AGGREGATIBACTER ACTINOMYCETEMCOMITANS is a bacterium with an array of diverse potential virulence characteristics, including multiple immune evasion mechanisms and novel mechanisms for binding to host matrices and invading host cells, any one of which may play a crucial role in the local tissue pathology of Localized aggressive periodontitis.

1. AGGREGATIBACTER
ACTINOMYCETEMCOMITANS
1
DR.VIDYA VISHNU
SENIOR LECTURER
MALABAR DENTAL COLLEGE AND RESEARCH CENTRE

2. INTRODUCTION
 Periodontal diseases are infections caused by micro-
organisms that colonizes the tooth surface at or below the
gingival margin.
 Above 700 different species are capable of colonizing the
mouth and any individual may typically harbor 150 or more
different species.
2

3.  The oral cavity should be considered as an ‘open growth
system’ with uninterrupted ingestion and removal of
microorganisms and their nutrients.
 Bacteria may attach to tooth surface, epithelium, CT and
microbial surfaces.
 Tooth surface provides stable base for the microbes and
held in immediate proximity to soft tissue of
periodontium thus microbial colonization is facilitated.
3

4. ASSOCIATION OF PLAQUE MICROBES WITH
PERIODONTAL DISEASES
Susceptible host
Presence of
pathogenic species
Absence /small
proportion of
beneficial bacteria
Periodontal disease
4

5. PATHOGENIC BACTERIA
■ Bacteria accumulates on tooth surface as the organized
structure called dental plaque.
■ Broadly the periodontal bacteria are classified into
1. those associated in health.
2. those associated with disease.
5

6. Associated with Disease
▪(mostly Gm +ve, facultative species)
S.sanguis
S. mitis
A.viscosus
A. naselundi
▪(small proportion Gm –ve species)
P.intermedia
F. Nucleatum
Capnocytophaga
Nisseria & Veilleonella
 Beneficial species
Pseudomonas
S. Sanguis
V. Parvula
C. Ochraceus
 A.Actinomycetemcomitans
 P.Gingivalis
 P.intermedia
 T. Forsythia
 Fusobacterium species
 Peptostreptococcus mutans
 Eubacterium species
 C.rectus
 Treponema species
 Enteric rods and
 Candida species
Classification
Micro-organisms
Associated with Health
6

7. HISTORY
 The first isolates were recovered from cervicofacial
actinomycosis, in 1912 by Klinger R.
 Initially known as Bacterium actinomycetemcomitans.
 In 1921, the organism was referred to as Bacterium
comitans by Lieske.
7

8.  King & Tautam (1962) described the close phenotypic
relationship of A.A. with H. aphrophilus .
 A.A. was subsequently reassigned to genus
Hemophilus. (Pott’s et al 1985)
8

9.  First identified as periodontal pathogen in studies of LJP by
Newman et al & Slots et al 1976.
 Baehni et al 1979
important virulence factor- leukotoxin
 Genco et al 1980, Ebersole et al 1987
Elevated serum antibody response to the species
9

10.  Slots & Rosling 1983
Species eliminated in successfully treated sites.
 Preus 1988, Mandell et al 1986
Treatment failures associated with failure to lower the number
of organisms in treated sites.
10

11.  Sreenivasan et al 1993
Invasion of A.a in gingival epithelial cells.
 Bueno et al 1998
subjects harboring JP2 clonal type A .a with 530 bp deletion
in the leukotoxin operon were 22.5 times more likely to
convert to LAP.
11

12.  Scheiken et al 2000
Invasion of A.a in vascular endothelial cells
 Rudney et al 2001
Invasion of A.a in buccal epithelial cells in vivo.
 Named as ‘Aggregatibacter actinomycetemcomitans’ by
Norskov, Lauritsen & Kilian in 2006.
12

13.  The name actinomycetemcomitans arises from its
association with an actinomycete and frequent isolation
from actinomycotic lesions along with Actinomyces
israelii.
 The genus name refers to the star-shaped inner structure
sometimes seen in colonies on selective medium.
actin- star shaped
bacillus- rod shaped
14

14.  A conspicuous growth in broth as small granules
adhering to walls of the test tubes was noted in the
original description of Bacterium
actinomycetemcomitans.
 Accordingly the name Aggregatibacter was proposed
(rod shaped bacterium that aggregates with others).
15

15. Taxonomy of A. actinomycetemcomitans
Scientific classification
Kingdom Bacteria
Kingdom Bacteria
Phylum Proteobacteria
Class Gammaproteobacteria
Order Pasteurellales
Family Pasteurellaceae
Genus Aggregatibacter
Species Actinomycetemcomitans
16

16. MORPHOLOGICAL CHARACTERISTICS
 Small gram-negative rod {0.4–0.5 μm x 1.0–1.5 μm in
size}
 micro aerophillic & facultatively anaerobic
 ▪ Is Capnophillic requiring atmosphere containing 5-10%
CO2 for good growth.
 non sporulating, non-motile, non-hemolytic,
saccharolytic, oxidase & catalase positive.
17

17.  Closely related to members of the Pasteurellaceae
 Microscopically cultures appear predominantly bacillary
with few coccal forms.
 Cells may appear coccobacillary, particularly if the
colonies have been directly isolated from solid
medium.
18

18.  Longer forms may be seen in Gram stains of cultures
older than 3 days, or from cells grown in a glucose-
containing liquid medium.
19

19. ▪ Fermentative ability to utilize galactose, dextrin,
maltose, mannitol and xylose permits the biotyping of
this organisms, and to distinguish from other organisms
of oral cavity. (Zambon JJ 1985)
 Upon primary isolation, colonies are small
approximately 0.5-1.0 mm in diameter, translucent
/transparent with irregular edges & appear smooth,
circular and convex.
20

20.  The colonial morphology of fresh isolates is distinctive
with the internal star-shaped or crossed cigar
morphology form embedding in the agar that gives AA
its name (Zambon JJ 1985).
 Fresh colonial isolates are rough-surfaced.
21

21. ▪ Repeated subculture yields two types of colonial
variants; one is smooth-surfaced and transparent, the
other is smooth-surfaced and opaque.
▪ The colonial variation is associated with fimbriation.
22

22. BIOCHEMICAL CHARACTERISTICS
▪ King & Tatum provided the first detailed biochemical and
serological description of A.A.
▪ A.A. is closely related to H. aprophilus than
Actinobacillus lingeresii.
▪ However unlike the genus Hemophillus that require either
Factor X (hemin), or factor V (Nicotinamide adenin
dinucleotide ), A.A. can grow in absence of these factors.
(Slots J 1982)
Periodontology 2000, Vol. 20, 1999, 14-52
23

23. ▪ AA can be distinguished from H. aprophilus by its ability
to ferment starch, sucrose, trehalose and production of
specific enzymes such as B-glucosidase & B-
galactosidase (Zambon 1985).
24

24. - Decompose H2O2 (catalase +ve)
- Benzidine +ve
- Reduce nitrate to nitrite
- Produce strong alkaline and acid phosphatases
- Ferment fructose, glucose and mannose
25

25. SURFACE ULTRASTRUCTURE OF A.A.
▪ A significant feature of A.A. is its surface ultra
structure
1. Fimbriae
2. Vesicles
3. Extracellular amorphous material
26

26.  The expression of these material appears to be a
function of the strain as well as culture conditions, such
as broth, agar, aerobicity, anaerobicity, or nutrients, and
they helps in functioning of bacteria.
Periodontology 2 000, Vol. 20, 1999, 136-167
27

27. 1. Fimbriae
 It is small filamentous, cell surface appendage
associated with bacterial colonization at host.
 Appears in peritrichous areas; may be more than
2µm in length and 5nm in diameter; often occurs
in bundles.
28

28.  Freshly isolated strains are fimbriated but in vitro
subcultures results in organism to lack fimbriation.
 Fimbriated strains produce colonies with star shape -
star positive.
 Non fimbriated strains -star negative.
29

29. - Number of studies indicate that colonial variation and
fimbriation are associated with adhesion of bacteria.
- The fimbriae associated protein gene is strongly
expressed in fimbriated strain than non-fimbriated
strain.
- Overall these observations demonstrate the positive
correlation between fimbriation and adhesion.
30

30. 2. Vesicles
 Other prominent feature of surface is vesicles (blebs).
 originate from and are continuous with outer membrane
protein.
 Almost all strains of Aa extrude membrane vesicles.
 Growth conditions alter the formation and morphology of
the vesicles.
 Are lipopolysaccharides in nature, Also released in external
environment in large number .
 Highly leukotoxic strains have an abundance of extracellular
membranous vesicles in contrast to non-leukotoxic strains.
(Lai C H 1981)
31

31. - Other biologically active components of vesicles are:
(1) leukotoxin
(2) endotoxin
(2) bone resorption activity &
(3) bacteriocin( actinobacillin)
- It also shows the adhesive properties.
- These observations prompted that vesicles function as
delivery vehicles of A.A.
32

32. 3.Extracellular amorphous material (EAM)
- Associated with the surface of certain AAC cells is an
amorphous material that frequently embeds adjacent cells
in a matrix.
- Some studies demonstrate that cells grown in liquid
medium lacked this material.
- In contrast others did observe this material on cells grown
in liquid culture, but not on all strains.
33

33.  Production in part may be associated with growth in
‘tryptone’- based medium.
- The material is protein, most likely glycoprotein & has
shown to exhibits both bone-resorbing activity and
adhesive properties
- Can be removed by washing the cells with phosphate
buffered saline.
34

34. - Bacteria from which the amorphous material has been
removed exhibit reduced adhesion to epithelial cells.
- A.A. strains, which normally exhibit low levels of
adhesion, exhibit increased levels of adhesion when
suspended in extracellular amorphous material, a
phenomenon termed conveyed adhesion.
35

35. CLASSIFICATION
• King & Tatum 1962 :
Distinguished 3 serogroups, from non-oral A.
actinomycetemcomitans based on a heat stable
component.
Pulverer & KO 1972 :
Using tube agglutination assays, detected 24 groups
within A. actinomycetemcomitans.
36

36.  Three serotypes, a, b and c, were defined by Zambon et
al. (1981).
 Taichman et al. (1982) described four serogroups based
on surface antigens and proteinaceous leukotoxin.
37

37.  The no of serotypes in A.a was later extended to:-
 5 [Mombelli & Van Winkelhoff 1999]
 to total 6 [Kilian et al 2006]- a, b, c, d, e, f
38

38.  According to serological specificity defined by
structurally & antigenically distinct O-polysaccharide
components of their respective lipopolysaccharide
molecules the organism was classified into six
serotypes a – f .
(Page et al.1991; Perry et al.1996, Kaplan et al.2001)
39

39.  Serotypes a and b were most common in the oral
cavity, serotype c constituted 10% of the human oral
isolates.
▪ The most frequently associated serotype with LAP –
serotype b.
40

40. More commonly detected in adult periodontal diseases
samples – serotype a.
▪ More common in periodontal health – serotype c
41

41. Organism can be grouped into three major
phylogenetic lineages comprising
 (i) serotype b strains,
 (ii) serotype c strains, and
 (iii) serotype a, d, e and f strains
42

42.  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.a serotypes appears to vary
according to the person’s geographical location and
their ethnicity.
 Serotype alone does not appear to be a direct
indicator of strain virulence.
43

43.  Associations between a single serotype b clonal lineage
(JP2 clone) and the aggressive form of localized
periodontitis in adolescents have been the focus of
much investigation.
 The JP2 clone of A.a has a 530 bp deletion in the
leukotoxin promoter, which results in significant
enhancement of leukotoxin production &
alteration of iron acquisition mechanisms.
44

44.  The JP2 clone shows a limited geographical &
ethnic host range, predominating in subjects with
an African lineage but absent from non- African
populations from Northern Europe.
 It has been proposed that localized aggressive
periodontitis actually comprises two different diseases.
45

45.  In Northern European Caucasian populations, LAP is
associated with a non-JP2 oligoclonal population of
bacteria, resulting from indigenous A. a acting as an
opportunistic pathogen, whereas in adolescents
descended from North or West African populations, the
JP2 clone has been suggested to be the main cause of
LAP.
 This interpretation has been challenged by Kaplan et al.
46

46.  Subjects that harboured the JP2 clone alone had a
greater relative risk of CAL compared to subjects that
harboured either the JP2 clone in combination with a
non-JP2 clone, harboured non-JP2 clones only, or who
did not harbour A. a.
 The pathogenicity of the JP2 clone may be
moderated in the presence of other bacteria.
47

47.  These findings suggest that the presence or absence
of A. a may not be the only indicator of disease risk,
& that combinations of bacteria may also affect
the clinical severity of periodontal diseases.
 Virulence phenotype of A. a is enhanced by growth in
the presence of an oral streptococcus.
48

48.  A.A. is unrelated to H. Influenza, the type specific
genus of hemophilus and reclassification of A.A. is not
favored by ICSB Subcommittee on Pasteurellaceae and
related organisms (Frederiksen 1987).
 Accordingly Norskov-Lauritsen & M. Kilian 2006
proposed Aggregatibacter as the generic name for the
group.
49

49. The species coming under this genus are
1. Aggregatibacter actinomycetemcomitans
Basonym: Actinobacillus actinomycetemcomitans
(Klinger 1912) Topley and Wilson 1929.
50

50. 2. Aggregatibacter aphrophilus
Basonym: Haemophilus aphrophilus Khairat 1940
3. Aggregatibacter segnis
Basonym: Haemophilus segnis Kilian 1977
51

51. HOW IS AAC IMPLICATED IN DISEASE?
Oral infections
 A.A. is an important pathogen in severe and recurrent
forms of periodontitis.
 The prevalence is nearly 90% in LAP & 30-50% in
severe adult periodontitis .
Periodontology 2000, Vol. 20, 1999, 136-167
52

52. Localized Aggressive Periodontitis
 Microbiologic, clinical and immunological evidence as
described below have been used to implicate this as causative
agent:
 Association:- Large numbers of AAC are routinely isolated
from localized juvenile periodontitis lesions, whereas the
isolation of the bacterium from healthy sites is low.
 Elimination:- The eradication of AAC from diseased sites is
usually correlated with recovery from clinical symptoms of
disease.
53

53.  Host response:- The presence of large numbers of A.A. in the
periodontal pocket is correlated with a significant humoural
immune response.
 Virulence Factors:- AAC produces a wide array of potent cell
bound and secreted virulence factors that have been implicated
in the pathogenesis of disease.
 Animal studies:- Induced disease in animal models like rats &
mice.
54

54. Rapidly progressing periodontitis
 Severe and rapid loss of the bone is the hallmark of RPP,
affecting 25 to 30yr age group adults.
 The isolation of A. actinomyceterncomitans, P gingivalis,
B. capillus, P. intermedia, E. corrodens and
Campylobacter rectus from disease sites has been reported
either singly or in combination.
 Also has been associated with periodontal lesions that are
refractory to standard therapy.
55

55. Extraoral infections
 It has been reported to cause serious infections at several sites
outside the mouth.
 They have been reported from brain, meninges, septicemia,
urinary tract infections, vertebral osteomyelitis and abscess of
the abdomen, brain, face, hand, and thyroid gland.
56

56.  Among these the endocarditis and soft tissue abscesses
remain the most common infections, with the latter
syndrome generally occurring in association with
Actinomyces.
 AA-mediated endocarditis is a chronic syndrome with a
sub-acute course including fever, chills, anorexia, weight
loss, heart murmur and night sweats (Kaplan 1989).
57

57.  Patients usually present clinical symptoms of
hepatosplenomegaly and peripheral evidence of
endocarditis.
 Laboratory investigation usually reveals anaemia, elevated
ESR and microscopic hematuria.
58

58.  Combination therapy using an aminoglycoside along
with ampicillin has been most often used to treat
endocarditis.
 Frequently identified complications of A.
actinomycetemcomitans mediated endocarditis include
embolism and congestive heart failure
59

59.  Three lines of evidence have led to conclusions that
associate and implicate A.A. in oral and systemic diseases:
1 Clinical and microbiological data linking A.A. to the
initiation, progression and recurrence of disease in
localized aggressive periodontitis. (Zambon et al 1985).
61

60. 2 Genetic and experimental data linking reputed virulence factors
possessed by A.A. to pathogenic events known to occur in LAP.
(Fives-Taylor PM, Meyer DH, Mintz KP, Brissette C. 1999)
Periodontology 2000, Vol. 42, 2006, 114–157
62

61. 3 Reports indicating that organisms of the HACEK group
(Haemophilus sp., A. A., Cardiobacterium hominis,
Eikenella corrodens and Kingella kingae), and A.A, in
particular, are associated with systemic diseases distant from
the oral cavity.
(Das M, Badley AD, Cockerill FR, Steckelberg
JM, Wilson WR, 1997)
63

62.  Contamination
- first step
 Incubation
- time required to reach the appropriate concentration
-minimal concentration required for disease by A.A.
1 x 106 per ml.
 Prodromal period
- subclinical infection, only detected by sophisticated tools.
64

63.  Period of disease
- frank tissue-destructive activity takes place & the host is
compromised
 Recuperation period
- In the best-case scenario, recuperation ends the cycle of
disease activity and indicates that the host has successfully
mounted an authentic immunological response.
65

64.  A.a is also associated with periodontitis lesions of
Papillon-Lefevre syndrome patients; virally mediated
host defense impairment may cause overgrowth of
subgingival A.a.
66

65. Modes of Transmission
Vertical transmission:
 With the exception of one study (Tinoco et al in 1998)
in developing countries, the result agree that if children
harbour A.A. the parent usually harbor the same species.
67

66. Horizontal Transmission
 Horizontal transmission may occur between siblings or
between spouses.
 Siblings can harbor the similar strains of A.A. in their oral
cavities. (DiRenzo et al in 1994, Tinoco et al in 1998)
 Transmission of A.A is rather uncommon in between
spouses in regard to their intimate cohabitance for several
years.
68

67. Saliva is a likely transport vehicle for transmitting strains of
A.A.
 Subgingival and salivary strains within the same individual
were of the same serotype and same genotype.
(Asikainen et al 1997)
 These results suggest that saliva can be used as
representative sampling material in searching for A.A. in
transmission studies.
69

68. VIRULENCE FACTORS OF A.A.
 Virulence factors are attributes of a microorganism that
enable it to colonize a particular environment in its host,
overcome the host defenses and initiate a disease process.
 To act as a pathogen bacteria must colonize appropriate host
tissue & cause destruction.
70

69.  Process of tissue destruction results from interaction of
bacteria with host cells.
 Organisms with many virulence factors are more
pathogenic.
71

70. Virulence factors of A.A
Factors that promote colonization & persistence in the oral cavity
•Adhesins
•Invasins
•Bacteriocins
•Antibiotic resistance
Factors that interfere with host’s defenses
•Leukotoxin
•Chemotactic inhibitors
•Immunosuppressive proteins
•Fc-binding proteins
Factors that destroy host tissues
•Cytotoxins
•Collagenase
•Bone resorption agents
•Stimulators of inflammatory mediators
Factors that inhibit host repair of tissues
•Inhibitors of fibroblast proliferation
•Inhibitors of bone formation
72

71. Polysaccharide
and
lipopolysacchar
ide
Leukotoxin
Extracellula
r proteolytic
enzymes
GroEL heat
shock
protein
Fimbriae
Capsule
Lipopolysacc
haride
Fimbriae
Extracellul
ar
proteolytic
enzymes
Heat shok
protein
Haemaggluti
nins
Antigenic
outer
membrane
proteins
A ACOMITANS P GINGIVALIS
73

72. ADHESION OF A. A
 The first challenge any microorganism faces is the ability to
adhere tightly to a specific substrate. Without adhesion, the
organism is quickly cleared from the surface.
 The oral cavity poses distinct problems for microbes, as
eating, drinking, talking and salivary flow induce many shear
forces that remove microorganisms not firmly attached.
74

73.  The bacterial surface components involved in adhesion are
called Adhesins.
 Proteinaceous structures found on the surface of the
bacterial cell.
75

74.  They interact and bind to very specific receptors in
saliva, on the surface of the tooth, on extracellular matrix
proteins and on epithelial cells.
 Attachment is favored by the surface structure -fimbriae,
extracellular amorphous material and extracellular
vesicles.
76

75. ATTACHMENT TO EPITHELIAL CELLS
 Most A.A. strains adhere strongly to epithelial cells.
 Binding occurs very rapidly, reaching saturation levels
within 1 hour after infection.
 Fimbriae most probably function in adherence of rough
variants, whereas non fimbrial components like vesicle are
probably involved in adherence of smooth, highly invasive
strains.
77

76.  Trypsin and protease reduces ability to adhere, suggesting
some adhesins in the outer membrane are protein in
nature.
 AA also produces poly-N-acetylglucosamine (PGA), a
surface polysaccharide that mediates intercellular
adhesion, biofilm formation & detergent resistance.
78

77.  Other components for adhesion are ‘extracellular
amorphous protein’ and ‘surface vesicles’.
 Extracellular amorphous material has both a protein and
carbohydrate moiety.
79

78. Attachment to Extracellular Matrix Proteins
 In order to initiate disease in extraoral sites, AA must
bind to the extracellular matrix, the complex network of
proteins & polysaccharides that is secreted by and
underlies epithelial and endothelial cells and surrounds
connective tissue.
 The major component of ECM is collagen.
80

79.  Collagen types I, II, III, V and XI are predominant in CT.
 Type IV is the major component of the basement membrane.
 Less abundant, but equally important, are the
noncollagenous glycosylated proteins, fibronectin and
laminin.
81

80.  AA binds to immobilized collagen types I, II, III and V
but not to type IV collagen.
 Does not bind to any of the collagens in soluble form.
 Outer membrane proteins on the bacterial cell surface are
essential for binding.
82

81.  It also binds to fibronectin, but not to the plasma
protein, fibrinogen.
 Binding is highly specific.
 Binding to the insoluble form of proteins aid the
organism in its spread and colonization of oral sites and
extraoral sites.
83

82. VIRULENT TRAITS RELEVANT TO
COLONIZATION
Autotransporter proteins
 Autotransporters constitute the largest known family of
bacterial extracellular proteins (Pallen MJ 2003).
84

83.  Autotransporters are produced almost exclusively by
members of the family Proteobacteria and are being
increasingly recognized as virulence factors in
pathogenic species (Ray SK 2002).
85

84.  Bacterial autotransporters display a variety of effector
functions, including adherence, invasion, proteolysis and
serum resistance (Henderson 2001)
 Recent studies have shown that AA produces three
autotransporter proteins: Aae , EmA and ApiA (also
known as Omp100)
 All three autotransporters have been immuno localized
either to the cell surface or to the outer-membrane cell
fraction.
86

85. Surface components that may be involved in the colonization of host tissue
Name Structure Functions
Aae surface protein Attachment to
epithelium
ApiA surface protein Serum resistance
Attachment to
epithelium
EmaA surface protein Attachment to collagen
Attchment to
epithelium
PilA Pili Thin Pili DNA Uptake
DNA Binding
Flp-1 pili Bundle-forming pili Autoaggregation
Attachment to abiotic
surfaces
Attachment to
epithelium
Biofilm formation
PGA Surface polysaccharide Autoaggregation
87

86. Antibiotic Resistance
 Tetracyclines are frequently employed in localized aggressive
periodontitis.
 Roe DE et al 1995 showed that 82% of 19 clinical isolates of
AA were resistant to tetracyclines and carried the tetB
resistance determinant.
 tetB determinant was capable of being transferred by
conjugation to other AA and to H. influenzae.
 Antibiotic resistance in Aa is on the rise and likely to be
responsible for treatment failures in the future.
88

87. Bacteriocins
 Bacteriocins are proteins produced by bacteria that are
lethal for other strains and species of bacteria.
 Confer a colonization advantage for the bacterium by
lessening the ecological pressures associated with
competition by other organisms for both nutrients and
space.
89

88.  Actinobacillin, a bacteriocin that is active against S.
sanguis, Streptococcus uberis and A. viscosus, has been
identified and purified.
 Actinobacillin is associated with both the bacterial cell
surface and extracellular vesicles.
90

89.  Increase the permeability of the cell membranes of
target bacteria.
 Leakage of DNA, RNA and macromolecules essential
for growth.
 Actinobacillin may be responsible for the clinical
observation that a reciprocal relationship occurs
between AA and S. sanguis and/or A. viscosus in plaque
and in patients with LAP.
91

90. Bone Resorption
 Aa stimulate bone resorption by several different
mechanisms:
 Lipopolysaccharide (Kiley P, Holt SC 1980)
 Proteolysis-sensitive factor in microvesicles
(Nowotny A, Behling UH, 1982) &
 Surface -associated material. (Meghji S, Henderson
B, 1995) tumor necrosis factor-a, interferon-c
interleukin-12, metalloproteinase 8 , matrix
metalloproteinase II).
92

91. • Surface-associated material has been identified as the
molecular chaperone, GroEL.
• The chaperone appears to act in a direct way with the
major bone-resorbing cell population, the osteoclast.
93

92.  AA LPS are very effective bone resorption mediator, has been
shown to cause the release of calcium from fetal long bones in
the Ca2+- fetal bone resorption assay.
 The bone resorption activity is completely inhibited by
dexamethasone and which states that probably it involves PG
and IL-1.
 This is in direct contrast to GroEL, whose bone absorption
activity is not inhibited by interleukin-1 receptor antagonist
protein.
94

93.  Generation and activation of osteoclasts is under the
control of a tripartite cytokine signalling system.
 LPS of AA can promote osteoclast formation.
 The capsular-like polysaccharide material can induce
osteoclastogenesis, inhibit osteoblast proliferation by
inducing apoptosis and stimulate macrophage cytokine
synthesis.
95

94. Collagenase
 A major feature of periodontal disease is a marked
reduction in gingival collagen fibre density.
 Collagenases are enzymes that break the peptide bonds in
collagen.
 Collagenase activity is associated with two important
periodontal pathogens, Aa & Pg.
 Some of the reduction in collagen density may also be due
to tissue collagenases induced in periodontal disease.
96

95. Fc-binding Proteins
 The Fc region of an antibody molecule is important in the
binding of the antibody to specific receptors on
polymorphonuclear leukocytes.
 If other proteins compete for binding to this region of
PMN, binding of the antibody may be inhibited and,
thereby, inhibit phagocytosis.
 Tolo & Hegland 1991, demonstrated that molecules on the
surface of AA that are associated with capsular material
and secreted into the medium bind to the Fc portion of
IgG;
97

96.  The binding inhibits the ability of opsonizing antibodies
to bind PMN and reduces phagocytosis by 90%.
 It is believed that the Fc receptors also play a role in
complement activation.
 The ability of this organism to make proteins that bind
to the Fc receptors would inhibit all of these functions.
98

97. LEUKOTOXIN
 This toxin is a 114-kDa protein produced by 56% of strains
isolated from LAP patients. (Zambon JJ 1983)
 Member of the RTX (Repeats in Toxin) family of toxins
that produce pore-forming hemolysins or leukotoxins.
(Lally ET 1989)
 Name derives from the characteristic calcium-binding
motif that is repeated in the carboxy terminal of such
proteins (Chenal A 2009)
99

98.  LtxA can block the processes of bacterial uptake into
phagocytes and the associated processes of bacterial killing.
 Killing of leukocytes has been shown to be due to induction
of the processes of apoptosis.
100

99.  LtxA induces apoptosis by the mitochondrial pathway,
(Korostoff 2000) involves activation of caspases.
 LtxA is the major factor of A.a that is able to induce
interleukin-1β synthesis (Kelk P 2008).
101

100. 102

101. CYTOLETHAL DISTENDING TOXIN
 The most fascinating and well studied cell cycle-
blocking bacterial toxin.
 First discovered in entero pathogenic E. coli
 Three genes, designated cdtA, cdtB and cdtC
 It was found to block eukaryotic cell division in G2
(1997)
 Cause cell distension and death
 The production of Cdt by AA was reported by Sugai et
al (1998)
103

102.  The AA protein dimerizes, and this is associated with
rapid aggregation and loss of activity in buffered solution.
 The holotoxins bind to cholesterol within the cell
membrane(Guerra 2005).
 The toxin is reported to be internalized through the Golgi
complex, and then undergoes retrograde transport to the
endoplasmic reticulum.
104

103. Role of Cdt in pathogenesis of periodontal diseases
 Shenker BJ in 1990 reported that AA produced an
immunosuppressive factor that could block T-cell proliferation.
 This immunosuppressive factor was the Cdt B component of
CDT, and was able to block cell-cycle progression in human T
lymphocytes (Shenker BJ 1999,2000) and induce apoptosis in
these cells (Shenker BJ 2001).
105

104.  CDT may contribute to the immunosuppressive
phenotype of AA, adding to, or synergizing with, the
effects of AA leukotoxin.
106

105. ACTIONS OF CDT
 Deoxyribose nuclease activity
 immunosuppressive factor- able to block cell-cycle
progression in human T lymphocytes and induce
apoptosis in these cells.
 AA Cdt B has phosphatidylinositol-3,4,5-trisphosphate
phosphatase activity.
107

106.  CDT can stimulate the generation of a key osteoclast
regulatory protein, receptor activator of NF-kB
ligand(RANKL), in gingival fibroblasts and periodontal
ligament cells (Belibasakis GN et al 2008,2005)
 CDT inhibits nitric oxide production by activated
macrophages.
( Fernandez 2008)
108

107. LIPOPOLYSACCHARIDE
 LPS (endotoxins) have a high potential for causing
destruction of an array of host cells and tissues.
 It causes skin necrosis (Schwartzmann reaction), bone
resorption and platelet aggregation, and it activates
macrophages.
 Low concentrations of AA LPS stimulate macrophages to
produce IL (lα, lβ) and TNF, cytokines involved in tissue
inflammation and bone resorption.
109

108.  Macrophages that migrate to gingival sites of AA
infection will be stimulated to produce these cytokines
 Both LPS & these cytokines, which are known to play an
important role in atherosclerosis and coronary heart
disease, also appear systemically, where they may
function in initiating and/or exacerbating conditions
associated with cardiac disease.
110

109. IMMUNOSUPPRESSIVE FACTORS
 Host defense mechanisms play a major role in controlling
concentrations of bacterial communities in dental plaque.
 AA suppresses these host defense mechanisms.
 The organism produces a protein that inhibits DNA, RNA and
protein synthesis in mitogen-activated human T cells.
 60-kDa protein secreted shown to inhibit IgG and IgM
synthesis by human lymphocytes.
111

110.  AA affects IG production by activating B cells that
downregulate the ability of B and T cells to respond to
mitogens.
 Leukotoxin impairs ability of lymphocytes to respond to
mitogens by inhibiting DNA, RNA, protein, IgG and IgM
synthesis.
112

111. Inhibitors of PMN Function
 The host’s first line of defense against invading bacteria
is the recruitment of phagocytes to the area.
 The ability to disrupt chemotaxis permits the invading
organism to survive this major challenge from the host.
 AA secretes a LMW compound that inhibits PMN
chemotaxis.
114

112.  Activity of PMN is the killing of bacteria by a wide variety
of potent antibacterial agents that are gained when the
PMN fuse with lysosomes.
 Organisms that can inhibit this fusion or resist the
antibactericidal action of these agents are protected from
this detrimental host activity.
115

113.  AA is capable of inhibiting PMN from producing some
of these compounds, and it is intrinsically resistant to
others.
 A heat-stable protein in AA inhibits the production of
H2O2 by PMN and many strains are naturally resistant to
high concentrations of H2O2.
 Resistant to several of the cationic peptides, known as
defensins that are found in neutrophils.
116

114. Penetration of Epithelial Cells
 Bacteria penetrate and survive within eukaryotic
cells.(Finaly BB 1997)
 AA can penetrate the gingival epithelium
 AA was the first invasive periodontopathogen to be
reported.
 Smooth colony variants of Aa invaded more efficiently
than rough variants.
117

115.  Meyer et al. reported that strain JP2, a high level
leukotoxin producer, was not as invasive as SUNY 465,
the invasion prototype.
 This uptake into cell confers an advantage to the
bacterium.
118

116.  It permits them to either transit the epithelial cell barrier
or persist and grow in a protected cellular
environment.
 Bacteria are protected from immune defenses and from
antibiotics when they are within cells (Wilson 2002).
119

117.  Studies showed that AA occurs in very specific
intracellular locations and that it exhibits a definite
pattern of penetration.
 AA invasion of epithelial cells is a highly dynamic
complex process. (Mayer DH 1997)
120

118. attachment of organisms to the host cell with initiation
of some form of signalling,
binding to a receptor,
entry in a vacuole,
escape from the vacuole,
rapid multiplication,
intracellular spread,
exit from the cell
cell-to-cell spread.
121

119.  Meyer et al. 1997 suggest that the primary receptor for
AA invasion is the transferrin receptor.
 Data suggest that integrins, transmembrane proteins
involved in the adhesion of eukaryotic cells to the
extracellular matrix, also mediate entry.
 Few isolates also enter via receptor-mediated
endocytosis.
122

120.  Invasion is an active process requiring protein synthesis and
energy generation.
 Inhibition of either oxidative phosphorylation or glycolysis
inhibits invasion, and it does not occur at 4°C.
 Not inhibited by endosomal acidification in the host cells.
 Microbial uptake by non-professional phagocytes depends
on the rearrangement of the host cytoskeletal network.
123

121.  Invasion process is a rapid mechanism involving the
formation of cell-surface craters or apertures with lip-like
rims 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 AA.
 It was postulated that entry of AA is rapidly followed by
cell division and then transcytosis of bacteria via the
protrusions.
124

122.  Invasion by the majority of AA isolates is inhibited by
cytochalasin-D, demonstrating a role for actin in the
invasion process.
 AA is taken up in a host derived membrane bound
vacuole, which it lyses and enters the cytoplasm.
 AA possesses phospholipase C implicated in vacuole
lysis.
125

123.  Intracellular AA is not quiescent.
 It transits through the cell to neighbouring cells via bacteria-
induced protrusions that appear to be extensions of the host
cell membrane.
 If neighbouring cells are lacking, bacteria are simply released
from the host cell via rudimentary protrusions.
 Bacteria can be seen within these protrusions by scanning,
transmission and fluorescent microscopy.
126

124.  Microtubules are strongly implicated in the intra- and
intercellular spread & its release into the extracellular
environment.
 Microtubule inhibitors, Colchicine and Nocadozole,
which bind to and inhibit microtubule polymerization
and Taxol, which binds to and stabilizes microtubules,
increase the number of intracellular AA and prevent its
release from the host cell.
127

125. IRON ACQUISITION
 There is an evolutionary struggle between bacteria and
their hosts for the metal iron.
 Iron is essential for bacterial growth, excess iron uptake
can produce toxicity, and iron uptake is consequently
subject to rigorous control.
 The best known iron-responsive regulator is the ferric
uptake regulator Fur.
128

126.  AA has a LPS that binds to haemoglobin and could play a
role in iron acquisition.
 AA can utilize haemoglobin as an iron source, and
bacterial binding to haemoglobin depends on two cell-
surface proteins with molecular masses of 40 and 65 kDa.
 Iron binding proteins of AA - a 35 kDa iron-repressible
protein (termed afuA) that was located in the periplasm
 fbpA iron binding protein- expressed by AA
129

127.  Another iron acquisition system used by AA is the
afeABCD system.
 This gene system was present and expressed in three
serotype strains of AA, including the JP2 clone.
 Under iron-limiting conditions, there is increased
transcription and translation of all biofilm
determinants.
130

128. AA & systemic diseases
 Organisms of HACEK group & AA in particular, are
associated with systemic diseases distant from the oral
cavity (Fine et al 2006).
 As AA is an organism that avidly attaches to both soft
& hard tissues of tooth, & positions itself adjacent to
permeable junctional & pocket epithelium, AA has
been isolated from a no. of organs distant from the oral
cavity & is capable of causing serious infections in
humans.
131

129.  Such infections include facial plane infection, heart
infection, endocarditis, pericarditis, lung infection,
necrotizing pneumonia, mediastinitis, mediastinal
abcess, transdiaphragmatic infection,
endophthalmitis, skin infection, vertebral
osteomyelitis, cervical lymphadenitis,
submandibular space abcess & UTI. (Fine et al 2006,
Kaplan et al 1989).
132

130. Microbiologic tests for AAC
▪ CULTURE
Selective Media:
- Are widely used to isolate pathogenic microorganisms from
clinical specimens.
- Employs antimicrobial agents to suppress non-targeted
microorganisms and thereby improve the detection limit.
133

131.  MGB, a selective medium was one of the earliest media
developed by (Mandell / Socransky 1981).
- The medium consisted of Trypticase soy agar 40 gm/liter,
bacitracin 128 μg/ml, malachite green 8 μg/ml and 5%
defibrinated sheep blood.
134

132. - This was followed by the development of a medium
composed of trypticase soy agar and serum with
bacitracin and vancomycin added as selective agents
(TSBV) (Slots 1982).
 TSBV agar is an excellent primary selective medium for
A.A. that can detect the microorganism in levels as low as
20 viable cells per ml (Slots 1982).
135

133.  Another medium was developed based on blown antibiotic
susceptibility of periodontal flora.
 This medium known as ‘A’ medium is essentially TSBV
supplemented with spiromycin, fusidic acid and
carbenicillin (Holm et al 1987).
136

134. Defined Media
 Use to determine the precise nutritional requirement of
microbe.
 Also useful in genetic analysis and isolation of metabolic
mutants.
 A.A. is a fastidious organism; hence the development of the
defined media is difficult.
137

135.  More recent study utilized tissue culture medium as the
defined medium (Sreenivasan PK, Meyer DH, Fives-
Taylor PM. 1993).
 RPMI- 1640 and Dulbecco's modified Eagle medium are
commonly used tissue culture medium to support growth of
A.A.
138

136. Effect of supplements
1. Yeast extract - addition of yeast extract to trypticase
soy broth enhances growth of most strains of A.A.
(Sreenivasan PK 1993).
2. Cystine - Cystine and thiamine promoted growth of
A.A. (Sreenivasan PK 1993).
Increased the growth of all strains of AA
3. Hormones - steroid hormones (estrogen, progesterone, and
testosterone) enhanced growth of AA (Sreenivasan PK,
Meyer DH, Fives-Taylor 1993).
139

137. 4. pH – A.A. demonstrates optimal growth at pH 7.5 (Sreenivasan
PK1993).
5. Salt concentration – A.A. demonstrates optimal growth
between 85.1mEq/L and 170mEq/L (Sreenivasan PK1993).
6. Iron - AA expresses iron- binding proteins and has hemin-
binding activity.
 Addition of iron salts and protein-bound iron to trypticase soy
broth did not influence the growth or protein profiles of AA.
140

138. Immunodiagnostic methods
 Employ antibodies that recognize specific bacterial Ag's to
detect target microorganisms.
 The methods do not require viable bacteria and are less
susceptible to variations in sample processing.
 less time-consuming and easier to perform than culture.
141

139.  However, the accuracy of immunodiagnostic tests
depends greatly on the quality of the reagents used.
 Poorer detection limits than nucleic acid probe or PCR
assays.
142

140.  Bonta et al. (1985)
described an indirect
immunofluorescence
identification method of
AA with a detection limit
of 500 cells/ ml.
143

141.  Evalusite Test is a
commercially
developed, antibody
based sandwich
enzyme-linked
immunosorbent assay
for the detection of
A.a and P gingivalis
144

142. Radiolabelled DNA probes
 DNA probes entail segments of single-stranded nucleic acid,
labeled with an enzyme or radioisotope, that can locate and
bind to their complementary nucleic acid sequences with low
cross-reactivity to non-target microorganisms.
 DNA probe may target whole genomic DNA or individual
genes.
145

143.  Whole genomic probes are more likely to cross-react with
non-target organisms due to the presence of homologous
sequences between different bacterial species.
 Based on 16S RNA genes, & showed a detection limit of
1000 cells with no evidence of cross reactivity (Dixit et al
1990).
146

144. A. actinomycetemcomitans cloned probe and P gingivalis whole
genomic probe comprise the basis of the commercial DMDx detection
method (Omnigene, Cambridge, MA).
147

145.  The detection limit is approximately 1000 cells.
 Is able to simultaneously screen for the presence of upto 43
different bacterial species in a specimens using a single
nitrocellulose membrane.
 Particularly suitable for large scale, microbiological studies
of the subgingival microbiota.
149

146. PCR
 PCR involves a reiterate amplification of a region of DNA
flanked by a selected primer pair specific for the target
species .
 The presence of the specific amplification product indicates
the presence of the target microorganism
 Displays the best detection limits, identifying as few as 3-5
cells and shows no cross reactivity under optimal condition.
150

147. EFFECTS OF PERIODONTAL TREATMENT ON AA
 AA may survive in untreated periodontal lesions for a
considerable period of time.
 In 1983, Slots & Rosling showed that scaling and
root planing alone was unable to remove AA from
LAP periodontitis lesions.
151

148.  Gunsolley in 1995
Nonsurgical therapy had the least effect on AA counts in
heavily infected periodontal lesions, due to the ability of
the organism to involve gingival tissue and thereby evade
the effect of mechanical debridement and periodontal
healing (Saglie 1991).
152

149.  AA cells in gingiva may constitute a reservoir for
repopulating periodontal pockets.
 Modified Widman flap surgery may suppress AA to
below detectable levels in about 50% of localized
juvenile periodontitis lesions.
153

150.  Tuan et al. (1999) found that an apically positioned flap
with osseous recontouring is more effective than an
apically positioned flap without osseous recontouring in
reducing the pocket depth and levels of subgingival AA.
 Resective type of periodontal surgery is more effective
than access flap surgery due to the excision of infected
gingival tissue and to pocket depth reduction to levels
permitting adequate cleaning by tooth brushing, flossing
or other oral hygiene measures.
154

151.  Systemic antibiotic therapy has the potential to
eradicate AA residing in periodontal pockets and
gingival tissue.
 Tetracyclines combined with scaling or root planning
(Slots 1991) or with P.D. surgery (Mandell 1986) may
markedly suppress or eliminate subgingival AA in LJP.
 Systemic metronidazole has demonstrated good anti-
AA activity in LAP patients (Saxen / Asikainen 1993)
but not in adult periodontitis patients (Niemisen et al
1996).
155

152.  Systemic use of Amoxicillin- Metronidazole has shown
striking clinical results in the treatment of AA associated
-LAP, adult periodontitis and refractory periodontitis
even in the absence of other P.D. therapy (van
Winkelhoff 1996).
 The antibiotic recommendation is a course of amoxicillin
and metronidazole, 250-mg each, three times daily for
8 days (adult dosage).
156

153.  Combination therapy is warranted in mixed periodontal
infection because:
1) each drug is aimed at one or several important
pathogenic microorganisms
2) delay emergence of microbial mutants resistant.
3) bactericidal synergism can be achieved reducing the
dose required and shortening the course of therapy
and avoiding toxicity.
157

154. Disadvantages of combination therapy:
o Greater risk of adverse reaction.
o Antagonism may take place.
o Superinfection with resistant organism
158

155.  Systemic antibiotics should only be administrated as an
adjunct to mechanical debridement because in
undisturbed subgingival plaque sample the target
organisms are effectively protected.
159

156.  Antibiotics have been used in essentially two ways:
1) In combination with intensive instrumentation over a
short period of time after achievement of adequate plaque
control in pre treatment motivation period.
2) As staged approach after completion of initial therapy.
160

157. CONCLUSION
 AA is a bacterium with an array of diverse potential virulence
characteristics, including multiple immune evasion
mechanisms and novel mechanisms for binding to host
matrices and invading host cells, any one of which may play a
crucial role in the local tissue pathology of LAP.
 Technologies have not really touched AA, although it is clear
that this organism is capable of causing marked alterations in
host as a result of its powerful toxins and its ability to adhere
to host cells and to enter into them and travel through them.
161

158.  AA has clearly adapted well to its environs; virulence
factors ensures its survival in the oral cavity and enables it
to promote disease.
 AA also orchestrates its own survival by elaborating
factors that interfere with the host’s defense system
 It is critical that in-depth investigations into the
biochemistry, genetic expression, regulation and
mechanisms of action of these factors be initiated to
eradicate this pathogen.
162

159. References
1. Newman, Takei, Klokkevold, Carranza- Clinical
Periodontology.11thed. Elsevier publication; 2011
2. Jan Lindhe, Niklaus P Lang, Thorkild Karring -Clinical
Periodontology & Implant Dentistry .
3. Paula M. Fives-Taylor, Diane Hutchins Meyer, Keith P. Mintz &
Catherine Brissette; Virulence f actors of A.A: P 2000, Vol. 20,
1999, p 136-167
4. Ingar olsen Harounn N. Shah& Saheer E. Gharbia; Taxonomy
and biochemical characterhtics of Actinobacillus
actinomycetemcomitans and Porphyromonas gingivalis
Periodontology 2000, Vol. 20, 1999, 14-52
5. Slots J; Actinobacillus actinomycetemcomitans and
Porphyromonas gingivalis in periodontal disease: introduction
Periodontology 2000, Vol. 20, 1999, 7-13
163

160. 6.Brian Henderson, John M. Ward & Derren Ready ;
Aggregatibacter (Actinobacillus) actinomycetemcomitans: A
tripleA periodontopathogen? Periodontology 2000, Vol. 54,
2010, 78–105
7.Casey chen & Jorgen slots; Microbiological tests for
Actinobacillus actinomycetemcomitam and Porphyrornonas
gingivalis. Periodontology 2000, Val. 20, 1999, 53-64
8. Brian Henderson, Michael Wilson, Lindsay Sharp and John
M. Ward. Actinobacillus actinomycetemcomitans. J. Med.
Microbiol. — Vol. 51 (2002), 1013–1020
164

161. THANKYOU…
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