description about red complex bacteria and Aacomitans.

1. KEY CHARACTERISTICS OF
SPECIFIC PERIOPATHOGENS
-SHEETHALAN.M.S.R

2. CONTENTS
 INTRODUCTION ABOUT MICROBIOLOGY
-KOCH’S POSTULATES AND ITS APPLICABILITY IN PERIODONTAL DISEASE
-SOCRANSKY’S CRITERIA FOR PERIO PATHOGENS
-BACTERIAL MORPHOTYPES
-BACTERIA IN ORAL HEALTH
-SHIFT FROM HEALTH TO PERIODONTAL DISEASE
-BACTERIAL COMPLEXES DESCRIBED BY SOCRANSKY
 SPECIFIC PERIOPATHOGENS
1.ACTINOBACILLUS ACTINOMYECETUM COMITANS
-INTRODUCTION
-GENERAL CHARACTERISTICS
-COLONY CHARACTERISTICS
-VIRULENCE MECHANISM
-MODE OF ADHERENCE
-TOXINS

3. 2.PORPHYROMONAS GINGIVALIS
-INTRODUCTION
-COLONY CHARACTERISTICS
-VIRULENCE FACTORS
-Lipopolysaccharides (LPS),
-Capsular polysaccharide,
-Fimbriae
-Enzyme activity
-Outer membrane vesicles,
-Hemagglutinin , and
-Protein antigens all potentially contribute to its pathogenicity in
periodontal disease
-THERAPHY
3.TANERALLA FORSYTHIA
-INTRODUCTION
-BACTERIAL CHARECTERISTICS
-COLONY CHARECTERISTICS
-VIRURENCE MECHANISM
-LITRATURE REVIEW

4. 3.TREPONEMA DENTICOLA
-INTRODUCTION
-GENERAL CHARECTERISTICS
-BACTERIAL MORPHOLOGY
-VIRULENCE FACTORS
-ADHERENCE TO THE HOST
-IMMUNO MODULATION
-ROLE IN PERIODONTAL DISEASE
-THERAPHY
 REFERENCES

5. BREIF INTRODUCTION ABOUT
MICROBIOLOGY
 Microbiology can be defined as the biology of microscopic organisms, or
life too small to be seen with the naked eye.
 Microbiology covers several disciplines, including bacteriology (study of
bacteria), virology (study of viruses), mycology (study of fungi), and
parasitology (study of parasites).

6.  The human fetus inside the uterus is sterile , but after passing through the
birth canal, fetus acquires vaginal and faecal microorganism.
 Within hours after birth, the sterile oral cavity becomes colonized by low
numbers of mainly facultative and aerobic bacteria.
 2nd day-anaerobic bacteria develops.
 Within 2 weeks-mature microbiota established in the gut of new born.
 After 2yrs, the entire human microbial flora is formed by a complex
collection of 1014 microorganisms with more than 400 different types of
bacteria.

7. Criteria for identification of pathogens
Koch’s postulates

8. Applicability of Koch postulate in
periodontal disease
 In periodontitis 3 main problem existed –
1.inability to culture all the organism
2.difficulties in defining and culturing sites of active disease
3.lack of animal model system
 Sigmund socransky, a researcher at forsyth dental center ,boston, proposed
criteria by which periopathogens can be identified.

9. Socransky’s postulates
 Must be associated with the disease evident by organism must be found in
relatively high numbers in proximity to the periodontal lesion;
 Must be eliminated or decreased in sites that demonstrate clinical
resolution of the disease with treatment.
 Must demonstrate a host response, in the form of an alteration in the
cellular or humoral immune response.
 Must be capable of causing disease in experimental animal models.
 Must demonstrate virulence factors responsible for enabling the
microorganism to cause destruction of periodontal tissues.

10. Diversity of intraoral surfaces for
bacterial adhesion :
5 basic Ecosystems / Niches
 Intraoral supragingival hard surfaces (teeth, prosthesis , restorations )
 Periodontal pocket , periimplant pocket
 Buccal epithelium , palatal epithelium, floor of the mouth
 Dorsum of the tongue
 Tonsils

11. Bacterial Morphotypes…
Cocci - spherical
Bacilli-rods,rectangle
Filaments - threadlike
Fusiforms - threadlike with tapered ends
Spirochetes - spiral with axial fibrils in cell walls

12. Bacterial characteristics based on Oxygen
Environment….
Aerobes - require oxygen for growth
Anaerobic - do not require oxygen
Facultative anaerobic - use oxygen when it is present
Obligate Anaerobic - Cannot survive in presence of oxygen
Aerotolerant anaerobes - do not use oxygen but can tolerate
oxidizing environments

13. Bacteria in oral health
Facultative gram-positive bacteria such as :
Streptococcus species :
 S.sanguis (produces H2o2 – lethal to A.a)
 S. mitis
Actinomyces species
 A.naeslundii
 A. viscosus
Gram negative species :
• Prevotella intermedia
• Fusobacterium nucleatum
• Capnocytophaga
• Neisseria
• Veilonella species (increased in inactive sites , prevents colonization and proliferation of
pathogenic organisms )
• Few spirochetes and motile rods

14. Shift from health to Periodontal
disease:
In the process…..
 Gram positive to Gram negative
 Aerobic to Anaerobic
 Facultative to Obligatory
 Fermentive to Proteolytic
 Non-motile to motile

15. Bacterial complexes as described by
Socransky et al :
 5 complexes:
 Primary colonizers:
 Secondary colonizers:
Streptococcus
species
Actinomyces
odontolyticus
• Eikenella corrodens
• A.a comitans
serotype a
• Capnocytophaga
species
• P.gingivalis
• Tannerella forsythia
• Treponema
denticola
• Fusobacterium
• Prevotella
intermedia
• Camplylobacter sp.

16. KEY PERIODONTAL
PATHOGENS

17. Actinobacillus
Actinomycetemcomitans

18. INTRODUCTION
 The term Actinobacillus actinomycetemcomitans was coined by Topley
and Wilson.
 Member - Actinobacillus
 Family - Pasturellaceae
 Given the name Actinobacillus actinomycetemcomitans, that is, derived
from the Greek words, actes- meaning ray , because of the star on the top
of the agar colony and, Mycetes meaning fungus, because Actinomyces
was originally thought to be a fungus.
 The Latin word Comitans, meaning in common with, or accompanying
Actinomycetes spp.

19. Historic review ….
 A. actinomycetemcomitans was first reported in a publication by Klinger in
1912, and also isolated A.a from a Actinomycotic lesion .
 Thjotta and Sydnes in 1951, were the first to report that
A. actinomycetemcomitans could act as the sole infecting agent in
humans.
 In 1951 – Home put forward that A.a could cause disease in humans
 In 1959 Heinerish suggested that it a part of normal flora.
 In 1976 , Newman, Socransky and Slots – related A.a to Juvenile
periodontitis
 In 1979 , Tsai et al, discovered A.a leukotoxin

20.  Page et al1991,perry et al 1996,Kaplan et al 2001- Six serotypes, a–f, have
been described. The serological specificity is defined by six structurally and
antigenically distinct O-polysaccharide components of their respective
lipopolysaccharide molecules.
 In 2004 , Roe et al – identified the complete A.a genome
 In 2006- studies have shown a phylogenetic similarity of A.
actinomycetemcomitans and Haemophilus aphrophilus, H. paraphrophilus,
and H. segnis, suggesting the new genus Aggregatibacter.

21. General characteristics of A.
Actinomycetemcomitans:
 Fastidious
 Facultatively anaerobic
 Non-motile
 Non-sporing
 Non-hemolytic
 Small gram-negative rod, 0.4–0.5 lm x 1.0–1.5 lm in size
 Oxidase +ve and Catalase +ve

22. Microscopy ...
 After sample collection it should be immediately placed into a pre-
reduced transport medium such as reduced transport fluid (RTF) and taken
to the laboratory for processing, ideally within 30 min of collection
 Blood agar or chocolate blood agar supplemented with 5–7% defibrinated
horse blood if incubated in an aerobic atmosphere supplemented with 5–
10% carbon dioxide.
 Faint colonies may be visible after 24 h incubation.
 However, 48–72 h incubation is required for colonies with a diameter of 1–3
mm to appear.

23. Colony characteristics :
 Colonies are generally round with an irregular edge, domed and colourless
in appearance, and may have central wrinkling and adhere to the agar
surface.
 The pale colour of the agar allows light to be transmitted through the agar,
and lighting the agar plate from underneath allows easy discrimination of
the internal structures.
 Adherent, catalase-positive colonies, with star-like internal structures, that
reduce nitrates to nitrites are characteristic of A. actinomycetemcomitans.
 Colonial variants :1.Transparent rough,
2.transparent smooth and
3.opaque smooth

24. Selective medium:
 Tryptic soy–serum–bacitracin–vancomycin agar (contains 10% horse serum,
75 mg ⁄ l bacitracin and 5 mg ⁄ l vancomycin)
 The presence of these antibiotics suppresses the growth of gram-positive
bacteria.
 Colonies incubated for 4–7 days on a serum-containing medium will
develop star-like structures centrally.
 This rough star-like morphology may be lost on subculturing, producing
smooth isolates that are less adherent to the agar surface.

25. Three major phylogenetic lineages comprise (antigenic composition of A.a )
 (i) serotype b strains
 (ii)serotype c strains
 (iii) serotype a, d, e and f strains
 Most individuals harbour a single serotype that remains stable in the host
over time, although some patients have been shown to harbour two or
three serotypes of A.a
 The distribution of A. Actinomycetemcomitans serotypes appears to vary
according to the persons geographical location and their ethnicity.

26. PCR :
 For confirmation of strain
 Multiplex PCR - only allow determination of the presence or absence of A.
actinomycetemcomitans in clinical samples, and do not quantify the
numbers of bacteria present.
 Realtime PCR - used for the detection of periodontal pathogens, providing
valuable quantification data and reducing the time required for detection
of A. actinomycetemcomitans to hours rather than the days required by
conventional culture techniques .

27. Virulence mechanisms of bacteria
 Virulence is defined as the relative capacity of a microbe to cause disease
(Slots, 1999)
 Poulin and Combes (1999) defined the concept of virulence in terms of the
“virulence factors”, which are molecules or components from a microbe
that harm the host

28. Holt and Ebersole (2005) proposed that virulence factors have multiple
functions ...
 Adhesion to cells
 Invasion of cells
 Ability to evade host defence mechanisms, including the innate and
acquired immune systems.
 Enzymes such as proteases, which can directly damage host tissues.

29. 1) Adherance of A.a
 If a bacterium cannot adhere to a particular environment, it cannot
survive.
 So bacteria have evolved a range of adhesins, ranging from the long
distance adhesive systems of fimbriae and flagella to fibrils, to short-range
adhesive proteins attached to the bacterial cell wall
 Bacterial adhesins may also be the medium for invasion of bacteria into
cells of the host.

30. Mode of adherence ....
 The Transparent rough organisms are fimbriated with a protein that has a
subunit of molecular mass 54 kDa.
 Antibodies to synthetic A. actinomycetemcomitans fimbria blocked binding
of this organism to saliva-coated hydroxyapatite beads, buccal epithelial
cells and a fibroblast cell line, showing the importance of these cell-surface
fibrils for adhesion to multiple surfaces

31. a) Tight adhesion A–G operon (tadA–G) :
 Comprises seven adjacent genes (tadA–G).
 Responsible for bacterial tight adhesion.
 Mutants of tadA, tadC and tadG showed lower levels of fimbriae
expression.
 Mutations in tad genes also influence the release of leukotoxin

32. b) Fimbriae Associated protein ...
 Isolated A.a fimbriae contain a low-molecular-mass protein (approximately
6.5 kDa) that has been termed Flp, together with low amounts of a 54 kDa
protein termed Fup. This was later shown to be a low-molecular-mass
protein termed Fap (fimbriae-associated protein)
 The flp-1 gene lies upstream of the tad operon
 Gene inactivation resulted in failure to produce fibrils and a loss of
adherence.
 Mutations in this gene cluster block the bacteriums ability to form micro-
colonies in the presence of human fibroblasts

33.  In an oral colonization model, flp-1 and tadA mutants of A. a showed no
evidence of colonization of soft tissues or plaque from maxillary molars, and
no bone loss, demonstrating the importance of flp gene cluster for virulence
of this bacterium
 Mutants of flp-2 showed lower levels of fimbriae expression.

34. c) Colony proteins ....
The RcpA ⁄ B (rough colony proteins)
 The RcpA, B and C proteins, together with the putative lipoprotein tadD,
are found in the bacterial outer membrane
 RcpA,RcpC and tadD are also found in the inner membrane,
 RcpA forms a multimeric outer-membrane secretion channel (a so-called
secretin) for generation of the fimbriae
 The role of rcpB remains unclear in terms of adherence ⁄ biofilm formation

35. d) Adhesins ...
 A. actinomycetemcomitans binds to and invades cells via adhesins other
than the bundled fibrils produce by the flp operon.
 One of the most prevalent bacterial adhesins is the receptor for the host
glycoprotein fibronectin
 Named ComE1 and binds to a unique site in fibronectin – the FnIII9-10
domain
 The receptor for FN in turn binds to the cell-surface integrin α5β1 on the cell
being invaded, with the bacterium entering by receptor mediated
endocytosis.
 Inactivation of the comE1 gene completely abrogated bacterial binding to
FN, revealing that this is the major fibronectin adhesin.

36. e) Omp100
Omp100 is homologous to the family of adhesins
 A. Actinomycetemcomitans possesses several outer-membrane proteins, of
which Omp100 has adhesive properties .
 This protein is randomly localized on the bacterial outer surface, and an
antibody to the protein was able to inhibit binding (and invasion) of A. a to
human gingival keratinocytes.
 Inactivation of the gene decreased adhesion and invasion by 60%.

37. f) Collagen-binding proteins ...
 Similar to adhesins
 Mintz identified a gene encoding the protein EmaA (extracellular matrix
protein adhesin A), a collagen-binding adhesin.
 EmaA is the largest oligomeric coiled-coil adhesin protein (202 kDa)
 Electron microscopic examination of A.a identified antenna-like protrusions
on the bacterial surface.
 Such structures are absent in emaA mutants and collagen binding is
decreased
 It has been reported that, in a rabbit model of endocarditis this binded to
type I collagen

38. Biofilm formation by A.a ….
 The interaction of A.a within the biofilm is due to poly-N-acetyl-
glucosamine, which mediates intercellular adhesion and attachment of
cells to abiotic surfaces.
 It offers a high degree of protection of A. actinomycetemcomitans and
other bacterial biofilms against detergents like sodium dodecyl sulfate and
macrophage killing.

39. Invasion of A.a
 Bacteria are protected from immune defences and from antibiotics when
they are within cells, but are also likely to induce apoptosis or be subject to
intracellular defence mechanisms
 Bacteria interact with the intracellular actin cytoskeleton to invade and
move through cells
 A.a was the first invasive periodontopathogen to be reported.

40. How does it take place ...
 Invasion process is a rapid mechanism involving the formation of cell-
surface craters or apertures, with bacteria appearing in the host cell
cytoplasm within 30 min.
 Invasion was associated with protrusions from the host cells that formed
connections between cells and harboured A. a and entry of A.a is rapidly
followed by cell division.

41. Toxins of A. Actinomycetemcomitans....
 This organism expresses two toxins:
(i) An RTX (repeat in toxin) leukotoxin and
(ii) Cytolethal distending toxin (CDT)
(iii) A third potential toxin is that encoded by cytotoxin-associated gene E
(CagE)

42. A.actinomycetemcomitans leukotoxin:
 It is an example of RTX toxin, whose name derives from the characteristic
calcium-binding motif that is repeated in the carboxy terminal of such
proteins.
 They are produced by a variety of Gram negative bacteria.
 The classic RTX toxin is E. coli haemolysin (HylA).
 Gene ptsH is required for LtxA secretion. Mutants lacking ptsH failed to
produce LtxA

43. Secretion of Ltx:
 Microvesicles budding from the outer membrane of A.a have been shown to
contain leukotoxin , suggesting that this is a novel mechanism for producing this
toxin.
 However, vesicles lacking LtxA also interact with target cells, showing that the
toxin does not drive this process of leukocyte interaction
 A distinct 141 kDa protein, which has been named Morphogenesis protein C
(MorC) , has also been found to be involved in LtxA secretion.
 Inactivation of morC results in the changing from an irregular to a flat profile of
outer membrane of A.a
 This is associated with failure to secrete LtxA.
 Studies have suggested that human serum can cause the release of LtxA from
A.a and that secretion is blocked by the presence of free iron

44. The mechanism of Leukotoxin action :
 There are two leukotoxin mediated mechanisms of cell death namely
necrosis and apoptosis .
 Exposure of neutrophils and monocytes or macrophages to LtxA results in
killing within a short period of time
 It is due to formation of pores in the membrane of target cells leading to
osmotic lysis caused by water influx into cell
 Prolonged exposure of lymphocytes and NK cells to LtxA results in
apoptosis.
 Lower concentrations lead to apoptosis while higher concentrations lead
to necrosis .

45. Role of LtxA of A.a in the pathogenesis of
periodontal disease....
Periodontitis can be furthered by
i. the bacterium inducing pro-inflammatory factors and tissue-damaging
agents,
ii. inhibition of the killing actions of the key anti-bacterial components of
immunity (phagocytes), and
iii. protection of the bacteria from immune-mediated killing

46. Other functions of leukotoxin…
 LtxA kills polymorphonuclear leukocytes and monocytes, as well as
lymphocytes, Killing is through apoptosis .
 LtxA can block the processes of bacterial uptake into phagocytes and the
associated processes of bacterial killing
 There are marked differences in leukotoxin expression within the various A.a
strains.
 It has been reported that certain strains produce little leukotoxin and others
produce relatively moderate amounts, whereas strains such as JP2 produce
very large amounts of toxin.

47.  It has also been reported that JP2 clones of A.a are associated with severe
forms of periodontitis
 Cross-sectional studies have demonstrated a positive association between
the presence of this clone and the occurrence of early-onset periodontitis
(EOP) (Haubek et al).
 Moreover, patients who harbor the JP2 clone have more advanced stages
of the disease than patients without the clone - Haubek et al

48. Cytolethal distending toxin
 Cell Cycle-blocking bacterial toxin is cytolethal distending toxin (CDT)
 First discovered in enteropathogenic E. coli .
 CDT was found to block eukaryotic cell division in G2. This is part of the cell
cycle after the DNA has been replicated during which the quality of the
newly synthesized DNA is checked.

49. How does CDT function ?
 The toxin must enter the cell and then move to the nucleus where it exerts
its effect
 They appear to bind to cholesterol within the cell membrane, as removal of
this planar lipid results in loss of activity of the toxin or responsiveness of the
cells
 The toxin is reported to be internalized through the Golgi complex, and
then transported to the endoplasmic reticulum

50. Role of CDT in the pathogenesis of disease
associated with A.a :-
 A.a produce an immunosuppressive factor that could block T-cell
proliferation .
 It was subsequently reported that this immunosuppressive factor was the
CdtB component of CDT, and was able to block cell-cycle progression in
human T lymphocytes and induce apoptosis in these cells, and the activity
of CdtB was increased by addition of CdtA and CdtC
 Thus CDT may contribute to the immunosuppressive phenotype of A.a
synergizing with the effects of A.a leukotoxin

51.  Teng & Hu, have identified a CagE homologue in A.a
 The protein was shown to induce changes in cells similar to those reported
for the H. pylori protein
 H. pylori is well known for its possession of the Cag pathogenicity island, with
associated cytotoxin associated genes A and E (cagA ⁄ E), which are major
virulence factors that are injected into host cells and cause cellular
alterations such as increased cell proliferation, motility, apoptosis and
morphological changes
 This adds another toxin to the armamentarium of this oral bacterium

52. Cellular mechanisms responsible for bone
detruction :
 Components of A. actinomycetemcomitans inhibit osteoblast proliferation and
synthetic activity and also the activation of bone resorption and the induction
of osteoclast proliferation and activation
The various components are -
-Lipopolysaccharide
-Chaperonin 60 (Hsp60)
-Capsular-like polysaccharide
-Possibly other cell surface-associated proteins
 Cell-wall components of A. Actinomycetemcomitans has osteolytic signals.
These have included lipopolysaccharide and cell-surface capsular-like
polysaccharide.

53. Porphromonas
Gingivalis

54.  Porphyromonas gingivalis belongs to the phylum Bacteroidetes and is a
nonmotile, Gram-negative, rod-shaped, anaerobic, pathogenic
bacterium.
 It forms black colonies on blood agar
 Porphyromonas gingivalis has been considered as one of the putative
periodontal pathogen.
 P.gingivalis colonizes periodontal pocket and spreads into deeper tissues,
including connective tissue and bone
 P.gingivalis is frequently isolated from subgingival plaque samples in
patients with adult and other forms of periodontitis.
 Major oral ecologic niche for this species appears to be subgingival
plaque.

55.  P.g. possess significant proteolytic activity.
 P.g is at present the only known porphyromonas species isolated from
human that produces phenyl acetic acid as a metabolic end product.
 Key test for identification of species include haemagglutination &
proteinase activity.

56. Colony characteristics …
 Asaccharolytic and black pigmented rod that form greenish-black colonies
in blood agar plates (Haffajee and Socransky, 1994).
 Fresh clinical isolates of this organism have different colony morphologies,
ranging from smooth to rough colony morphotypes (Reynolds et al., 1989).

57.  In both periodontitis and healthy subjects, P. gingivalis can be recovered in
low frequency from the subgingival flora, tongue, buccal mucosa and
tonsils and saliva. The presence of P. gingivalis has also been correlated
with periodontal pocket depth (Dahlén et al)
 when found in healthy cases or sites P. gingivalis is present in low numbers,
while in deep periodontal pockets its level is significantly higher.
 Higher serum titers of antibodies against P.gingivalis in periodontitis patients
than in periodontally healthy have been demonstrated (Naito et al)

58. Virulence factor
P. gingivalis has a wide range of significant virulence factors such as (Haffajee and
Socransky, 1994).
1. Lipopolysaccharides (LPS),
2. Capsular polysaccharide,
3. Fimbriae ,
4. Enzyme activity
5. Outer membrane vesicles,
6. Hemagglutinin , and
7. Protein antigens all potentially contribute to its pathogenicity in periodontal
disease

59. Lipopolysaccharides (LPS):
 LPS are major surface components of Gram-negative bacteria and they
build up a complex consisting of the core polysaccharide and Lipid A.
 Two distinct lipopolysaccharide macromolecules have been identified in P.
gingivalis strain .
 One of these species contains an anionic polysaccharide with
phosphorylated branched mannose repeating units linked to lipid A (A-
LPS).
 The other is a polysaccharide with tetrasaccharide repeating units (O-
antigen) linked to lipid A (O-LPS) .

60.  Lipid A is the toxic part of LPS and has endotoxic activity and stimulates
host inflammatory response indirectly by host derived cytokines (Bartold et
al., 1991; Yamaji et al., 1995)
 The polysaccharide chain constitutes the O-specific antigen and has also
significant immunological activity (Takada et al., 1992).
 Kadano et al. demonstrated that P. gingivalis lipopolysaccharide can
inhibit the differentiation of rat osteoprogenitor cells into osteoblasts
 These results suggest that P. Gingivalis lipopolysaccharide is not only
capable of stimulating bone resorption, but is also capable of inhibiting
bone formation, which may significantly contribute to the loss of tooth-
supporting alveolar bone in periodontitis.
 Gingival fibroblasts have been reported to produce a range of
proinflammatory cytokines, including IL-1, IL-6, IL-8 and TNFa, upon exposure
to P. gingivalis lipopolysaccharide

61. Structure:
As with any prokaryotic cell, it has
 A cell envelope (capsule, cell wall, cell membrane)
 Cytoplasm (ribosomes, chromosomes)
 Appendages (fimbriae)

62. Capsule:
 Bacterial capsules- major virulence factors
 It is formed by a polysaccharide heteropolymer on the outer membrane of
the bacterial cell.
 It has various functions forming a physiochemical barrier for the cell
protecting against opsonization and phagocytic host cells e.g. neutrophils
(polymorphonuclear leukocytes) and from desiccation (Chen et al., 1987).
 Especially, the antiphagocytic activity against host cells is important for a
periodontal pathogen such as P. gingivalis in its penetration into the host
tissue in periodontal pockets, and survive and multiply in this area.

63. Fimbriae:
 Filament components of the cell surface structure with a diameter of 5 nm.
 They are highly antigenic and show high serum IgA and IgG antibody responses
(Ogawa et al., 1990; Yoshimura et al., 1987).
 Environmental factors like temperature, ph etc play important role in fimbriae
formation.
Functions ...
 The most essential role of fimbriae is the binding capacity to host cells including
the oral epithelial cells, gingival fibroblasts and endothelial cells, other bacterial
species, extracellular matrix protein and salivary proteins (Hamada et al., 1998).
 P. Gingivalis fimbriae have also been reported to mediate the coaggregation
of P. gingivalis and other plaque-forming bacteria such as Actinomyces
viscosus, Streptococcus gordonii and Streptococcus mitis
 In addition, minor (short) fimbriae induce production of several cytokines from
macrophages.(Hamada et al., 2002).

64. MAJOR fimbriae:
 First described by Yoshimura et al in 1984.
 Lee et al in 1991 described four types based on N terminal amino acid
sequences.
 Nagakawa et al in 2002 described 6 variants based on the nucleotide
sequences as type I, Ib, II, III, IV & V.
 Type II is the most virulent type followed by Ib, IV and V.
 Types I and III are avirulent / noninvasive.
MINOR fimbriae:
 First described by Hamada et al in 1996.
 Differs from that of major type in less antigenicity and decreased size.
 Production of both major and minor fimbriae is required for expression of
pathogenic traits

65. So Functions of fimbriae as a whole…
 mediates adhesion
 stimulates intracellular signaling between bacteria and host during invasion.
 Modulates the production of proinflammatory cytokines.
 Induce inflammation through toll like receptors
 Wang et al. showed that mutant strains of P. gingivalis lacking Fimbriae
gene induced significantly less alveolar bone loss compared with wild-type,
thus indicating the importance of Fimbriae in the virulence of P. gingivalis

66. Extracellular proteolytic enzymes:
 P. gingivalis produces a wide variety of enzymes.
 Of these, the Arg-X and Lys-X specific extracellular cysteine proteinases can
degrade serum proteins including immunoglobulin as well as extracellular
matrix proteins.
 This family of cysteine proteinases have been given the name “gingipains”
(Curtis et al., 1999).
 The gingipains constitute a group of cysteine endopeptidases that are
responsible for at least 85% of the general proteolytic activity (Potempa et
al., 1997) and 100% of the ‘‘trypsin-like activity’’ produced by P.gingivalis
(Potempa et al., 1995).
 Therefore, gingipains are important virulence factors in the periodontal
infection.

67. Proteinases :
They can be classified as
A. Trypsin like proteinase
B. Collagenolytic proteinase
C. Other proteinase- Dipeptidylpeptidases
 A significant proportion of the trypsin like activity is associated with bacterial
membranes.
 These enzymes are located at the inner cell membrane and the cell surface.
High activity has also been found in extracellular outer membrane vesicle.

68. A. Trypsin like proteinases
 Porphyromonas gingivalis was found to have a particular ability to cleave
peptide substrates with arginine terminal groups such as benzoyl – arginine
– 2-napthylamide (BANA) or benzyoyl-arginine-p-nitroanilide (BAPNA). This is
called trypsin like activity.
 Four proteinases was found
-Three cysteine proteinases, two of which cleaved arginine bonds one of
which cleaved lysine bonds

69.  Trypsin like activity from P.gingivalis from both outer membrane vesicles and
culture supernatant.
 They showed that this was a cysteine proteinase which cleaved synthetic
arginine substrates.
 They proposed the name gingivain for this enzyme.
 Similarly, an identical proteinase was isolated from the culture supernatant
and they gave it the name of gingipain.

71. B. Collagenolytic proteinases :
 Collagenolytic activity has frequently been reported in porphyromonas
gingivalis.
 One group of workers purified the collagenolytic activities from
porphyromonas gingivalis using electrophoretic techniques.
 The enzyme was capable of cleaving basement membrane type IV
collagen and synthetic substrates from bacterial collagenase.

72. C. Other proteinases :
 Two other protease coding genes, tpr and prtT, have been isolated from
P.gingivalis and they translate to low-level protease activity.
 The resultant enzyme was active against general protein substrates but not
collagen.
 In addition two proteases degrade fibrinogen and fibronectin, and
collagenase like neutral protease has also been isolated from
Porphyromonas gingivalis.

73. Outer membrane vesicles (OMV):
 Most gram-negative bacteria form small structures on the outer membrane
surface of bacteria named “outer membrane vesicles”.
 This OMV are released from the outer membrane during growth (Handley
and Tipler, 1986).
 The OMV of P. gingivalis may contain several virulence factors including
gingipains (Marsh et al., 1989).

74. End-products of metabolism:
 The bacterial metabolic end-products (e.g. volatile short chain fatty acids,
sulfur products and ammonia) can contribute to the nutritional resources
and support other bacteria within biofilm, as well as toxicity to host cells
(Holt et al., 1999)
 The short-chain fatty acids such as succinate,isobutyrate and isovalerate
can inhibit the function of neutrophils and T-lymphocyte
 Hydrogen sulfide and methyl mercaptan have been detected in significant
phagocytes ,gingival fibroblasts and periodontal ligament cells amounts in
periodontal pockets (Persson, 1992).
 Ammonia is strongly cytotoxic to neutrophils and gingival fibroblasts
 Since all these bacterial metabolites are smaller molecules than other
cytotoxic factors e.g. Proteases and lipopolysaccharides, they may more
easily penetrate into the periodontal tissues at an increased bacterial
metabolic activity and growth.

75. Coaggregation:
It is a highly specific stereochemical interaction of protein and carbohydrate
molecules located on the bacterial cell surfaces.
P.gingivalis coaggregate with,
 S.oralis – G3P dehydrogenase of S.oralis is needed.
 S.gordonii – 40 kDa OMP of P.g is needed
 A.naeslundi – gingipains of P.g is needed
 F.nucleatum – needs galactose binding adhesion site.

76. Therapy …
Gingipains as targets for periodontal therapy
(i) Vaccines
 The first possibility is a vaccination therapy using gingipains for periodontitis.
 Immunization gingipain R resulted in protection from P.gingivalis invasion.
 Antibodies directed against gingipain R are capable of inducing protective
immune response against P.gingivalis infection.

77. (ii) Inhibitors for gingipains
 Another possibility is the development of inhibitors for gingipain.
 A proteinase inhibitor reduced Porphyromonas gingival growth, suggesting
the potential therapeutic effect of gingipain inhibitors in periodontitis.

78. Tanerella forsythia

79. History…
 T. forsythia was first isolated at The Forsyth Institute from subjects with
progressing advanced periodontitis in the mid-1970s and was described as
fusiform Bacteroides by Tanner et al.
 Around the same time, T. forsythia was isolated as one of the Bacteroides
group from the extensive cultural studies of periodontal infections by Moore
and Holdeman-Moore at the Anaerobe Laboratory of the Virginia
Polytechnic Institute (VPI).
 The species was subsequently detected by culture from oral samples at the
Forsyth and VPI laboratories from progressing periodontitis ,endodontic
infections , gingivitis and early periodontitis , refractory periodontitis , and
peri-implantitis

80. Bacterial characteristics …
 Gram –negative obligate anaerobe
 Non-motile ,
 spindle-shaped,
 highly pleomorphic rods
 In case of advanced Active periodontitis, it is found along with P.gingivalis.
 It is much higher in subgingival plaque than supragingival plaque.
 Most detected species in Refractory periodontitis.

81. Characteristics of isolates
 Growth of T.forsythia is stimulated by N-acetyl-muramic acid
 Shape of T.forsythia cells and colonies varies depending on the growth
condition
 Cells of cultures grown on agar media without N-acetyl muramic acid
a. Large
b. Filamentous
c. Tapered (fusiform)

82.  Colony morphology changes in presence of N–acetyl- muramic acid or
growth-stimulating species.
 Colonies are
a. tiny and opaque
b. pale, pink and speckled circular
c. slightly convex may have depressed centre (donut shape)
 Also cells become regularly shaped, short ,gram-negative rods.

83. Distinctive ultrastructure -:
1. Inner-membrane
2. Outer-membrane
3. Distinctive outer layer.

84. Characteristics ...
 T. forsythia has the ability to metabolize a range of substrates and, in
common with many enteric Bacteroides species, to hydrolyze esculin.
 In contrast to the enteric species, however, T. forsythia is not resistant to bile,
thus it does not grow on Bile Esculin Agar
Growth media ..
 Porphyromonas gingivalis and T. forsythia will grow on media supplemented
with vitamin K for P. gingivalis, and N-acetylmuramic acid for T. forsythia,
each species is inhibited by the growth requirement of the other, so
compromising primary isolation.
 Both T. Forsythia and P. gingivalis are anaerobes but it also has been
suggested that they may , be able to grow in the presence of low levels of
oxygen.

85. Antibiotic sensitivity...
 Generally sensitive to antibiotics that are active against anaerobes.
 Most active antibiotics were amoxicillin with clavulanate (100%), ampicillin
(98%), doxycycline (98%), amoxocillin (96%),tetracycline (90%), and
clindamycin (86%).
 Fairly active antibiotics were penicillin (70%) and spiramycin (68%)
 Poorly active antibiotics were erythromycin (54%) and ciprofloxacin (46%).
 In addition, T. forsythia was found to be sensitive to metronidazole

86. Antibiotic resistance ...
 tet(Q), a gene encoding the ribosome protection protein resulting in
antibiotic resistance to tetracycline.
 In addition to tet(Q) genes, strains of T. Forsythia contained erm(F) which
codes for erythromycin resistance

87. Identification in periodontium ....
 The ability of T. forsythia to hydrolyze the trypsin like benzyol-DL-arginine-2-
naphthylamide, BANA, has been incorporated in the test for periodontal
pathogens pioneered by Loesche .
 Trypsin-like activity is shared by P. gingivalis and T. denticola of the
Socransky red complex .
 Other subgingival species are also BANA-positive, but not as strongly as P.
gingivalis, T. denticola, and T. forsythia, thus making this biochemical marker
a test for the presence of these species

88. Immunological assays :
 Antibody raised against T. forsythia cells and used for species identification, did
not cross-react with P. gingivalis, Prevotella intermedia.
 Antibody to T. forsythia was serologically distinct from other oral and clinical
Bacteroides species (now Bacteroides, Porphyromonas, and Prevotella) .
 Monoclonal antibodies were also produced against T. forsythia that were strain
and species specific and that did not react with other subgingival species .
 They were subsequently used in enzyme-linked immunosorbent assays and
immunofluorescence assays
Molecular identification …
 PCR
 DNA probes

89. Biofilm formation …
 T. forsythia can form biofilms in vitro with F. Nucleatum .
 The thickness and structure of T. Forsythia biofilms is influenced by F.
nucleatum.
 Both species co-aggregate when in a planktonic form, and this interspecies
binding appears to be critical in the formation and structure of T. Forsythia–
F. nucleatum biofilms, and favors T. forsythia growth
 This type of interaction was not observed for the biofilms formed by T.
denticola with P. Gingivalis and by Streptococcus oralis with Actinomyces
naeslundii
 Both F. nucleatum and T. denticola can bind to T. forsythia

90. Virulence mechanisms of T.Forsythia:
Surface components …
Surface-layer associated glycoproteins
Leucine-rich repeat BspA protein
Surface lipoproteins
Glycosidic activity

91. Surface-layer associated glycoproteins :
 The S-layer structure is the first cell ultrastructure encountered by the
environment and the host.
 The S-layer has been shown to comprise at least two high-molecular-mass
glycoproteins of 220 and 210 kDa encoded by the tfsA and tfsB genes
respectively
 Provides a protective shield.
 Promotes epithelial cell adherence and invasion.
 S-layer proteins are immunogens in periodontitis patients .
 The S-layer also mediates hemagglutination, which is inhibited by N-
acetylglucosamine, and heat denaturation of the proteins.
 Pre-immunization of mice with S-layer protected these animals against
abscess formation

92. 2) Leucine-rich repeat BspA protein
 A surface-associated as well as secreted protein BspA (Bacteroides surface
protein A) belonging to the leucine- rich repeat family has been identified
in T. Forsythia
 Analysis of the carboxy-terminal region of BspA revealed it to be involved in
trafficking of bacterial proteins to the outer membrane and secretion.

93. Function of BspA protein
 BspA protein could be involved in the protein–protein interactions that are
important in mediating T. forsythia interactions with the factors and ⁄ or
components of other bacteria
 BspA has been shown to bind to the extracellular matrix component
fibronectin and the clotting factor fibrinogen
 BspA has been shown to trigger the release of bone-resorbing
proinflammatory cytokines from monocytes

94. 3)Surface lipoproteins :
 Activates host cells to release proinflammatory cytokines
 Induce cellular apoptosis
 Lipoprotein fractions of T.forsythia – stimulate fibroblasts and monocytic
cells to release IL-6 and TNF-α

95. 4) Glycosidic activity :
 T. forsythia has been shown to express a variety of glycosidases
 In principle, these glycosidases can hydrolyze terminal glycosidic linkages in
the complex oligosaccharides and proteoglycans that are abundant in
saliva, gingival crevicular fluid and periodontal tissue.
 Degradation of oligosaccharides and proteoglycans will affect the
functional integrity of the periodontium and may promote disease
progression.
 The degradation of host oligosaccharides and proteoglycans by these
glycosidases can also provide nutrients for other community bacteria.

96. Treponema Denticola

97.  The genus Treponema belong to a phylum of distinctive Gram-negative
bacteria called Spirochaetes.
Spirochete:
 Defined as a spiral-shaped bacterium with periplasmic flagella that
originate at opposite poles of the bacterium and overlap near the middle
of the cell
 The phylum consists of Spirochaetaceae, Serpulinaceae and
Leptospiraceae
 The first group consists of Spirochaeta, Borrelia, Clevelandina, Cristispira,
Diplocalyx, Hollandina, Pillotina and Treponema.
 Among these, only the genus Treponema has been isolated from the oral
cavity.

98. Treponema species in the oral cavity of humans
 Treponema amylovorum,
 Treponema denticola,
 Treponema lecithinolyticum,
 Treponema maltophilum,
 Treponema medium,
 Treponema parvum,
 Treponema pectinovorum,
 Treponema socranskii
 Treponema vincentii
 Out of which only T.denticola was isolated from periodontally diseased than
healthy sites and more common in subgingival than supragingival plaque

99. Bacterial Morphology :
 Dark field microscope Spirochetes present a long and slightly helically
coiled (spiral, helical or serpentine-shaped) - this particular morphology
causes a twisting motion which allows to penetrate into dense media
 In particular, family of Spirochaetes are distinguished from other bacterial
phyla by the location of their flagella, axial filaments, which run lengthwise
between the cell wall (peptidoglycan layer) and outer membrane.
 Flagella filaments wind around the body toward the center of the cell and
may or may not overlap with one another in the center. The overall
diameter of a filament is usually within the range of 16-25 nm.

100. Characteristics of T. Denticola
A) Envelope like structure
 T. denticola are covered with an outer sheath consisting of a fragile
envelope like structure
 Periplasmic flagella are located on the cytoplasmic membrane and are
covered with the outer sheath.
 T. denticola typically produces four flagella, which are intertwined around
the cell
 Two originate at each end and they usually overlap in the middle of the
bacterium.

101. B) Major outer sheath protein (Msp)
 The is the predominant protein in the outer sheath
C)Tpr proteins
 Tpr proteins are candidate rare outer membrane proteins and have been
proposed to endow T. pallidum with the ability to alter its surface antigenicity
D) Dentilisin :
 A prolyl phenylalanine-specific peptidase dentilisin (chymotrypsin-like protease)
is located on the surface of T. Denticola
 The protease domain is encoded by prtP gene

102. E) The periplasmic filament
 It is located just beneath the cytoplasmic membrane.
 A periplasmic filament-deficient mutant exhibited chains of treponemal
cells and highly condensed chromosomal DNA, which contrasted with the
homogeneous distribution of DNA throughout the cytoplasm in the wild-
type organism.
 This suggests that these cytoplasmic filaments are involved in chromosome
structure, segregation, or the cell-division process

103. Epidemiology:...
 The association of oral spirochetes with chronic periodontitis , acute ulcerative
necrotizing gingivitis has been reported
 T. denticola was detected in the deciduous dentition and the detection rate
was found to increase with age
 T. denticola was also reported to increase susceptibility to gingival inflammation
 It is co-isolated with P. gingivalis and Tannerella forsythia .
 The consortium of these species is known as the red complex
 Recent epidemiological data further suggested its potential involvement of
periodontitis in cardiovascular disease .
 In this regard, T. denticola was detected from atherosclerosis lesions

104. Virulence factors:
The potential virulence factors of this microorganism include
 adherence factors,
 motility,
 evasion mechanisms from host defenses and
 cytotoxic factors for host tissues.

105. Adherence to host:
 T. denticola has no specific adherence structures, such as fimbriae, but has
the ability to adhere to various surfaces of the oral cavity, such as tooth,
host cells and other microorganisms in dental plaque
 This adherence ability is a result of the variety of adherence factors on its
surface.
 Collagen and cell-adhesion molecules, such as fibronectin and laminin, are
receptors for the bacterial colonization
 A 27 kDa type IV collagen-binding protein of T. denticola has also been
reported

106. 1) Msp:
 The ability of T. denticola to bind to human gingival fibroblasts is suggested
to be caused by lectin-like proteins or mediated by host proteins.
 Antibody against the Msp of T. denticola strain inhibited the adherence of
this strain to human gingival fibroblasts
 These results revealed that Msp is a fibronectin-binding protein involved in
binding to fibroblasts.

107. 2) OppA:
 Ortholog of oligopeptide transporter unit –OppA
 A 70 kDa protein, has also been reported to bind to fibronectin
 The protein-encoding gene was isolated and designated oppA
 An oppA deficient mutant was not affected in binding to epithelial cells,
and native OppA did not bind to immobilized substrates or to epithelial
cells, suggesting that this protein does not participate in direct adherence
to cell-bound receptors, although binding activity was shown at the protein
level

108. 3) Factor H-like protein-1 binding proteins
 Factor H is a crucial negative regulator of the alternative pathway for
complement activation.
 It acts as a competitor for factor B, binding to C3b to form convertases,
and is an essential cofactor for factor I-mediated degradation of C3b to
iC3b
 In addition to factor H, this protein is involved in the down-regulation of C3b
production
 The protein is located on the surface of T. denticola and its levels were
decreased with repeated washing.

109. 3) Dentilisin for Coaggregation:
 Coaggregation is a major strategy for colonization into dental plaque
biofilms and it occurs via the adherence of cells to the surface of biofilms.
 T. denticola was co-isolated with P. gingivalis and T. forsythia, suggesting
that coaggregation and synergy among these species are important in
colonization by these microorganisms.
 In addition, enhancement of virulence by this consortium was reported.
 T. denticola has been reported to co-aggregate with several members of
dental plaque, such as P. gingivalis, T. forsythia and F. nucleatum
 Msp was reported to be a candidate ligand for coaggregation reactions
between T. denticola and P. gingivalis or F. nucleatum
 Dentilsin was also suggested to be involved in coaggregation, Long
fimbriae (FimA) of P. gingivalis reacted with a 72 kDa T. denticola protein,
and this protein was identified as dentilisin

110. Immunomodulation:
 T. denticola was reported to induce a strong antibody response in young
adult patients with localized juvenile periodontitis
 Mangan et al. reported that spirochetes comprised 20% of the total
number of microorganisms detected in plaque from these patients,
whereas no spirochetes were observed in plaque from the controls
 Several proteins, including Msp and dentilisin, and the flagella of this
microorganism, were reported to be antigenic
 Mice actively immunized with formalin-killed treponemes developed serum
antibody levels seven to eightfold greater than those in animals after
primary infection.

111. 1) Immunosuppressive factors:
 Fractions from T. denticola, with molecular mass values of approximately
100 and 50 kDa, suppress human peripheral mononuclear cells and
fibroblasts, respectively
 Spirochete immunoinhibitory proteins of 50 and 53 kDa inhibited activation
of human peripheral blood mononuclear cells in a dose-dependent
manner.

112. 2) Dentilisin:
 Treponema denticola was reported to induce cytokine production which
was induced by cell-surface components, such as peptidoglycan and
other components of outer membrane
 Peptidoglycan isolated from T. denticola induced the release of
interleukin-1b, interleukin-6, tumor necrosis factor-a , interleukin-8, matrix
metalloproteinase-8 and prostaglandins from macrophages.
 Monocyte chemoattractant protein-1 and interleukin-8 were also
degraded by dentilisin , which may be associated with evasion of the host
defense by T. denticola.
 These properties of T. denticola may ultimately contribute to tissue
destruction.

113. 3 & 4 ) Msp and lipooligosaccharide
 Several reports have indicated that T. denticola activates host cells via Toll-
like receptor
 T. denticola activates the immune system via two different pathways and
that certain cytokines modulate this activation.
 T. denticola cells and Msp induced innate immune responses of
macrophages through Toll like receptor 2, whereas liposaccharide induced
a macrophage response through Toll-like receptor 4

114. 5) Resistance to defensins :
 Antibacterial peptides play an important role in the initial host defense
against microbial attack.
 Epithelial cells produce antibacterial peptides, such as beta-defensin,
which protect against colonization by microorganisms.
 These small cationic peptides interact with negatively charged cell wall
components of bacteria and fungi, disrupting their membrane integrity .
 Beta-defensins are also active against oral microbes, including
periodontitis-related bacteria .
 However, T. denticola is resistant to human betadefensins 1 and 2

115. Chemotaxis and gene expression
 The motility of T. denticola is dependent upon genes coding for periplasmic
flagella and chemotaxis. The flagellar filament of T. denticola consists of three
core proteins (FlaB1, FlaB2 and FlaB3) and a major sheath protein, FlaA .
 Extracellular stimuli are sensed by membrane-spanning methyl-accepting
chemotaxis proteins (Mcps) and the signals are transformed into appropriate
motor responses.
 Controls the direction of flagellar motor rotation.

116. Acquisition of Fe+
 Acquisition of the Fe ion is essential for the growth of microorganisms.
 Lactoferrin- binding proteins of 50 and 35 kDa, and heminbinding proteins of 47
and 44 kDa, were reported in T. denticola .
 These proteins play an important role in the acquisition of Fe ions because these
ions are sequestered by lactoferrin or transferrin

120. Role in Periodontal Disease :
 In the experimental animals, the spirochetes were able to penetrate the
normal sulcular epithelium and were subsequently found, in considerable
numbers, associated with ulcerated sites and within cellular exudates.
 Evidently, the combination of immune suppression by corticosteroids and
inoculation with spirochete-rich debris was sufficient to initiate NUG lesions
in beagle dogs, which experimentally mimicks the combination of stress,
poor hygiene, and infection that predisposes some humans to periodic
necrotic ulcerative gingivitis

121.  In another study by Okuda - Co-infection with T. denticola enhanced the
virulence of low dose P. gingivalis challenges.
 He used a similar approach and demonstrated that mixed infections of T.
denticola and P. gingivalis could still form abscesses in immunized mice ,
implying that the combination of species found in proteolytic complexes in
clinical studies may form mutually protective bacterial communities in vivo
that would be expected to persist in local lesions like abscesses.

122. Treatment
 Treatment of subjects with periodontitis by scaling and root planning or by
this therapy combined with either systemic Amoxicillin or metronidazole led
to a reduced frequency of detection and mean bacterial count of the
treponemal species.
 Therapeutic suppression of the treponemes was long-lasting.
 But , T. Denticola was also found more frequently and with a greater mean
count in individuals who were refractory to periodontal treatment

123. REFERENCES
 CLINICAL PERIODONTOLOGY,CARRANZA,TENTH EDITION
 Periodontology 2000 Vol. 54, 2010, 45–52
 Periodontology 2000, Vol. 52, 2010, 218–237.
 Periodontology 2000, Vol. 38, 2005, 72–122
 Periodontology 2000, Vol. 5, 1994, 78-11 1

124. THANK YOU