Microbiology of the oral cavity, types of organisms, plaque, biofilm

1. PLAQUE AS A BIOFILM
AND
MICROBIOLOGY OF PERIODONTAL
DISEASES
Navneet Randhawa
MDS Final year

2. INTRODUCTION
• It is estimated that about 700 different spp. are capable of colonizing the
mouth.
• An individual may harbor 150 or more different spp.
• The ecological relationship between periodontal microbiota & host –benign .
• A subset of organisms either introduced, overgrow or exhibit new properties –
leading to periodontal diseases.

3. EVIDENCE OF INFECTIOUS NATURE
OF PERIODONTAL DISEASE
1. Acute periodontal infections: - alleviated by anti microbial therapy.
2. Positive co-relation of plaque levels to gingivitis and amount of bone loss. (Loe et al
1965)
3. Efficacy of antibiotics in treatment of periodontitis:
4 HOST IMMUNOLOGIC RESPONSE: ↑serum antibody response (Taubman et al 1994 )
5. Toxic products (endotoxins, H2S , NH3, indole, amines, leucotoxins ) - demonstrate
pathogenic potential.
6. STUDIES IN EXPERIMENTAL ANIMALS: Induce periodontal disease

4. UNIQUE FEATURES OF PERIODONTAL
INFECTIONS
• Mineralized tooth structure passes through the integument so that part of it is
exposed to the external environment while part is within C.T.
• Non shedding tooth surface.- provides ‘sancturies’ in which micro-organisms can
hide.
• Causative agent reside in biofilms outside the body.
• Formidable problem for host and therapist.

5. HISTORICAL PERSPECTIVE

6. • 1683 Antonie van Leeuwenhoek
• “Poor oral hygiene – large no. of bacteria”
“I didn’t clean my teeth for three days and
then took the material that had lodged in
small amounts on the gums above my
front teeth…. I found a few living
animalcules…”

7. The search for the etiological agents of periodontal diseases started
in the ‘golden age of microbiology’ [app. 1880-1920]

8. • 1890 W.D. Miller “pyorrhoea alveolaris is not caused by a specific bacterium”
• Invesigators from the period of 1880-1930 suggested four distinct groups of micro-
organisms. Viz; amoeba,spirochetes,fusiforms,sreptococci.
• 1925 -1950
Decline of interest in microorganisms
Bacteria : secondary invaders/contributors to inflammation
Emphasized on occlusal factors,calculus & poor host defense

9.  1950 Waerhaugh : Dental plaque in the initiation & progression of pdl
diseases
• 1950-1975 : Non specific hypothesis
• 1970s : Improvement in anaerobic culture technique.Distinct microfloras in pdl health
&gingivitis-Specific plaque hypothesis
• 1980s : A.a , P.g, P.intermedia , T.forsythus  periodontitis

10. These classic studies provided
the initial impetus to perform
large scale studies attempting to
relate specific microorganisms to
the etiology of periodontal
disease

11. PLAQUE AS A BIOFILM

12. BIOFILM
Biofilm
 Microbial biofilm: a structured community of bacterial cells
enclosed in a self-produced polymeric matrix and adherent
to an inert or living surface.
‘ Matrix enclosed bacterial populations adherent to each other
and to surfaces or interfaces’
Costerton et al,1994

13. • Biofilms are ubiquitous and form on virtually all surfaces immersed in natural aqueous
environment

14. • Biofilms are the preferred method of growth for most bacteria.

15. • Protection from :
- competing microorganisms
- environmental factors
-toxic substances
• Facilitate :
-processing and uptake of nutrients
-cross feeding
-removal of potentially harmful metabolites
-development of appropriate physicochemical
environment

16. PLAQUE
• Dental plaque as a naturally occuring microbial deposit represents a ‘true
biofilm’
 “Highly specific variable structural entity formed by the sequential
colonization of microorganism on tooth surface, epithelium & restorations”.
(WHO)
 “Clinically as a structured resilient, yellow greyish substance that adheres
tenaceously to the intraoral hard surfaces, including removable & fixed
restorations”.

17. COMPOSITION OF DENTAL PLAQUE:
• Composed primarily of microorganisms within an intercellular matrix
• 1g 2 * 10 bacteria
Polysaccharides
Proteins
Organic Glycoproteins
Lipid
Intercellular matrix
Calcium
Inorganic Phosphorus
Na,K,F

18. FORMATION OF DENTAL PLAQUE
• Formation of pellicle on tooth surface
• Initial adhesion & attachment of bacteria
• Colonization & plaque maturation

19. FORMATION OF PELLICLE
 Initial phase of plaque formation
 Forms with in nanosecond - vigorously polished teeth
 Derived from components of saliva + crevicular fluid +bacterial & host tissue cell
products Components-
 Glycoprotein (mucin)
 Proline rich protein
 Enzymes (α-amylase)
 Other molecules

20. • Studies of early (2-hour) enamel pellicle reveal that its amino acid composition differs
from that of saliva, indicating that the pellicle forms by selective adsorption of the
environmental macromolecules(Scannapieco1990).
• The mechanisms include
• Electrostatic,
• Van der Waals, and
• Hydrophobic forces.

21.  hydroxyapatite surface - negatively charged phosphate groups
 Interact with positively charged components of salivary and crevicular fluid
macromolecules.
 Pellicles function as-
 a protective barrier,
 providing lubrication
 preventing tissue desiccation.
 provide a substrate to which bacteria in the environment attach.

22.  The specific component of pellicle depend on underlying surface.
 Thus the characteristic of the underlying hard surface are transferred through the
pellicle layers and influence initial bacterial adhesion.(Pratt et al 1991)
 Absolom et al (1987) even observed a clear relationship between the type of
proteins adsorbed in the pellicle & the free energy of the substratum surface.

23. 2]Initial adhesion and attachment of bacteria:
Phase 1: Transport to the surface
-Brownian motion
-Sedimentation of bacteria
-Liquid flow
-Active bacterial movement

24. Phase 2: Initial adhesion
• Initial adhesion- reversible.
• Initiated by the interaction between the bacterium and the surface.
• Long-range (>50nm):non-specific interaction : Van der Waals
attractive forces.
• Shorter-range (10-20nm) interactions: Van der Waals attraction
forces + electrostatic repulsion →weak area of attraction
→reversible adhesion

25. PHASE 3: ATTACHMENT
• After initial, a firm anchorage -specific interactions (covalent, ionic or hydrogen
bonding)
• This follows direct contact or bridging- true extracellular filamentous appendages
(10nm)
• On a rough surface bacteria are better protected against shear forces so that a
change from reversible to irreversible bonding occurs more easily & more frequently.

26. SECONDARY COLONIZATION & PLAQUE
MATURATION
 Secondary colonizers
 Prevotella intermedia,
 Prevotella loescheii,
 Capnocytophaga spp.,
 Fusobacterium nucleatum, and
 Porphyromonas gingivalis.
adhere to cells of bacteria already in the plaque
COAGGREGATION

27.  Highly specific stereochemical interaction -protein and carbohydrate molecules
located on the bacterial cell surfaces
 Eg
 Fusobacteria coaggregate with all other human bacteria.
 Veillonellae, capnocytophagae & prevotella bind to streptococci & actinomycetes.
(Kolenbrader et al1995)

28.  Most coaggregation
 Mediated by lectinlike adhesins
 Inhibited by lactose & other galactosides.
 Well characterized interaction include the coaggregation of–
 Fusobacterium nucleatum with S.sanguis,
 Prevotella loescheii with A. viscosus
 Capnocytophaga ochraceus with A. viscosus
 Streptococci show intrageneric coaggregationbind to the nascent monolayer of
already bound streptococci.

29. CORN COB TEST TUBE BRUSH

30. • Sequential colonization :‘bridging species’.
• Thus as plaque matures an ecologic shift occurs in the biofilm from the
early aerobic environment characterized by gram positive facultative
species to a highly oxygen deprived environment where gram negative
anaerobic organisms predominate

31. CLASSIFICATION OF PLAQUE
SUPRAGINGIVAL SUBGINGIVAL

32. Supragingival
 Direct contact with the gingival margin is referred to as marginal plaque.
 Demonstrates stratified organisation of bacteria.
 Gram +ve cocci & short rods predominate at the tooth surface.
 Gram -ve rods & filaments as well as spirochetes - outer surface of mature plaque
mass.

34. Subgingival:
• Formed below the gingival margin,
• Present between the tooth & sulcular tissue
• Subgingival microbiota differ in composition: Local availablity of blood product
Low redox potential  anaerobic environment.

35. • Subgingival plaque can be divided in to
• Attached/tooth associated
• Unattached plaque
• Epithelial associated
• Within CT
• On bone surface

36. PLAQUE HYPOTHESIS
NON SPECIFIC PLAQUE HYPOTHESIS:
• In 1900s Any accumulation of microorganisms at or below the gingival margin would produce
irritants leading to inflammation
• Periodontal disease results from the ‘elaboration of noxious products by the entire plaque flora’.
When only small amount of plaque  the noxious product neutralized by
host.
Similarly large amounts of plaque noxious product overwhelm the host
defense.
Limitations
• Some individuals with considerable amount of plaque & gingivitis never developed destructive
periodontitis.
• Site specificity in the pattern of disease

37. SPECIFIC PLAQUE HYPOTHESIS
[WALTER LOESCHE 1979]
• Only certain plaque is pathogenic & its pathogenicity depends on the presence of or
increase in specific microorganism.
• This concept predicts that plaque harboring specific bacterial pathogens results in a
periodontal disease because these organism produce substances that mediate the
destruction of host tissue.
• Acceptance of this hypothesis was spurred by recognition of A.a as a pathogen in
localized aggressive periodontitis.

38. ECOLOGICAL PLAQUE HYPOTHESIS [MARSH 1991]
• A change in a key environmental factor will trigger a shift in the balance of the resident
microflora & this might predispose a site to disease.
Increased Increased High GCF flow, bleeding
plaque inflammation temp
Overgrowth of gram –ve
anaerobes
• Microbial specificity in disease would be due to the fact that only certain species are
competitive under the new environmental condition
ECOLOGICAL
SHIFT

40. Special Bacterial Behaviour in a biofilm:
1)Antibiotic resistance:
a] Slower rate of growth within the biofilm
b] Matrix resists diffusion of antibiotics
acts as an ion exchange resin
c] Beta lactamase,formaldehyde lyase & formaldehyde dehydrogenase may
become conc.
d] Super resistant bacteria with ‘multi drug efflux pumps’
3)High density of cells facilitate exchange of genetic information:
-conjugation
-transformation
-plasmid transfer
-transposon transfer

41. • When within a biofilm bacteria exhibit both favourable & antagonistic relations with the other species
Favourable interactions:
- provides of growth factors
-facilitates attachment
Antagonistic interactions:
-enzymes which inhibit binding
-factors that kill other species

42. QUORUM SENSING
• Quorum sensing in bacteria "involves the regulation of expression of specific
genes through the accumulation of signaling compounds that mediate inter
cellular communication” (Prosser 1999).
• Quorum sensing - dependent on cell density
• Few cells: signaling compounds at low levels
• Auto induction ↑concentration
• Once the signaling compounds reach threshold level (quorum cell density),
gene expression is activated.

43. FACTORS AFFECTING THE COMPOSITION
OF BIOFILM
• Disease status- ↑red and orange complex
• Pocket depth: red and orange complex↑ with ↑ pocket depth
• Transmission:
Two types of transmission are recognized:
• "vertical“- that is transmission from parent to offspring, "horizontal”- passage of an
organism between individuals outside the parent—offspring relationship.
• Acquisition of new strains of pathogenic species can occur at both young and older ages.
If the newly acquired strain is more virulent than the pre-existing strain of that species,
then a change in disease pattern could occur

44. • Host susceptibility
HIV infection and diabetes
• Studies such as these suggest that altered host susceptibility → change the rate of
disease progression(periodontal pathogens –same)
Smoking
• ↑red and orange complex
• Tobacco- diminish the local and systemic immune response

45. PERIODONTAL MICROBES

46. ACQUISITION OF MICROFLORA
• Mouth of fetus- sterile.
• No. of organisms
rapidly increase
following 6- 10 hrs
after birth.
• Anaerobic bacteria -
2nd day
Birth
• >2 yrs complex
microflora is formed
• More than 400
different types of
bacteria
Infancy and
early childhood • Greatest increase with
the eruption of
permanent teeth.
• Bacteria occupy
several niches
Adolescence

47. CLASSIFICATION OF ORAL MICROBES.
bn

49. GRAM + VE GRAM - VE
PROKARYOTES FACULTATIVE ANAEROBES OBLIGATE ANAEROBES FACULTATIVE ANAEROBES OBLIGATE ANAEROBES
COCCI
STREPTOCOCCUS
S.MUTANS
S.SANGUIS
S.ORALIS
S.MITIS
S.INTERMEDIUS
PEPTOSTERPTOCOCCUS NISSERRIA VEILLONELLA
V.PARVULA
RODS
ACTINOMYCES
A.NAESLUNDI
A.VISCOSUS
A.ISRAELLI
A.ODONTOLUTICUS
LACTOBACILLUS
L.ORIS
L.ACIDOPHILLUS
L.SALIVARIUS
L.BUCCALIS
EUBACTERIUM
ACTINOBACILLUS :A.a
EIKENELLA:E.corrodens
CAPNOCYTOPHAGA
CAMPHYLOBACTER
PORPHYROMONAS: P.g
PREVOTELLA :P.i
BACTEROIDES : T.f
FUSOBATERIUM : F.n
SELENOMONAS
SPIROCHETES &
MYCOPLASM
MYCOPLASM
SPIROCHETES OF ANUG
T.Denticola
T.Socranskii
EUKARYOTES CANDIDA
CANDIDA ALBICANS
ETNAMOEBA TRICHOMONAS

50. CURRENT CONCEPT OF THE ETIOLOGY OF
PERIODONTITIS:
SUSCEPTIBLE PRESENCE OF
HOST PATHOGEN
ABSENCE OF
BENEFICIAL Spp.

51. ROLE OF BENEFICIAL SPECIES:
• Occupies the niche
• Limits pathogens’ ability to adhere
• Affects vitality & growth of pathogen
• Affects ability of pathogen to produce virulence factors
• Degrades virulence factors produced by pathogens

52. CURRENT SUSPECTED
PATHOGENS OF
DESTRUCTIVE PERIODONTAL
DISEASE.

54. DEFINING PERIODONTAL
PATHOGENS…
• Periodontal disease occurs in area normally inhabited by many bacteria,
hence – difficult to identify specific microbes.
• KOCH’S POSTULATES 1870
1. The causative agent must be routinely isolated from diseased individuals
2. Be grown in pure cultures in lab.
3. Must produce a similar disease when inoculated in susceptible lab animal.
4. Be recovered from lesions in a diseased lab animal.

55. PROBLEMS IN APPLYING THESE
POSTULATES IN PERIODONTAL
DISEASES…
i. In ability to culture all the micro-organisms that have been associated with the
disease.(eg: oral spirochetes).
ii. Difficulty in defining and culturing the sites of active disease.
iii. Lack of animal model system to study periodontitis.

56. SOCRANSKY (1977) PROPOSED AN ALTERNATIVE CRITERIA
TO IDENTIFY KEY ORGANISMS IN PERIODONTAL
INFECTIONS…
i. Organism must be associated with disease as evident by increase in no. of organism at
disease sites.
ii. Must be eliminated or decreased in sites that demonstrate clinical resolution of disease
with treatment.
iii. Must demonstrate a host response in form of an alteration in the host cellular and
humoral immune response.
iv. Must be capable of causing disease in experimental animal model
v. Must demonstrate virulence factors responsible for enabling the micro organism to cause
destructive periodontal disease.

57. SOCRANSKY ET AL 1987 REVIEWED REASONS
FOR UNCERTAINITY ON DEFINING
PERIODONTAL PATHOGENS…1. Over 500 spp may be cultured from periodontal pockets of different individuals
and one may recover 30-100 spp from a single site.
2. Many spp- difficult to grow and identify.
3. Physical contraints of pocket makes it diffcult to collect a representative sample.
4. Site within a subject do not appear to be progressing equally at all times- time of
sampling may play a critical role.

58. 5. there appears to be multiple destructive periodontal disease that mostly cannot be
differentiated on a clinical basis.
6. opportunistic spp my grow as a result of disease rather than as the cause.
7.some of the infections are mixed- difficult to evaluate the role of single spp.
8.strains of putative pathogens may differ from virulence.
9.more virulent strains of a spp may harbor plasmids that might confer virulence
properties.

59. HILL’S POSTULATES
• Given current obstacles etiology of periodontitis – pathogenic microbial
cummunity concept +hills criteria of causality

60. MICROBIAL COMPLEXES
• Socransky et al 1998 examined over 13000 sub gingival plaque samples from 185 adults and
recognized 6 closely associated groups of bacterial spp using DNA hybridization technique.

61. • Kolenbrander et al 2006 cell to cell recognition in oral bacteria is not random and
each strain has a defined set of partners.
• Functionally similar adhesins found on bacteria of different genera may recognise
the same receptors on other bacterial cells helping in coaggregation.

62. DESCRIPTION OF MICRO
ORGANISMS

63. AGGREGATIBACTER
ACTINOMYCETEMCOMITANS
 Actinobacillus actinomycetemcomitans.
• Klinger (1912) first isolated from cervicofacial actinomycosis lesions.
• Henrich and Pulverer(1959) were the first to demonstrate that A. a was
part of the normal oral flora and indicated that it could colonize teeth,
mucosa and the oropharyx.
• With respect to periodontal disease, A. a was first implicated as the cause
of juvenile periodontitis in 1976 by Newman et al. and by Slots .

64.  Isolated together with A.israelii, hence the name
which means ‘together with actinomyces’.
 Gram –ve, small, non-motile, saccharolytic,
capnophilic, round-ended rod .
 Culture condition & identification: grows as white,
translucent, smooth, non hemolytic colony on
blood agar with star shaped internal
structure(Actino=star)

65. • The primary oral ecological niche for Aa is dental plaque.
• High levels- in pockets
• Low levels- in other oral surfaces, tongue, saliva
• It has not been cultured from edentulous mouths
• It does not belong to indigenous microbiota of any other body site.
• Can cause non oral infections such as endocarditis, abscess in lungs, head & neck and
abdominal areas.
• Distribution pattern- generally isolated areas, as IgG response to Aa is protective and limit
infection.

66. • Conventional srp is not effective in eliminating Aa due to ability of Aa to invade the
gingival c.t. (christerson et al)
• Hence effective therapy involves antibiotics alone or in combination with surgery (zambon
et al 1986)

67. Adhesin- Aae
binds to CHO
receptor on buccal
epithelial cell
Moves to
supragingival
plaque by Flp 1
Fimbrae along with
CHO polymer PGA
mediate attachment
to hard surface
Moves to sub
gingival plaque
From here may
attach to & invade
epithelial lining of
pocket & enter c.t
Finally may leave
oral cavity &
contribute to or
cause endocarditis

68. SEROTYPES• 6 serotypes- a,b,c,d,e and f.
• Most subjects infected with only one serotype
• Serotype ‘a’ and ‘b’ are most common in oral cavity while serotype ‘c’ is important in
extra oral infections.
• While serotype ‘b’ was more common in LAP individuals serotype ‘a’ was common in
chronic periodontitis.
• Intrafamilial transmission- family members seem to be infected by the same serotype

69. TRANSMISSION OF A.A
• Vertical or horizontal
• Route of transmission: Salivary and mucosal contact or on inanimate object.

71. VIRULENCE FACTORS PRODUCED BY
A.A…
• Can be classified as_
i. Factors that assist in colonizing the dental plaque and gingival sulcus-bacterial capsule
and fimbrae
ii. Factors helping it to evade host defence mechanism-leukotoxin
iii. Factors causing tissue destruction-LPS endotoxin(bone resorption), Collagenase(
connective tissue breakdown) Reduction in collagen density

72. VESICLES
• Blebs-LPS in nature
• Originate & continuous with outer membrane.
• Vesicles function as delivery vehicles for A.a toxic materials
• vesicles

73. ADHESINS
 Among adherence factors there are bacterial capsules and fimbriae .
FIMBRIAE
• Associated with adhesion
 Non fimbriated also exhibit adhesive properties
 Anaerobically grown Aa produce more fimbriae than the Aa grown in
aerobic environment. (scannoapieco et al)

74. SUSTENINS
BACTERIOCINS
• Proteins produced by bacteria…..lethal to other bacteria
• Active against S.sanguis/A.viscosus
• MOA : permeability of cell membrane of target bacteria
• Leakage of DNA/RNA/macromolecules essential for growth

75. EVASINS
CYTOTOXINS
• (-) fibroblast proliferation
• Leukotoxin : heat labile factor, that can destroy PMNs
• Aa strains from LAP pts exhibited higher leukotoxin production than from
healthy subjects
• More common in young patients

76. LEUKOTOXIN
Species specific / cell specific
Binds to neutrophils/monocytes/lymphocytes
Pores in the membrane of target cells
Hampers the ability of cell to sustain osmotic
homeostasis

77. IMMUNOSUPPRESSIVE FACTORS
• A.a produces protein
• Inhibits DNA/RNA synthesis in T cells
• Inhibits IgG & IgM synthesis by lymphocytes

78. RED COMPLEX BACTERIA
T . Denticola
T. Forsythia
P.Gingivalis

79. PORPHYROMONAS GINGIVALIS
• Gram-negative, anaerobic, non-motile, asaccharolytic rods - member of the much
investigated "black-pigmented Bacteroides" group.
• Initially grouped into a single species, B. melaninogenicus.
• At present only known porphyromonas spp isolated from humans that produce
phenylacetic acid as metabolic end product.
• Non-oral infections- endocarditis

80. • Habitat :
• Mouth with poor oral hygiene preferred over clean tooth surface, need G+ve bacteria.
• Not seen in edentulous mouth
• Levels increase with age so infrequent in children, acquired in later life
Association:
• Commonly encountered in sites with destructive periodontal disease
• Refractory sites (haffajee et al, 1988)
• Advanced periodontal disease (Slots 1977)
• Highly inflamed gingivitis lesions (mayrand 1981)
• Acute periodontal abscess (newman and sims 1979)
• Able to invade human gingival epithelial cells (duncan et al 1993) -found in higher number in epithelial cells
recovered from periodontal pocket (dzink et al, 1989)

81. VIRULENCE FACTORS
1. Capsule: polysaccharide capsule helps in protection against phagocytosis.
2. Fimbriae: helps in binding to host cells and saliva coated tooth
3. Proteinases: a large no. of hemolytic, proteolytic and lipolytic substances are produced.
• 4 proteolytic substances are recognized.
• Serine
• Aspartate
• Thiol
• Metalloprotienases
• Out of these collegenase, aminopeptidase, trypsin like protease are critical.

82. Cysteine proteinases (gingipains)
• Cleave polypeptides after arginine and lysine residue and classified as
• Arginine (Arg-) specific proteinases
• Lysine (Lys-) specific proteinases
Effects:
• ↑ vascular permeability ---↑ GCF
• Chemotactic for PMNL so ↑ conc.of PMNL at sites of tissue destruction
• Arg- gingipain distrupts oxidative burst mechanism of PMNLs.
• Only known prokaryote to inhibit this PMN function.

83. PATHOGENICITY OF P.GINGIVALIS
ADHESION & COAGGREGATION
• 1st step
• Fimbriae(major adhesive determinant)
• Proteases
• Hemagglutinins
• LPS
• Arg – gingipain also helps in binding of fimbriae to host cells
• Capable of coaggregating- strept. Spp & actinomyces spp.
• Vesicles- bridges non aggregating spp like S.aureus, certain types of candida

84. SUSTENINS• Sustenance of spp is crucial for its survival
• Biomolecules released to physiologic requirements of these bacteria
• Critical virulent factors
• Proteases- nutrition for bacterial growth in confines of their ecological environ.
• Important for survival of bacterias and collateral damage to host.

85. • Gingipains-originally considered as trypsin like protease
• Comprises of a group of cysteine endopeptidases that account for 85% of
proteolytic activity of P. gingivalis.
• Soluble and cell associated and product of 3 genes rpg A, rpg B, kpg encoding
these proteinases.

86. END PRODUCTS OF METABOLISM
• P.g utilizes peptides
• Produce ammonia & organic acids
• Used by other members in subgingival biofilm
P.g peptidylarginine deiminase
NH4
• –ve impact on neutrophil function
• Pathogen growth

87. • P.g- H2S, CH3SH
• Halitosis
• Toxic to host cells
• Degrade disulfide bonds

88. EVASINS
• P.g alters the secretion & accumulation of selected chemokines
• Interefers with MCP-1
• IL-8 expression
• chemokine level : Ability of host to recognise bacterial species
• Ability of PMNs to remove bacterial cell
• Destruction of neutrophils before it can act-----this provides the bacteria with
proteolytic nutrients for metabolism & growth
• Attack & degrade C3a & C3b

89. • Directly activates C5  C5a
• P.g forms small outer membrane vesicles that contains bacterial proteases
• Degrade host C5a receptors that are present on the surface of attacking neutrophils
• Neutrophil loses its phagocytic function
• PMNs death & degranulation
• Release PMNs associated hydrolytic enzymes
• Further affect host cell & tissue destruction

90. • Gingipain R from P.g
• Cleaves CD14
• LPS induced IL-8 secretion from gingival fibroblast
• Immune evasion by

91. • P.g secretes heat stable molecule ( Geatch et al )
• Inhibit host cell growth / proliferation
• Promote apoptosis (lymphocytes)
• P.g metabolism
• Butyric acid
• Inhibit Immune cell function &Lymphocyte proliferation

92. • LPS of P.g causes increase in IL1,TNFα, PGE2 capable of activating osteoclasts.
• Putrify salivary glycoproteins
• Undermining their function in maintaining homeostasis
Effects on blood factors , coagulation and clotting
• Gingipain –activates blood clotting pathways
• PMNs accumulation in actively resorbing periodontitis site

93. • LPS thrombin & IL-I in GCF tissue destruction
• Gingipain R activates factor IX, X, prothrombin
• Gingipain degrade fibrinogen very rapidly (Pike et al)
• Results non clotting host condition
• Active against fibrin gel

94. OTHER VIRULENCE FACTORS….
• Various other factors are collagenases, endotoxins, trypsin like enzyme,
fibrinolysin, proteases that destroys Ig, fatty acids, NH3, hydrogen
sulphide.
• Induces elevated systemic and local immune response in periodontitis
subjects.not seen in normal individual

95. TANERELLA FORSYTHUS
• Previously known as B. forsythus
• First described in 1979 (Tanner et al. 1979) as a "fusiform" Bacteroides at The Forsyth Institute.
• Gram-negative, anaerobic, spindle-shaped, highly pleomorphic rod.
• Difficult to grow, often requiring 7–14 days for minute colonies to develop.
• The growth of the organism was shown to be enhanced by co-cultivation with F. nucleatum and
commonly occurs with this species in subgingival sites (Socransky et al. 1988).
• The species was shown to have an unusual requirement for N-acetylmuramic acid (Wyss 1989).

97. ASSOCIATION:
• Found in higher numbers
• in sites of destructive periodontal disease or
• periodontal abscesses than in gingivitis or healthy sites (Lai et al. 1987).
• in active periodontal lesions than inactive lesions (Dzink et al. 1988)
• Refractory periodontitis cases. (Listgarten et al,1993).
• Subjects who harbored T. forsythus were at greater risk for alveolar bone loss, attachment
loss and tooth loss (Machtei et al. 1999

98. • Studies using checkerboard DNA–DNA hybridization techniques to examine subgingival
plaque samples demonstrated that T. forsythus was the most common species detected from
periodontal pockets (Dibart et al. 1998).
• Listgarten et al 1993 found that T. forsythia was the spp most frequently detected in refractory
periodontitis.
• Serum Ab to this organism was often extremely elevated in subset of refractory periodontitis
subjects.

100. S LAYER- MEDIATES HEME AGGLUTINATION, ADHESION/INVASION OF
EPITHELIAL CELLS, MURINE SUBCUTANEOUS ABSCESS FORMATION.

101. ADHESINS
• Coaggregates with F.nucleatum
• Increase colonisation in subgingival biofilms
SUSTENINS
• Produces enzymatic peptidase
• Degrades BANA
• Described as trypsin like proteases : ( - ) by serine protease

102. • Sialidase from T.f
• Cleaves α-ketosidic linkages between sialic acid & glycosyl residues of host
glycoproteins/glycolipids
• Modified / degraded proteoglycans : nutrition for members of oral microbiota

103. EVASINS
• T.f produce lipoproteins
• Activates gingival fibroblasts
• increase IL-6 & TNF-alpha
• Also kappa B production by fb

104. TREPONEMA DENTICOLA
ADHESINS
• Coaggregation b/w P.g & T.d is mediated by fimbriae binding protein-DENTILYSIN
• Helps transport of Pg to deeper regions
• Binds to ECM proteins like laminin fibronetin /heparin
• And host cells like gingival fibroblast
• Collagen binding proteins of T.d binds Type I,IV,V----------adherence /colonozation (Li et al)

105. • Produces major outer sheath proteins
host cells
• lectin like proteins
• Binding activity (-) mannose/galactose residues
• This protein might have porin activity –enables transportation of molecules into /out of host
cells

106. SUSTENINS
• Possess peptidases associated with its outer sheath
• Chymotripsin like protease ( Uilto et al )
• T.denticola + erythrocyte---resulting in cell lysis & competing with host for available hemin derived iron
• Synthesize 2 low iron induced outer membrane proteins : HbpA & HbpB—binds hemin

107. EVASINS
• Chymotrypsin like protease + proteins on outer sheath
• Chymotrypsin like protease complex
• Adhesion
• Degradation of humoral proteins ( basement membrane components)
• Activates MMPs
• Forms vesicles with hyaluronidase

108. Effects on host innate & immune mechanism
• T.denticola on fibroblast
• Cell rounding
• Formation of surface blebs
• Detachment from cell surface (Weinberg & Holt)

109. • Major sheath protein complex of T.d interferes with collagen attachment via β-integrin
• Direct effect on host response
• T.d produces immunosuppressive proteins (Sip)
• Lymphocyte proliferation
• By inducing an arrest at G1 phase in human T-cells
• Irreversible
• Activates apoptotic pathway in these cells
• May be resistant to β-defensins -1 & -2

110. PREVOTELLA INTERMEDIA
• Second black-pigmented Bacteroides to receive considerable interest
• Gram-negative, short, round-ended anaerobic rod
• Association:
• acute necrotizing ulcerative gingivitis ( Loesche et al. 1982),
• certain forms of periodontitis (Tanner et al. 1979, Herrera et al)
• progressing sites in chronic periodontitis (Tanner et al)
• rapidly progressive periodontitis subjects (Hillmann)
• Elevated serum antibodies to this species have been observed in some but not all subjects
with refractory periodontitis (Haffajee et al. 1988b).

111. Elimination:
Berglundh et al. (1998) demonstrated that improved clinical parameters after the use of
mechanical therapy and systemically administered amoxicillin and metronidazole were
associated with a decrease of periodontal pathogens including P. intermedia.
Successful treatment of peri-implantitis with local delivery of tetracycline also significantly
decreased the frequency of detection of P . intermedia (Mombelli et al. 2001)

112. • This species appears to have a number of the virulence properties exhibited
by P . Gingivalis
• shown to induce mixed infections on injection in laboratory animals (Hafstrom
& Dahlen 1997).

113. Pregnancy gingivitis
Physiological level of steroid hormone increases
vascular permeability increases
E/P substitute menadione as a growth factor in P.i
• P. intermedia can utilize albumin, mucin and glucose as sources of energy
• Modify its pathogenic factors (proteolytic activity, depending on the nutrients in the
environment)
• This nutritional flexibility may explain why P.intermedia resides in both subgingival
/supragingival areas

114. SPIROCHETES
• Gram-negative, anaerobic, helical shaped, highly motile microorganisms
• implicated as the likely etiologic agent of acute necrotizing ulcerative gingivitis by
its presence in large numbers in tissue biopsies from affected sites (Listgarten &
Socransky 1964, Listgarten 1965)
Cultural studies suggested that T. denticola and a "large treponeme" were found
more frequently in patients with severe periodontitis than in healthy or gingivitis
sites (Moore et al. 1982).

115. FUSOBACTERIUM NUCLEATUM
• Gram-negative, anaerobic, spindle shaped rod
• Association:
• periodontitis (Papapanou,Socransky)
• periodontal abscesses (Herrera et al. 2000).
• Elimination: Successful treatment of peri-implantitis with local delivery of tetracycline was associated
with a significant reduction in frequency of detection in several species including F. nucleatum
(Mombelli et al. 2001).
• Invasion of this species into human gingival epithelial cells in vitro was accompanied by an increased
secretion of IL-8 from the epithelial cells (Han et al. 2000).

116. CAMPYLOBACTER RECTUS
• Earlier called Wolinella rectus
• Gram-negative, anaerobic, short, motile vibrio.
• Utilizes H2 or formate as its energy source.
• It was first described as a member of the "vibrio corroders", a group of
short nondescript rods that formed small convex, "dry spreading" or
"corroding" (pitting) colonies on blood agar plates.

117. • Association:
• Higher numbers in disease sites as compared with healthy sites (Moore et al. 1983)
• In sites exhibiting active periodontal destruction (Dzink et al. 1985)
• In periodontitis patients with NIDDM.
• Elimination: found in lower numbers
• After successful periodontal therapy (Tanner et al. 1987)
• Treatment of peri-implantitis with local delivery of tetracycline (Mombelli et al.)
• Shown to produce a leukotoxin (Gillespie et al. 1992)
• Capable of stimulating human gingival fibroblasts to produce IL- 6 and IL-8

118. EIKENELLA CORRODENS
• Gram-negative, capnophilic, asaccharolytic, regular, small rod with blunt ends
• Also found in osteomyelitis, RC infections, CNS infections
• Association:
• periodontal destruction as compared with healthy sites (Savitt & Socransky 1984)
• active sites (Dzink et al. 1985, Tanner et al. 1987)
• in sites of subjects who responded poorly to periodonal therapy (Haffajee et al. 1988)
• with Aa in some lesions of LJP (Mandell 1984)
• Stimulate the production of matrix metalloproteinases (Dahan et al. 2001) and IL-6 and IL-8 (Yumoto et al.
1999).

119. PEPTOSTREPTOCOCCUS MICROS
• Gram-positive, anaerobic, small, asaccharolytic coccus.
• associated with mixed anaerobic infections in the oral cavity and other parts of the body (Finegold
1977).
• Two genotypes
• smooth genotype (associated with periodontitis lesions)
• rough genotype
• Association:
• periodontal destruction as compared with gingivitis or healthy sites (Moore et al. 1983)
• actively breaking down sites (Dzink et al. 1988).

120. EUBACTERIUM SPECIES
• G+ve, strictly anaerobic, small, somewhat pleomorphic rods
• Possible periodontal pathogens due to their increased levels in disease sites, particularly those of severe
periodontitis (Moore et al. 1982)
• Difficult to cultivate, particularly on primary isolation, and appear to grow better in roll tubes than on blood
agar plates
• Elicited elevated antibody responses in subjects with different forms of destructive periodontitis (Tew et al.
1985a,b, Vincent et al. 1986, Martin et al. 1988)

121. OTHER SPECIES
• All periodontal pathogens have not yet been identified.
• Particularly in individuals who have responded poorly to periodontal
therapy, there are chances of infection with unusual species.
• Emphasis has been placed on enteric organisms, staphylococcal species

122. MICRO ORGANISM
ASSOCIATED WITH
SPECIFIC PERIODONTAL
DISEASE

123. MICROBIAL SHIFT DURING DISEASE:
HEALTH GINGIVITIS PERIODONTITIS
Gram +ve Gram –ve
Cocci Rods
Non motile Motile
Facultative anaerobes Obligate anaerobes
Fermenting Proteolytic

124. Gingivitis
Actinomyces species
Streptococcus
species
Veillonella species
Fusobacterium
species
Prevotella
intermedia
Health
Streptococcus
sanguis
Streptococcus mitis
Veillonella
parvula
Actinomyeces
naeslundii
Actinomyces
viscosus
Periodontitis
Porphyromonas gingivalis
Tanerella forsythus
Treponema denticola
BACTERIAL SPECIES ASSOCIATED WITH DIFFERENT PERIODONTAL CLINICAL
STATES

125. PREGNANCY GINGIVITIS• Dramatic increase in levels of P.intermedia.
• Other microbes reported : P.melaninogenica, F.nucleatum, T. forsythia.
• Periodontal abscess
• F. nucleatum, P. intermedia, P. gingivalis, P. micros, and B. forsythus

126. CHRONIC PERIODONTITIS• High percentages of anaerobic (90%) gram-negative (75%) bacterial species
• Bacteria most often cultivated at high levels include P. gingivalis, B. forsythus, P. intermedia, C. rectus, Eikenella
corrodens, F. nucleatum, A. actinomycetemcomitans, P. micros, and Treponema and Eubacterium
• C. rectus, P. gingivalis, P. intermedia, f. nucleatum, and B. forsythus were found to be elevated in the active sites
• Recent studies have documented an association between chronic periodontitis and viral microorganisms of the herpes
viruses group, most notably Epstein-Barr Virus-1 (EBV-1) and human cytomegalovirus (HCMV).

127. AGGRESSIVE PERIODONTITIS
• predominantly gram-negative, capnophilic, and anaerobic rods.
• almost all LJP sites harbor A. actinomycetemcomitans, which may comprise as much as 90% of the total
cultivable microbiota.
• Other organisms found in significant levels include P. gingivalis, E. corrodens, C. rectus, E nucleatum, B.
capillus, Eubacterium ,and Capnocytophaga spp. and spirochetes
• Herpes viruses, including EBV-1 and HCMV, also have been associated

128. NECROTIZING PERIODONTAL
DISEASE:
• Microbiologic studies indicate that high levels of P. intertmedia and spirochetes in necrotizing
ulcerative gingivitis lesions.
• Spirochetes are found to penetrate necrotic tissue and apparently unaffected connective
tissue.
• In NUG F. nucleatum is also found in association

129. PERIODONTITIS IN MEDICALLY COMPROMISED
PATIENTS
• HIV PERIODONTITIS
• HIV periodontitis lesions reveal spirochetes, Fusobacterium species, A.a, W.recta, P.micros and P.intermedia each averaging
5-20% of the subgingival microflora.
• Some lesions also yield B.fragilis, F.necrophorum, E.aerofaciens, Clostridium species, enterococci and P.aeuroginosa and
C.albicans.
• DIABETES MELLITUS
• Capnocytophaga species and P. intermedia have been related to the initial breakdown in periodontium.
• W. recta, A.actinomycetemcomitans and P. gingivalis may also play a role

130. PERIODONTAL ENDODONTIC
LESION
• Reflects microbiota of the separate endodontic and periodontal lesions
(Legoff 1997)
• Accordingly it might be impossible to obtain a sample that is
representative of either of the lesion.

131. PERIIMPLANTITIS
• Healthy perimplant pockets : high proportions of coccoid cells, a low ratio of anaerobic/aerobic
species, low detection frequencies for pathogens.
• Around failing implants : A.a., P.gingivalis, T. forsythia, P.micros, C.rectus, Fusobacterium and
Capnocytophaga
• Implant associated with refractory periodontitis : P.aeruginosa, C.albicans, staphylococci aureus

132. PARASITES
Entamoeba gingivalis
• Protozoan & normal commensal
• ↑elderly individuals and in diseased mouths.
• Earlier- critical role
Now- opportunistic pathogen
• Found in soft calculus, periodontal pockets and with diseased tonsils.
Trichomonas tenax
• Only parasitic flagellate found in the oral cavity.
• Numbers are seen to increase in periodontal pockets.

135. VACCINE ???
• Conventional therapy : painful & frequently unsuccessful
• Recurrence of destructive process
• Prevention & control of periodontitis

136. • The three types of vaccine that were employed for the control of
periodontal disease-
1. Vaccines prepared from pure cultures of streptococcus & other oral
micro organism
2. Autogenous vaccines prepared from plaque of patients with
destrucive periodontal disease
3. Stock vaccines such as Van Cott’s vaccine, Goldenberg’s vaccine,
Inava endocorp vaccine-adminstered systemically/locally.

137. • A recent report suggested that the hemagglutinin domain was implicated in the nuclear targeting of this
protease
• Vaccines are mainly peptide & DNA vaccines (Scragg 2002)
• DNA vaccine induces cellular immunity & humoral immunity
• Peptide vaccine induces humoral immunity

138. • Further analysis is required to evaluate at which stage these immune
responses prevent P. gingivalis infection
• Recent data indicate that the gingipains (RgpA and Kgp) of P.
gingivalis are potential candidates for a vaccine

139. CLINICAL IMPLICATIONS• Aid in designing the measure to prevent these diseases
• Determination of risk factors,
• e.g. high levels of p.gingivalis in a smoker
• To monitor and evaluation of treatment outcome
• Declining level of suspected pd pathogen
• To target therapy
• Development of vaccine
• Use of antimicrobials

140. ADVANCES IN MICROBIOLOGY
• DNA based methodology for identification and detection of specific
bacteria and virus offers remarkable advantage
• No. of samples examined and no of micro organisms identified have
increased dramatically
• Also recognition of beneficial activity of several groups such as
probiotics might open new strategies in Pdl therapy.

141. CONCLUSION
• A full understanding of the microbial factors, their
pathogenicity as well as host factors are of the essential importance for
pathogenesis of periodontal disease
• Microbial diagnosis may play a vital role in treating cases of refractory
periodontitis / periodontitis related to specific microorganisms

142. REFERENCES1. Contemporary Oral Microbiology and Immunology – Slots & Taubman.
2. Clinical periodontology and implant dentistry: jan lindhe 4th edition
3. Clinical periodontology: carranza 10th edition
4. Periodontology 2000, vol 38
5. Periodontology 2000, vol 55
6. Text book of Microbiology : Ananth Narayanan, C.K. Jayram Paniker.

143. THANK YOU