presentation includes various endodontic microrganisms

1. ENDODONTIC
MICROFLORA
Presented By:
Dr MEENAL ATHARKAR
MDS
DEPT OF ENDODONTICS AND
CONSERVATIVE DENTISTRY

2. Flow of Presentation
 Introduction
 Did You Know?
 Classification Of Microorganisms
 Morphology
 Theory of Focal Infection
 Association of Microbes with Endodontic Infections Association of Microbes with Endodontic Infections
 Microbiological Diagnostic Techniques
 Common microbes in Endodontic infection
 Conclusion
 Biofilm
 References

3.  The crown of this tooth was nearly all decayed, while the
roots consisted of two branches, so that the very roots were
uncommonly hollow and the holes in them stuffed with a soft
matter. I took this stuff out of the hollows in the roots and
mixed it with clean rain water, and set it before the
magnifying glass so as to see if there were as many living
creatures in it as I had a foretime discovered; and I must
confess that the whole stuff seemed to me to be alive. . .
Introduction
confess that the whole stuff seemed to me to be alive. . .
F Antonin Van Leeuwenhoek

4.  Microbiology, so called because it primarily deals with
organisms too small for the naked eye to see, encompasses the
study of organisms that cause disease, the host response to
infection and ways in which such infection may be prevented.

5.  Over 700 bacterial species found in oral cavity (Paster BJ et
al, Perio 2000, 2006;42(1):80-7)
Did You Kow?

6. Cellular Organisms
Prokaryotes Eukaryotes
Eubacteria Archaebacteria
Eukaryotes & Prokaryotes

7.  Based On:
 Shape and size: (0.2 – 5 µm)
Cocci (spherical)
Bacilli (rod-shaped)
Spirochaetes (helical)
 Arrangement:
Pairs (Diplococci)
Classification of Micro-organisms
Chains (Streptococci)
Grape like clusters (Staphylococci)
Angled pairs or palisades (Corynebacteria)

8.  Gram-staining characteristics
Gram Positive
Gram Negative
 First developed by Danish Physician, Christian Gram

10. A simple classification of Gram Positive bacteria
Clusters Staphylococcus S. aureus
Aerobes S. epidermidis
Chains/pairs Streptococcus S. pyogenes
Cocci Viridans group
Anaerobes Chains/clusters Peptostrptococcus P. anaerobius
Sporing Bacillus B. anthrax
Aerobes Non-sporing Corynebacterium C. diptheriaeAerobes Non-sporing Corynebacterium C. diptheriae
Lactobacillus L. acidosis
Nocardia N.
asteroides
Bacilli Sporing Clostridium C. tetani
Anaerobes C. perfringes
Non-sporing Propionibacterium P. acnes
Actinomyces A. israelli

11. A simple classification of Gram-negative bacteria
Anaerobes Neisseria N. meningitidis
Cocci N. gonorrhoeae
Aerobes Veilonella V.parvula
Aerobes Pseudomonas P. aeurginosa
a.Parvobacteria Haemophilus, Brucella, Bordetella,
Pasturella, Yersinia
b.Enterobacteria Escheria, Klebsiella, Proteus,
Aerobes/ Serratia, Salmonella, ShigellaAerobes/ Serratia, Salmonella, Shigella
Bacilli Facultative c.Vibrios Vibrio, Campylobacter
Anaerobes d.Legionella Legionella
Anaerobes Bacteroides, Porphyromonas, Prevotella
Aerobes Leptospira
Spirochaetes
Anaerobes Treponema

12.  Focus of infection contains pathogenic organisms & can occur
anywhere in the body.
 First reported claim of cure associated with focus of infection was
by Hippocrates
 1904, F Billings series of cures of afflictions by tonsillectomies &
dental extractions
Theory of Focal Infection
 E.C. Rosenow (1909) described “Theory of Focal Infection” as a
localized or generalized infection caused by bacteria traveling
through the blood from a distant source of infection

13.  1910, British Physician, William Hunter in McGill University
condemned practice of dentistry in US stating “a veritable
mausoleum of gold over a mass of sepsis”
 He believed it to be cause of American illness.
 However, in 1930 & 1940s editorial & research refuted theory
of focal infection & called for constructive rather thanof focal infection & called for constructive rather than
destructive dental treatment

14.  1940, a critical publication by Reimann raised several issues
regarding theory of focal infection, includes:
 Theory not proved
 Infectious agent unknown
 Large group of people with tonsils were no worse than those
with tonsils removed
 Having teeth extracted or tonsils removed were no better offHaving teeth extracted or tonsils removed were no better off
after surgery
 Surgery seldom beneficial
 Adverse effect of surgeries often outweighed
 Foci of infection often heal after recovery from systemic
disease or improved hygiene & dietary measure

15.  1939, Fish recognized four zones of reaction to bacteria
implanted in jaws of guinea pigs.
 Includes:
 Bacteria confined by PMN leukocytes: Zone of Infection
 Inflammatory cells with no bacteria: Zone of Contamination
 Histocytes & osteoclasts: Zone of Irritation
Fibroblasts: Zone of Stimulation Fibroblasts: Zone of Stimulation
 This theory became the basis for successful endodontic
treatment

16.  Endodontics has survived theory of focal infection because of
recognition by the scientific community that successful root
canal treatment is possible without endangering systemic
health.

17.  Bacteria are ubiquitous, but the environment selects
 In the oral cavity, there are an estimated 1010 bacteria
consisting of more than 700 different kinds of
microorganisms, all seek a niche and nutrition.
 One of the primary functions of tooth enamel is to exclude
these microorganisms from the underlying dentine–pulp
Association of Microbes with Endodontic
Disease
these microorganisms from the underlying dentine–pulp
complex

18.  W. D. Miller 1st person observed microorganisms in plaque.
 1890, He authored “Microorganisms of the Human Mouth”
 True significance of bacteria in endodontic disease was shown
by classic study by Kakehasi et al. in 1965.
They found no pathologic changes in exposed pulp ofThey found no pathologic changes in exposed pulp of
gnotobiotic rats but pulp necrosis & periapical lesion in
conventional animals.

19.  For any pathogen to establish itself it needs to Colonize 1st,
Multiply & then induce pathologic activity.
 An endodontic pathogen is defined as a microorganism
capable of inducing the tissue destruction of apical
periodontitis.
Virulence describes degree of pathogenicity. Virulence describes degree of pathogenicity.

21.  Lipopolysaccharide/endotox
in
 Peptidoglycans
 Lipoteichoic acid
 Fimbriae
 Capsules
 Chondroitin sulfatase
 Hyaluronidase
 Fibrinolysin
 Gelatinase
 Protease
 Hemolysins
Bacteria brings about destruction with virulence factors that it
possess like:
 Capsules
 Extracellular vesicles
 Exotoxins
 Extracellular proteins
 Short chain fatty acids
 Polyamines
 Superoxide anions
 Hemolysins
 Leukotoxin
 Coagulase
 Elastase
 Acids
 Alcohols

22.  Most of endodontic pathogens are strict anaerobes &
polymicrobial
 Prior 1970, very few strains identified as their isolation &
identification was difficult per inadequate culturing
techniques.
 Anachoresis is a process by which microbes may be
transported in the blood or lymph to an area of inflammation
such as tooth with pulpitis, where they may establish an
infection

23.  Anaerobes: Grows in absence of O2 ; Lacks enzymes
superoxide dismutases & catalase
 Microaerophilic Bacteria: Can grow in presence of O2 but
predominantly drive energy from anaerobic pathway
 Facultative Anaerobes: Grow in presence of O2 & usually
possess superoxide dismutase & catalase.possess superoxide dismutase & catalase.
 Obligate Aerobes: Require O2 for growth & possess both
superoxide dismutase & catalase

24.  Life is not easy for an endodontic pathogen.
 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.
Life as an endodontic pathogen
 During that time they have to compete in a limited space with
other microbes for the available nutrition.

25.  It is no accident that microbes berth in a particular
environment – there are ecological advantages for them to
establish and flourish if conditions are favorable.
 Through genetic exchange and mutation, microbes have
developed specialized systems that facilitate their ability to
find, compete and survive in these very specific environments.

26. Challenges for microbes to establish in the untreated root
canal

27.  Nutrients may be derived from the oral cavity, degenerating
connective tissue, dentinal tubule contents, or a serum-like
fluid from periapical tissue.
 These factors in the root canal environment permit the growth
of anaerobic bacteria capable of fermenting amino acids and
peptides, whereas bacteria that primarily obtain energy by
fermenting carbohydrates may be restricted by lack offermenting carbohydrates may be restricted by lack of
available nutrients.
 This is the likely reason why the flora is dominated by
facultatively anaerobic bacteria, such as streptococci, in the
coronal section of root canals exposed to the oral cavity, and
anaerobic bacteria dominate in the apical section

28.  The succession of strict over facultative anaerobes with time
is most likely due to changes in available nutrition, as well as a
decrease in oxygen availability.
 Facultatively anaerobic bacteria grow well in anaerobiosis;
however, their prime energy source is carbohydrates.
A decrease in availability of carbohydrates in the root canal A decrease in availability of carbohydrates in the root canal
occurs when there is no direct communication with the oral
cavity, which severely limits growth opportunities for
facultative anaerobes.

29.  Whilst the microbial flora in an untreated infected root canal
may experience feast, in the well-filled root canal there is
predominantly famine.
 Most or all of the original necrotic pulp will have been
eliminated leaving dry, barren conditions for surviving
microbial cells.
 These microbes would experience a static environment and
starvation, but with some luck may encounter a serum-like
Ecological differences between untreated and root-
filled root canals
starvation, but with some luck may encounter a serum-like
fluid transudate from the periapical tissue.

30.  The species that persist here are those that have either survived
the antimicrobial treatment and are the last ones remaining, or
have entered during treatment and found it possible to
establish where others cannot do so.
 For microbes to maintain apical periodontitis and cause post-
treatment disease, they must do more than just survive in the
root-filled canal; they must also possess the pathogenicroot-filled canal; they must also possess the pathogenic
properties necessary to perpetuate inflammation external to the
root canal system.

31.  In general, microorganisms involved in persistent infections
implement one of three strategies to evade the immune
response – sequestration, cellular or humoral evasion.
 Sequestration involves a physical barrier between the microbe
and the host.
 Cellular evasion means that microorganisms avoid leukocyte- Cellular evasion means that microorganisms avoid leukocyte-
dependent antibacterial mechanisms.
 Humoral evasion means that extracellular bacteria avoid the
host’s antibodies and complement.

32. Challenges for microbes involved in persistent infection.

33. Persistent infection requires not one, but a series of coupled characteristics.
Bacteria must possess an ability to survive the stages of treatment, ‘persistence’
characteristics and an ultimate ability to inflame host tissue. The location of
bacteria is critical for them to source nutrients and inflame tissue. The
concomitant interaction of these characteristics with the host defense results in
failure to heal.

34.  Interest in endodontic microbiology boomed after recognition
that apical periodontitis lesions are inflamatory diseases
caused by microorganisms.
 However, interest in this field has become more pronounced
with recent fast advances in methods based on detection &
analysis of microbial nucleic acids
Microbiological Diagnostic Techniques

35. Microbiological Diagnostic Technique
Cultural Immunological methods Non-cultural
Solid/liquid Media 1. Microscopy
2. Molecular Biology
. PCR & Its Derivatives. PCR & Its Derivatives
.DNA-DNA Hybridization
. FISH

36. The cycle of important events in diagnostic microbiology, depicting the
interaction between the clinician and the microbiology laboratory

37.  Bacteria grow well on artificial media unlike viruses which
require live cells
 Blood agar most commonly used
 It is non selective media but when chemicals are incorporated
into media to prevent growth of certain bacterial species &
promoter growth of others, know as selective media
Cultural Methods
promoter growth of others, know as selective media

38.  Main constituents are:
 Water
 Agar: a carbohydrate obtained from seaweed (as agar melts at
900 and solidifies at 400 , heat sensitive nutrients can be added
before the medium solidifies)
Growth-enriching constituents: e.g. yeast extract, meat extract
Bacteriological media
 Growth-enriching constituents: e.g. yeast extract, meat extract
(these obtain carboydrates, proteins, inorganic salts,vitamins,
and growth factors for bacterial growth)
 Blood: defibrinated horse blood or sheep blood

39. Methods of inoculating an agar plate to obtain discrete colonies of
bacteria (numbers indicate inoculation step)

40. Preparation of solid media & inoculation procedure
 When all necessary ingredients added to molten agar,
dispensed still warm, into plastic/glass Petri dishes.
 The agar gradually cools & sets at room temperature, yielding
a plate for inoculation
 The objective of inoculating on to a solid medium is to obtain
discrete colonies of organisms. Hence, standard technique
must be adhered

41.  Solid media are more useful than liquid media as they
facilitate:
 Discrete colony formation allowing subculture on secondary
plate. Pure growth can be used for identification of organism
using biochemical test,etc.
 Observation of colonial characteristics helpful in identification Observation of colonial characteristics helpful in identification
of organisms
 Quantification of organisms as colony-forming units (CFU) .

42. Liquid Media
 Are used to:
 Promote growth of small numbers of bacteria contaminated
with antibiotics. The antibiotic is diluted in fluid medium,
thereby promoting growth of the organism
 Promote preferentially growth of specific bacteria. These are Promote preferentially growth of specific bacteria. These are
called Enrichment media. (e.g. selenite F broth used for stool
cultures)
 test biochemical properties of bacteria for identification
purposes

43. Blood culture bottles; the bottle on the left contains the uninoculated
medium

44. Transport Media
Specimens transported in a transport medium to maintain its
viability in transit.
 Bacteriological transport media:
 Semisolid, non-nutrient agar such as Stuart transport medium
is widely used. Contains thioglycolic acid as reducing agent, &is widely used. Contains thioglycolic acid as reducing agent, &
electrolytes.
 Viral transport medium:
 A solution containing proteins & balanced salts
 Antimicrobials added to kill any bacteria present

45. Some selective media used in routine microbiology
Medium Selective Agents Differential
Substrate
(indicator)
Colonial Selected
Types organisms
I II
Major Organisms
Inhibited
MacConkey Bile salts Lactose
(neutral red)
Fermenter/Red
E. Coli
Klebsiella
Non-fermenters
Salmonella
Pseudomonas
Most cocci
Mitis salivarus Tellurite, crystal
violet
Sucrose (tryptan
blue)
Big>2mm Step
salivarus
Small<1mm
Step mitis Other
strep
Staph
enteric bacilli
Mannitol salt 7.5% NaCl Mannitol
(phenol red)
Big/Yellow
Staph aureus
Small/Pink
Staph epidermidis
Sterp
Enteric bacilli
Lowenstein-Jensen Malachite green - Rough
Mycobacterium
tuberculosis
Smooth/Pigmented
Atypical
mycobacteria
Cocci
TCBS Thiosulphate, citrate,
bile salts, high pH
(8.4)
Sucrose
(bromothymol blue)
Fermenter (yellow)
Vibrio cholerse
Aeromonas
Non-fermenter
Vibrio para-
hemolyticus
Cocci
Enteric bacilli
Thayer-Martin Antibiotics - Grey Colonies Gram Positive
Neisseria gonorrhoeae cocci
Neisseria meningitdis
Charcol yeast extract Cysteine, ferric
sulphate
- Cutglass Colonies Gram Positive
Legionella app cocci
Sabouraud Low pH (5.6) ±
antibiotics
Cream colonies Most Bacteria
Fungi

46. Atmospheric requirements and incubation
 Once inoculated the agar plates may be incubated:
 Aerobically: but addition of 10% CO2 enhances the growth of
most human pathogens
 Anaerobically: most bacteria, especially the oral pathogens are
strict anaerobes & only grow in the absence of O2 .
Anaerobic conditions can be produced in a sealed jar or inAnaerobic conditions can be produced in a sealed jar or in
large anaerobic incubators.
In either case, the environmental O2 is replaced by N2, H2 and
CO2
 At Body Temperature: 370 C (a few bacteria grow well at
higher or lower temperature; fungi usually grow at ambient
temperature)

47. Advantages and limitations of culture technique
 Advantages
• Broad range nature, identification
of unexpected species
• Allow quantification of all major
viable microorganisms in the
sample
• Allow determination of
antimicrobial susceptibilities of
 Limitation
• Impossibility of culturing a large
number of extant bacterial species
• Not all visible bacteria can be
recovered
• Once isolated, bacteria require
identification using a no. of
techniquesantimicrobial susceptibilities of
the isolates
• Physiologic studies are possible
• Pathogenicity studies are possible
• Widely available
techniques
• Misidentification of strains with
ambiguous phenotypic behaviour
• Low sensitivity
• Strict dependence on the mode of
sample transport
• Sample require immediate
processing

48. Advantages and limitations of culture technique contnd…..
 Limitation contd………..
• Specificity is dependent
on the experience of the
microbiologist
• Extensive expertise & specialized
equipment needed to isolate strict
anaerobes
• Take several days to weeks to
identify most anaerobic bacteria

49. Microscopic Methods
 Light Microcopy: To visualise bacteria clearly;
To categorize them according to staining
properties
 Bright-field or standard microscopy:
Stained smears from lesions examined with oilStained smears from lesions examined with oil
immersion(*100) using *10 eyepiece, yielding magnification
1000 times.
Wet films examined with dry objective(*40) to demonstrate
bacterial motility

50.  Dark-ground microscopy:
Specimen illuminated obliquely by a special condenser so
that light does not enter directly. Instead organisms appear
bright, as light rays hit them, against dark background
 Phase-contrast microscopy:
Although rarely employed, may be used to define detailed
structure of unstained microbesstructure of unstained microbes
 Fluorescence microscopy:
Fluorescence techniques widely used, especially in
immunology. It employs principle of emission of different
wavelength of light with when light strikes a fluorescent
object. UV light commonly used stained specimen with
fluorescent dye auramine

51.  Electron Microscopy:
Here, light waves are replaced by beam of electrons, allowing
resolution up to 0.001µm.
Can be used in diagnostic virology. Needs around 1 million
virus particles for such visualization. Clumps can be obtained
by reacting the sample with antiviral antibody- immuno-
electron microscopy

52. Immunological Methods
 Particularly useful to identify organisms and detect antibodies
in a patient’s body fluids, when the organisms cannot be
cultivated.
 Techniques
 Agglutination: 1. Slide Agglutination Agglutination: 1. Slide Agglutination
2. Latex Agglutination
 Immunofluorescence
 Enzyme-linked immunosorbent assay(ELISA)

53.  Agglutination
 Slide agglutination:
Antibodies against specific serotypes of organism (e.g.
Salmonella & Shigella species) can be used in identification
Suspension of organism when mixed with specific AB on a
glass slab, visible clumping occurs indicating +ve reaction
 Latex agglutination:
Agglutination of latex bead coated with specific antibody is
used. (e.g. Neisseria meningitidis, Haemophilus influenzae,
the yeast Crytococcus neoformans)

54. Latex agglutination test: latex beads coated with a known, specific
antibody is mixed with a suspension of unknown organism; visible
agglutination of the beads occurs instanteously if the identity is positive

55.  Immunofluorescence:
If Organism exposed to specific Ab tagged with fluorescent
dye , it binds to Ab & can be visualised in UV microscope.
Can be one step or two step
 ELISA:
In ELISA fluorescent dye tagged to Ab is replaced by enzyme,
& amount of bound enzyme can be demonstrated with the& amount of bound enzyme can be demonstrated with the
enzyme substrate

56. Principles of direct(1 step) & indirect (2 step) immunofluorescence
techniques. This eg illustrates detection of viral antigen; *
immunofluorescence label; V, viral antigen; Ag antigen; Ab antibody

57. Advantages & Limitations of Immunological methods
 Advantages
• Rapid- no more than few hours to
identify a microbial species
• Easily identified
• Low cost
• Detect dead organism
 Limitation
• Detect only the target specimen
• Low sensitivity
• Specificity is variable & depends
on the type of antibodies used

58. Molecular biology technique
 Rapid advances in molecular biology over the last 20 years
have provided a bewildering array of techniques aimed at
helping us to tease apart all aspects of biology.
 The discipline of microbiology has gained greatly from these
advances especially with respect to detection and identification
of micro-organisms.of micro-organisms.
 Indeed these molecular biology techniques have changed the
way we classy all life on Earth.

59.  All available surfaces in the oral cavity are colonized by
different and diverse microbial biofilms, but structures not
exposed to the microflora are usually sterile e.g. the
endodontium – the pulp and root canal system within teeth.

60.  From early microscopy studies it was shown 50% of the oral
microbiota was unculturable.
 It was very possible that unculturable taxa were present in root
canal infections and were potentially playing a role in the
disease initiation or progression or both.
 These unculturable taxa fall into two broad catagories. These unculturable taxa fall into two broad catagories.

61.  The first are taxa that need nutrients or other essential
components that conventional sampling techniques, transport
conditions or laboratory media do not provide.
 This could be sensitivity to oxygen (i.e. very strict anaerobes)
or the absolute requirement for products provided by other
taxa within the root canal.
 These taxa are therefore broadly unknown apart from
microscopy studies

62.  The second category contains those taxa that are known, and
very often common, but for some reason cannot be cultured,
i.e. they are in a dormant state and ‘non-culturable’. The term
‘viable but not culturable’(VBNC) was coined to describe this
state.
 It is thought that cells will go into this state as a protection
strategy in response to adverse environmental conditions.strategy in response to adverse environmental conditions.

63. PCR and its Derivatives
 Shortly after Kary Mullis described a polymerase chain
reaction (PCR) technique, for which he received the Nobel
Prize in 1993, the flood gates opened with respect to what was
possible in the world of microbial detection and identification.
 The application of PCR and sequencing (and associatedThe application of PCR and sequencing (and associated
database construction and searching software) revolutionized
the detection and identification of bacteria.

64.  The use of nucleotide sequence data from 16S ribosomal RNA
genes (among others), now makes it possible not only to
identify but to infer phylogeny for all organisms on Earth.
 Phylogeny is defined as the evolutionary relationships within
and between taxonomic levels, particularly the patterns of
lines of descent, in a sense a family tree spanning 3.5 billion
years.years.
 Therefore within reason a single methodology can be used to
identify any bacterial isolate from any environment.

65.  The 16S (small subunit) rRNA gene was selected as a
candidate molecule for a number of reasons:
• it is present in all organisms and performs the same function,
• its sequence is sufficiently conserved and contains regions of
conserved, variable and hypervariable sequence,
• it is of sufficient size (ca. 1500 bases) to be relatively easily• it is of sufficient size (ca. 1500 bases) to be relatively easily
sequenced but large enough to contain sufficient information
for the most commonly occurring species identification and
phylogenetic analysis.

66. Flow diagram showing the steps involved in bacterial
identification using a 16S rRNA sequencing approach.

67.  Touchdown PCR:
Touchown PCR is a strategy to increase the specificity of the
assay.
It have been considered useful to avoid the amplification of
spurious DNA fragments.
 Nested PCR:
Here, 2 sets of primers are used, 1st used for primaryHere, 2 sets of primers are used, 1st used for primary
amplification round; 2nd specifically chosen to anneal with an
internal sequence of the amplicon, re-amplifies the latter
‘specific’ sequence.
Has increased sensitivity

68.  Multiplex PCR:
This method simultaneously amplifies more than one locus of
nucleotide using multiple sets of primers, saving resources &
time.
 Real-time PCR:
Conventional PCR requires gel electrophoresis for analysis of
the amplicon.the amplicon.
This step is automatically performed in Real-time PCR within
closed system & identifies target sequence using either
labelled fluorophores or other similar labelled probes.

69.  Reverse Transcriptase PCR:
Developed to amplify RNA targets & to exploit the use of the
enzyme reverse transcriptase, which can synthesize
complementary strand of DNA.
Conventional PCR uses 2 steps.
RT-PCR may be modified into 1 step using it directly with
RNA as the template; uses both enzymes Reverse transcriptase
& DNA polymerase in this approach& DNA polymerase in this approach

70. Diagrammatic representation of the visualisation of polymerase chainDiagrammatic representation of the visualisation of polymerase chain
reaction products from 10 subjects where taxon ‘A’ was targeted with a
specific primer set. It shows a band present in subject samples 1, 2, 4, 5,
and 10. This indicates that taxon ‘A’ was detected in these five subjects
and not in subjects 3, 6, 7, 8, or 9.Cdenotes a control sample of target
taxon alone.

71. Diagramatic representation of the polymerase chain reaction (PCR)
cloning process used to singularise mixed PCR products. Broad Range
PCR

72. Strategy defining the unculturable microbiota in a root canal
sample.

73. DNA-DNA Hybridization
 Molecular techniques for bacterial detection and identification
are not restricted to PCR alone and a notable alternative
technique is checkerboard DNA–DNA hybridization &
microarray DNA-DNA hybridization
 This technique involves deposition of bacterial DNA from
clinical samples (root canal, plaque etc) in parallel (vertical)clinical samples (root canal, plaque etc) in parallel (vertical)
lines on a nylon membrane.
 Digoxigenin-labelled whole genomic DNA probes are run at
right angles to the samples (horizontal).
 Following washing the bound probe is detected and quantified.

74.  This method was pioneered and extensively used by Sigmund
Socransky in Boston, MA, USA.
 The technique utilizes whole genomic DNA for 40 bacterial
taxa and 28 patient samples per membrane this makes it a very
high throughput technique and thousands of samples can be
analyzed very quickly generating huge amounts of data
regarding the detection rates of the forty taxa in each sample.regarding the detection rates of the forty taxa in each sample.
 The technique is semi-quantitative and standards containing
known numbers of cells are used.

75.  A potential drawback is however the unknown cross reactivity
with unknown taxa present in the sample.
 Additionally the technique can only provide information on
known culturable taxa and while very valuable does not
address the unculturable proportion of any sample.
 The technique has been used to a limited extent in endodontic The technique has been used to a limited extent in endodontic
microbiology studies

76. Diagram of Checkerboard DND–DNA hybridization showing; A – vertical
lanes containing sample DNA and horizontal lanes with DNA from five
known taxa. B – following hybridization, washing and detection the
presence or absence of the various taxa in the patient samples can be
ascertained. Taxon b is present in all samples, taxon c is not detected in
any sample, taxon a is detection in samples 2 and 5, etc.

77. Diagrammatic representation of the visualisation of multiplex polymerase
chain reaction (PCR). Amplification products from 10 subjects where
three specific taxa ‘D’, ‘E’, and ‘F’ were targeted in one PCR reaction. All
three taxa are only detected in subject 2. None of the target taxa were
detected in subjects 6, 7, and 9.

78.  Fluorescence In-Situ Hybridization (FISH):
• It uses fluorescently labelled rRNA probes & fluorescence
microscopy to detect intact microbial cells.
• It gives additional information regarding presence,
morphology, number, organization, spatial distribution of
microorganisms.
• It includes 4 steps: 1)Fixation & permeabilization of sample;
2)Hybridization with repective probes for
detecting respective target sequences;
3)Washing steps to remove unbound
probe;
4)Detection of labelled cells by
microscopy or flow cytometry.

79. Fluorescence in situ hybridization using the Bacteria-specific probe EUB
338 on section from periapical granuloma. A large number of bacteria of
different morphotypes is present in limited area of the tissue. Bar, 10 mm.

80. Advantages and limitations of Molecular Biology methods
 Advantages
• Detect both cultivable &
uncultivable species or strains
• High specificity & accurate
identification of strains with
ambiguous phenotypic behaviour
• High sensitivity
• Detect species directly in clinical
 Limitations
• Most assays are qualitative or
semi quantitative (exceptions:
real-time PCR & microarray)
• Most assays only detect one
species or a few different species
at a time (exceptions: broad range
PCR, checkerboard, microarray)• Detect species directly in clinical
samples
• Rapid- most assays take no more
than minutes to few hours to
identify a microbial species
• Do not require carefully
controlled anaerobic conditions
during sampling and
transportation
PCR, checkerboard, microarray)
• Most assays detect only the target
species & fail to detect
unexpected species (exceptions:
broad range PCR)
• Some assays can be laborious and
costly (e.g. broad-range PCR)
•

81. Advantages and limitations of Molecular Biology methods
contnd….
 Advantages
• Can be used during antimicrobial
treatment
• Anaerobic handling & expertise
not required
• Samples can be stored frozen for
later analysis
• DNA can be transported easily
 Limitations contd….
• Biases in broad-range PCR
introduced by homogenization
procedures, preferential DNA
amplification, & differential DNA
extraction
• Hybridization assays using whole
genome probes detect only• DNA can be transported easily
between laboratories
• Detect dead organisms
genome probes detect only
cultivable species
• Can be very expensive
• Detect dead organism

82. Streptococci
 Streptococci comprise a relatively high proportion,
approximately 20%, of the microorganisms recovered from the
canals of teeth with post-treatment disease.
 However, the recovery of streptococci is less remarkable when
it is taken in the context of its high prevalence in untreated
Common Microbes in Endodontic Disease
it is taken in the context of its high prevalence in untreated
infected canals
 Streptococci have in common is a preferential capacity for
invasion of dentinal tubules, which should favor their ability to
enter and establish in the root canal system.
 Streptococcal surface adhesins mediate binding to dentin as
well as facilitating dentin invasion

83. General Characteristics
 Characteristics:
• Catalase negative, Gram +ve spherical/oval cocci in pairs and
chains;
• 0.7-0.9µm in diameter
• Chain formation best seen in liquid culture or pus
 Culture:
• Grow well on blood agar, may need enrichment of media with
glucose & serum

84. • Typical hemolytic reactions are produced on blood agar:
o ɑ-haemolysis: narrow zone of partial hemolysis & green
(viridans) discoloration around the colony. E.g. viridans
streptococci
o β-haemolysis: wide, clear, translucent zone of complete
haemolysis around the colony, e.g. Streptococcus pyogenes
o No haemolysis (γ-haemolysis), e.g. non-haemolytic
streptococci

85. Alpha & beta haemolysis: β-haemolytic colonies(eg. Streptococcus
pyogenes) produce complete translucence of blood agar, whereas ɑ-
haemolytic colonies(S. pneumoniae) do not.

86.  Serology
• Carbohydrate antigens found on the cell walls of the
organisms are related to virulence.
• Serogrouping termed, Lancefield grouping, is useful in the
identification of the more virulent β-haemolytic species.
• Currently 20 Lancefield groups recognized (A-H & K-V).
• Worth noting are:
o Group A: includes important human pathogen Streptococcuso Group A: includes important human pathogen Streptococcus
pyogenes
o Group B: contains one species S. agalactiae
o Group C: mainly causes disease in animals
o Group D: includes enterococci (S. faecalis), ranks next to
group A causing human disease

87. Gelatinous colonies of mutans streptococci mainly comrising extracellular
polysaccharide

88.  Actinomycetes
 The term ‘actinomycosis’ was introduced by Israel, in1878, in
his accurate description of a cervicofacial and thoracic case of
the disease.
 Additional clinical descriptions followed along with the
isolation of Actinomyces israelii by Bujwid in 1889.
 This species was then well described by Wolff and Israel, in
1891.

89.  The causative agents of this slowly progressive infection are
Gram-positive bacteria of the genera Actinomyces and
Propionibacterium, which are normal inhabitants of the oral
cavity, colon and vagina.
 A. israelii is by far the species most commonly involved in
causation of actinomycosis
 Actinomycosis is a chronic, granulomatous infectious disease
characterized by suppuration, abscess formation and draining
sinus tracts, which erupt to the skin or mucosal surfaces and
drain pus containing ‘sulfur granules’ (small colonies of
bacteria)

90. Sulfur granule from periapical lesion of tooth with refractory apical
periodontitis. The granule is soft, yellowish in color and 3–4mm in
diameter.

91.  The genus Actinomyces encompasses a heterogeneous group
of non-acid fast, non-motile, non-spore forming, obligately
anaerobic and facultatively anaerobic, Gram-positive rods.
 Early classification of Actinomyces was complicated by their
histological resemblance to fungi (Actinomyces: ray fungus),
which occurred due to the radial appearance of filaments in the
granules found in actinomycotic lesions.granules found in actinomycotic lesions.

92.  Actinomyces cells are 0.4–1 mm wide, short (1.5–5 mm long)
or longer (5–50 mm long).
 They can be straight, curved, branched or pleomorphic, and
they can occur singly, in pairs, clusters or short chains.
 Most of the species are facultative anaerobes, while some are
obligate anaerobes.obligate anaerobes.

93.  Grows slowly under anaerobic conditions, on blood or serum
glucose agar at 370C
 In about a week it appears as small, creamy-white, adherent
colonies on blood agar
 Actinomyces species are fermentative, generally utilizing
carbohydrates to produce formic, acetic, lactic and succiniccarbohydrates to produce formic, acetic, lactic and succinic
acids.

94. Molar tooth shaped colonies of Actinomyces israelii, on blood agar

95. P. propionicum
 P. propionicum was formerly assigned to the genus
Actinomyces, then transferred to the genus Arachnia and from
there to Propionibacterium on the basis of sequence homology
of ribosomal RNA.
 Further analysis of its fatty acid pattern supported transfer of
this species to the genus Propionibacterium.
 Cells are non-motile and may appear as irregular rods, 0.2–0.3
mm in diameter and 3–5 mm in length, which may or may not
be branched, often with swollen or clubbed ends.

96.  They can also occur as branching filaments, 5–20 mm in
length. Occasionally large round cells may be observed (5 mm
in diameter).
 P. propionicumis facultatively anaerobic, but best growth is
attained under anaerobic conditions.
 Propionic and acetic acids are the major end products of the Propionic and acetic acids are the major end products of the
anaerobic fermentation of glucose. CO2 and lesser amounts of
lactic and succinic acids are also produced.

97.  P. propionicum is a normal inhabitant of the human oral cavity,
and can be involved in several oral diseases.
 In addition, this species has been reported to occur in cases of
tympanomastoiditis, vertebral osteomyelitis, epidural abscess,
lacrimal canaliculitis, brain abscess, pulmonary infection in
patients with hairy cell leukemia, and actinomycosis.
 P. propionicum may produce disease clinically
indistinguishable from that caused by Actinomyces.
 Like Actinomyces species, P. propionicum is known to be
able to flourish in host tissues for long periods of time without
causing symptoms.

98. Enterococcus
 In the past few years, Enterococcus faecalis has been
mentioned with increased frequency with regard to teeth
with post-treatment disease (PTD), where it has also been
detected as monocultures.
 Enterococci are part of the normal flora in the oral cavity
and gastrointestinal tract.and gastrointestinal tract.
 They are recognized as potential human pathogens
causing 12% of the nosocomial infection.

99.  E. faecalis accounts for around 80% of all infections caused by
enterococci.
 Nosocomial and community-acquired infections caused by the
genus Enterococcus include urinary tract infections,
bacteremia, intra-abdominal infections and endocarditis.
 Enterococci are also frequently isolated from mechanically Enterococci are also frequently isolated from mechanically
ventilated (intubated) patients.

100.  Until the mid-1980s, enterococci were not allocated to a
separate genus, even though their unique characteristics were
recognized among streptococci.
 The basic observations on staining, cell shape and arrangement
as well as lack of catalase placed enterococci in the genus
Streptococcus.
 With the serological Lancefield’s classification and the
discovery of the group D antigen, enterococci were classified
as salt-tolerant group D streptococci

101.  However, the group D antigen is a lipoteichoic acid (LTA), one
of the class of compounds that is found in virtually all Gram-
positive bacteria and that is very different from the
carbohydrate group antigen of other streptococci.
 In 1984, enterococci were given a formal genus status after
DNA–DNA and DNA–RNA hybridization studiesDNA–DNA and DNA–RNA hybridization studies
demonstrated a more distant relationship with the streptococci,
and two new genera, Lactococcus and Enterococcus, were
introduced.

102.  Enterococcal cells are spherical or ovoid, occurring in pairs or
short chains in liquid media.
 Endospores are not formed and some species can be motile by
scanty flagella.
 They form creamy whitish colonies, are Gram-positive,They form creamy whitish colonies, are Gram-positive,
catalase-negative and able to grow in 6.5% NaCl, at
temperatures ranging from 100C to 450C, and they can survive
30 min at 600C and a pH over 9.6.
 Most enterococci are facultative anaerobes, but some species
are strict aerobes.

103. A thin sectioned cell of E. faecalis (TEM, 33 000).

104.  A wide range of carbohydrates are fermented in glucose broth
with the production mainly of lactic acid with a final pH of
4.2–4.6, sometimes with lower values.
 Enterococci do not normally reduce nitrate and do not digest
pectin or cellulose.
 They are ubiquitous and potentially pathogenic species that are They are ubiquitous and potentially pathogenic species that are
able to acquire an increased resistance or phenotypic tolerance
to many disinfectants or physical agents.
 Growth on bile–esculin is a useful characteristic to identify
enterococci.

105.  E. faecalis possess a group D carbohydrate cell wall antigen
(Lancefield antigen), which is an intracellular glycerol teichoic
acid associated with the cytoplasmic membrane.
 The cell wall contains a large amount of peptidoglycan and
teichoic acid.
 The peptidoglycan (cross-linked peptide sugar), which is The peptidoglycan (cross-linked peptide sugar), which is
found in most of the bacterial cell walls, helps to maintain the
microbe’s shape and has a polysaccharide backbone of
alternating N-acetylglucosamine (GlcNAc) and N-
acetylmuramic acids (MurNAc).

106. Smooth, shiny colonies of E. faecalis E.E. faecalisfaecalis and otherand other enterococcienterococci growgrow
on a horse blood agar plateon a horse blood agar plate on bileon bile––esculinesculin and stain the agarand stain the agar
characteristically browncharacteristically brown––black.black.

107.  The chemical and structural analyses of the capsular
polysaccharides have shown glycerol teichoic acid-like
molecules with a carbohydrate backbone structure and sialic
acid.
 These polysaccharides are cross-linked with peptide bridges
and contribute to the three-dimensional structure of
peptidoglycan.peptidoglycan.
 Because of the location of the peptidoglycan on the outside of
the cytoplasmic membrane and its specificity, the
transglycosylation step has been indicated as a potential target
for antibacterial medicaments.

108. Carbohydrate fermentation by E. faecalis (upper panel). Mannitol and
sorbitol fermentation (yellow color) are among the characteristics that
help to identify E. faecalis from other bacteria (lower panel).

109. Candida
 Candida albicans has been periodically reported in teeth with
persistent post-treatment apical periodontitis and yeasts have
also been observed by electron microscopy in such teeth.
 Yeasts are seldom seen in untreated root canals, unless canals
have been open to the oral cavity or there has been a history of
protracted treatment.protracted treatment.
 It is indigenous to oral cavity, G.I tract, female genital tract,
skin; hence infection is usually endogenous, although cross
infection can occur.

110.  Grows as spherical to oval budding yeast cells 3-5 * 5-10µm
in size
 Yeast-phase are also called blastopores.
 Pseudohyphae (elongated filamentous cells joined end to end)
are seen, especially at lower incubation temperatures & on
nutritionally poor medianutritionally poor media

111. Growth of different Candida species on Pagano-Levin agar exhibiting
varying colony colours and hues

112.  Grow on Sabouraud medium as creamy-white colonies, flat or
hemispherical in a shape with a beer-like aroma.
 C. albicans & C. dubliniensis can be differentiated from other
species by their ability to produce germ tubes &
chlamydospores
Yeast cells on incubation for 3h at 370C in serum, form germ Yeast cells on incubation for 3h at 370C in serum, form germ
tube
 Also, they form round, thick-walled, resting structures called
chlamydospores on incubation at 22-250C with decreased O2
on a nutritionally poor medium

113.  Biofilm biology has become an expanding field of research in
human, industrial and environmental ecosystems.
 The knowledge accumulated suggests that organisms growing in
biofilms develop properties different to those dwelling in the
planktonic state.
 Biofilm is a mode of microbial growth where dynamic communities
Biofilms
 Biofilm is a mode of microbial growth where dynamic communities
of interacting sessile cells are irreversibly attached to a solid
substratum, as well as each other, & are embedded in a self made
matrix of extracellular polymeric substances

114.  The microorganisms living in community :
• Must possess the abilities to self organize (autopoiesis)
• Resist environmental perturbations (homeostasis)
• Must be more effective in association than in isolation
(synergy)(synergy)
• Respond to environmental changes as a unit rather than single
individuals (communality)

115.  As far as endodontic infections are concerned, the biofilm
concept has thus far gained limited attention.
 Possibly the first identification of biofilm structures in infected
root canals was carried out by Nair
 It has been discussed mainly within the framework of
bacterial appearances on root tips of teeth with nonvital pulps.bacterial appearances on root tips of teeth with nonvital pulps.
 Such bacterial aggregations have been thought to be the cause
of therapy-resistant apical periodontitis

116.  Although the structural organization of biofilms, the
composition and activities of the colonizing microorganisms in
various environments may be different, the establishment of a
micro-community on a surface seems to follow essentially the
same series of developmental stages, :
a) including deposition of a conditioning film,
b) adhesion and colonization of planktonic microorganisms in
a polymeric matrix,
c) co-adhesion of other organisms, and detachment of biofilm
microorganisms into the surroundings.

117. Stages of Biofilm formation

118.  Quorum sensing, a bacterial cell-to-cell communication
mechanism for controlling cellular functions, is of particular
interest because it is known to be involved in the regulation of
several microbial properties, including virulence and the
ability to form biofilms, incorporate extracellular DNA and
cope with environmental stress
 There are reports showing that microorganisms grown in
biofilms could be two- to 1000-fold more resistant than thebiofilms could be two- to 1000-fold more resistant than the
corresponding planktonic form

119. Characteristics Of Biofilm
 Protection of Biofilm Bacteria from environmental threats
 Nutrient trapping & establishment of metabolic cooperativity
in biofilm
 Organized internal compartmentalization in biofilm
 Bacterial cells residing in a biofilm communicate, exchange
genetic materials, & acquire new traits

120.  It is reasonable to assume that the preconditions for biofilm
formation in the root canal vary depending on the cause of the
pulpal breakdown.
 An ischemic injury by trauma, leading to pulpal necrosis, is
likely to provide totally different prerequisites for the
colonization phase than in a caries exposure of the pulp.
 In the latter case, the inflammatory lesion front may recede
successively towards the apex, possibly in bursts, and provide
the fluid vehicle by which invading planktonic organisms can
multiply and start attaching to the root canal walls.

121. Endodontic Biofilms:
 Intracanal microbial biofilms:
Formed on the root canal dentine of endodontically treated
tooth
 Extra radicular biofilms:
Formed on the root (cementum) surface adjacent to the root
apex of endodontically treated tooth
 Periapical biofilms:
Biofilms found in the periapical region of endodontically
treated tooth
 Biomaterial centered infections:
BCI is caused when bacteria adheres to an artificial
biomaterial surface & forms biofilm structures

122. Display ofDisplay of biofilmbiofilm formations on the root canal walls of an extracted tooth withformations on the root canal walls of an extracted tooth with
attachedattached periapicalperiapical tissue lesion. The section has been stained with a Taylortissue lesion. The section has been stained with a Taylor--
modified Brown andmodified Brown and BrennBrenn method for bacterial identification. Bacteria are seenmethod for bacterial identification. Bacteria are seen
lining the walls of the root canal in what seems to be alining the walls of the root canal in what seems to be a biofilmbiofilm (A, B). Note absence(A, B). Note absence
of bacteria in the most apical portion of the root in (B). High magnification in (C)of bacteria in the most apical portion of the root in (B). High magnification in (C)
displays aggregation of numerousdisplays aggregation of numerous coccoidcoccoid and filamentousand filamentous
organismsorganisms

123.  Microorganisms cause virtually all pathoses of the pulp and
periapical tissues. To effectively treat endodontic infections,
clinicians must recognize the cause & effect of microbial
invasion of the dental pulp space & surrounding periapical
tissues.
 Thus, knowledge of the microorganisms associated with
endodontic disease is necessary to develop a basic
understanding of the disease process & a sound rationale for
Conclusion
understanding of the disease process & a sound rationale for
effective management of patients with endodontic infections.

124.  GO¨ RAN SUNDQVIST & DAVID FIGDOR, Life as an endodontic
pathogen. Endodontic Topics 2003, 6, 3–28
 LEIF TRONSTAD & PIA TITTERUD SUNDE, The evolving new
understanding of endodontic infections. Endodontic Topics 2003, 6, 57–77
 JOSE´ F. SIQUEIRA JR, Periapical Actinomycosis and infection with
Propionibacterium propionicum. Endodontic Topics 2003, 6, 78–95
 ISABELLE PORTENIER, TUOMOS M.T. WALTIMO & MARKUS
HAAPASALO, Enterococcus faecalis – the root canal survivor and ‘star’
in posttreatment diseaseEndodontic Topics 2003, 6, 135–159
References
in posttreatment diseaseEndodontic Topics 2003, 6, 135–159
 DAVID A. SPRATT, Significance of bacterial identification by molecular
biology methods. Endodontic Topics 2004, 9, 5–14

125.  Ingle’s Endodontics 6, Ingle, Bakland, Baumgartner
 Essential Microbiology for Dentistry, Lakshman Samaranayake
 Pathways of Pulp, Cohen 9th edition
 GUNNEL SVENSA¨TER & GUNNAR BERGENHOLTZ, Biofilms in
endodontic infections. Endodontic Topics 2004, 9, 27–36
References