ช่องปาก
 P.intermedia พบได้ใน periodontal
disease (Samaranayake

37. Anaerobes: Prevotella nigrescens
 ปัจจุบันแยกออกจาก Prevotella
intermedius, importance in
endodontic infection (Shah (1992), Bae
(1997), Dougherty (1998), Baumgartner
(1999))
 Endotoxin: lipopolysaccharide
(LPS) periapical lesion, pulp
necrosis, inflammation, bone
resorption, pain, edema (Schein and

38. Anaerobes: Prevotella nigrescens
 LPS+Peptidoglycans กระตุ้น hormone-
like cytokines tisssue destruction
(Henderson and Wilson (1998), Matsushita et.al.
(1998))
 กระตุ้น B lymphocytes และระบบ
complement cascade
 collagenase and interleukin
(macrophage cells)
 ผลิต pain mediators เช่น histamine,
bradykinin และ prostaglandin

39. Anaerobes: Actinomyces
 เป็น anerobic =microaerophilic gram-
positive filamentous bacteria, ไม่ก่อโรค
ในช่องปาก
 non motile, no spore
 Common in nasophalynx และ gingival
crevice
  70-80% chronic infection,
granulomatous และ endogenous
infection of oral cavity (Samaranayake
2002)

40. Anaerobes: Actinomyces
 found in endodoctic failure case
(resistant to routine antibiotic)
(Baumgartner 1991, Gohean 1990, Barnard
1996, Siqueira et.al.2002)
 sulfer granules exudates, in biopsy
branching filamentous form (acid-fast
staining)
 Tx: surgical curettage or resurgical with
long term antibiotic (Gohean, Pantera et al.
1990; Baumgartner and Falkler 1991; Barnard,

41. Fungi
 Candida albicans
 Debelian, Olsen et al. 1997 พบว่า
candida สามารถพบได้ในผนังคลอง
รากฟันในรูป blastospores และ
hyphal structures ซึงสามารถแพร่
่
ผ่าน dentinal tubule ได้

42. Fusobacteria: Fusobacterium
nucleatum
 เป็น a Gram-negative, non-
spore-forming, non motile,
obligatory anaerobic rod,
primary root canal infections.
(Moraes, Siqueira et al. 2002)
 All of F nucleatum associated
with severe pain, swelling and
flare-ups case (Chavez de Paz
Villanueva 2002)

43. Spirochetes
 Oral spirochaete (ซึ่งมีได้ทง เชื้อตัว
ั้
เล็ก กลาง และใหญ่) non culturable
 Common in root canal infections,
pericoronitis, gingivitis และ
periodontitis (10% in endodontic
abscesses). (Dahle, Tronstad et al.
1993)

44. Primary endodontic
infection
Primary infection is caused by microorganisms that initially
invade and colonize the necrotic tissue.
Primary infections are characterized by a mixed clostridium
composed of 10-30species per canal.
The number of bacterial cells in an infected canals varies from
103-108 cells.
Siqueira et al 2005
Sakamoto et al 2007
Vianna et al 2006

45. Primary endodontic
infection
Anaerobic bacteria
Prevotella,Porphyromonas, Actinomyces,Peptostrept
Tanerella, Fusobacterium, ococcus, Eubacterium,
Dialister, Camphylobactor, Filifactor,
Treponema Psudoramibactor
Sterptococci

46. Black pigmented bacteria
Saccharolytic;
Prevotella
Bacteroides
Asaccharolytic;
Porphyromonas

47. Black pigmented bacteria
In 1980, Griffee et al reported that B.melaninogenicus was
found to be significantly related to pain, sinus tract formation,
and foul odor.
Griffee et al 1980

48. Black pigmented bacteria
 Prevotella  Porphyromonas
P.intermedia P.endodontalis
P.nigrescense P.gingivalis
P.tannerae
P.multisaccharivorax
They seem to play important role in
etiology of both acute and chronic
apical periodontitis.
Siqueira et al 2001
Sundqvist et al 1989
Dougherty et al 1998

49. Fusobacterium nucleatum
Gram negative obligate nonmotile anaerobe bacteria.
Most common in symptomatic infection and abscess or
excarcerbation lesion than asymptomatic infection.
5 subspecies of F.nucleatum have been found and different
in genetic data but no study found that which subspecies
have more virulence factor.
Different type of subspecies are found in the same root
canals.
Moraes et al 2002
Siqueira et al 2005

50. Spirochete bacteria
Although spirochetes have been frequently observed in samples
by microscopy, they had never been identified to the species
level.
The application of molecular diagnosis to identification of spiral
bacteria has been overlooked the culture techniques.
All oral spirochetes are genus Treponema .
They can be classified in 2 group; saccharolytic and saccharolytic.
T.denticola, T.sokranskii have been detected in both symptomatic
and asymptomatic lesion.
Dewhirst et al 2000
Baumgartner et al 2007
Rocas et al 2003

51. Secondary endodontic infection
If microorganisms are allowed to remain at the time
of filling , there is increased risk of adverse outcome
of the endodontic treatment
Sjogren et al 1997
Waltimo et al 2005

52. Secondary endodontic infection
Create both excellent apical and
coronal seal.
Prevent bacterial nutrient
regain in to the canal.

53. Microbial in root filled teeth
Unlike primary infection, a more restricted group of
microbial species has been found in
persistent/secondary infection .
The prevalence of enterococci has been finding
in all studies that investigated flora in root-filled
teeth.
“ Enterococcus feacalis “

54. Microbial in root filled teeth
Recently findings from molecular studies have also
suggested that some anaerobic species commonly
found in primary infection.
T.forsythia, P.alactolyticus, F.acolis, D.pnuemosintes
Siqueira et al 2004,2005

55. Microbial in root filled teeth
Bacteria are secondary invaders that can gain entry in
to due to a breach in the aseptic chain during
intracanal intervention.
P.aeruginosa, Staphylococcus
Ranta et al 1988
Siqueira et all 2002

56. E.feacalis in secondary
infection
Facultative anaerobie, Gram
positive coccus.
This species has been found in low prevalence value in case
primary infection and more relate in asymptomatic cases than
symptomatic cases.
Rocas et al 2005

57. E.feacalis in secondary
infection
E.feacalis has been found in root filled teeth evincing
persistent apical periodontitis in prevalence values
ranging from 30-90% of the cases.
This species can be inhibited by other members of
mixed bacterial consortium commonly present in
primary infection.
Sedgley et al 2006

58. E.feacalis in secondary
infection
Virulence factor
Lytic enzyme, cytolysin, gelatinase,
hyaluronidase, pheromone, lipotheichoic
acid, adhesion molecules.
But cannot clarified
what factor play role in
pathogenesis. Kayaoglu et al 2004

59. E.feacalis in secondary
infection
Why E.feacalis can
survive in root filled teeth?

60. E.feacalis in secondary
infection
Studies have revealed that E.feacalis has ability to
penetrate far into dentinal tubules that can escape
from intracanal instrumentation and irrigants.
Haapasalo et al 1989
Siqueira et al 1996

61. E.feacalis in secondary
infection
E.feacalis has been shown to be able to form biofilms in
root canals and this ability can be important for
bacterial resistance and persistence in the
instrumented canals.
Distel et al 2002
The ability to resist high pH value seems to be related to
functional proton pump to acidfy. Which E.feacalis is
resistant to calcium hydroxide.
Evans et al 2002

62. E.feacalis in secondary
infection
E.feacalis can enter a VBCN state which can survive in
adverse environmental condition, including starvation.
They has the ability to survive in environments in scarcity
of nutrients and to flourish when the nutrient source is
reestablished and has a capacity to recover in root canal
treated teeth for 12 months without nutrients.
Figdor et al 2003
Sedgley et al 2005

63. Fungi in secondary
infection
The occurrence of yeasts were taken from samples that
not responding in favorably to conventional treatment
(72% of cases ).
Waltimo et al 1997
Candida albicans was the most common species.

64. Fungi in secondary
infection
By the new detection technology, C.albicans was
detected in primary infection in 21% too.
However the finding indicates that yeasts may be
present in low number at the start of treatment, and
they may reach higher proportion during root canal
procedures .
Buamgartner et al 2000

65. Fungi in secondary
infection
It is also possible that yeasts from oral cavity gain
access to root canal as contaminant during
endodontic procedures.
Siren et al 1997
Or they can overgrow after inefficient intracanal
antimicrobial procedures, which can cause imbalance
in microbiota.
Siqueira et al 2004

66. Fungi in secondary
infection
Virulence factor
Dentino phillic microorganism due to its ability
to colonize and invade to dentine by their
hyphae.
Resist to some intracanal medication such as
calcium hydroxide.
Release hydrolytic enzyme and can form
monoinfection biofilm in root canals.
Sen et al 1997
Waltimo et al 1999,1997
Matusow et al 1981

67. Biofilms
Definition
The colonization and proliferation of
microorganisms at surface and solution
interface; especially problematic in the
small-bore water lines of dental unit.
AAE,2004

68. SEM of bacterial cells
arranged in a biofilm
Siquera 2001

69. 1. Surface conditioning
2. Adhesion of ‘ pioneer ’ bacteria
3. Secondary colonizers
4. Fully functioning biofilm
1. A cooperative “consortia” of species
2. Biofilms grow and deattachment

70. A cooperative “consortia” of
species

71. Biofilms grow and spread

72. Cell-cell communication
 Communicate with one another in biofilm
communities via small diffusible molecules.
 Adapt & survive various environment stresses.
 Regulate expression of gene
 Ability to cause disease

73. Benefits
 A broader habitat range for growth
 A more efficient metabolism
 Increased resistance to stress and
antimicrobial agent
 Enhanced virulence

74. Microbial control in
biofilm
Bacteria in biofilm form is more resistance to anti-
microbial agent than planktonic form.
P.Gingivalis in biofilm is resit to amoxycillin,
metronidazole and doxycyclin when compare
to planktonic form. increased MIC & MBC
Larsen et al 2002

75. Irrigants for microbial
control
•Sodium hypochlorite
•Combination of sodium hypochlorite and
chlorhexidine
•Chlorhexidine
Concentration
•Povidone iodine
E.feacalis, P.micros,
F.nucleatum, S.intermedius
Spratt et al 2001

76. Er:YAG LASER
Er:YAG lasers had an anti-
biofilm effect at a low energy
and could reduce numbers of
the 6 species of biofilm-
forming cells examined.
Noiri et al 2008

77. Photodynamic with methylene blue
Photodynamic therapy (PDT) was developed as a
therapy for cancer.
Activated by light of the appropriate wavelength to
generate singlet oxygen and free radicals that are
cytotoxic to cells of the target tissue.

78. Photodynamic with methylene blue
Fimple et al 2008

79. Treatment of endodontics
infection
 Debridement of the root canal system
 Incision and drainage
 Intracanal medication
 Analgesics and antibiotics
 Follow up