The Gram-negative A. actinomycetemcomitans is assumed to be the primary etiologic agent of LAgP and has also been implicated in chronic periodontitis and severe non-oral infections.
2. Introduction
Historical perspective
Taxonomy
Morphologic and biochemical properties
Colony characteristics
Detection methods
Molecular detection
Genomics of a. a
Virulence factors
Systemic interactions
Diagnostic aids
Prevention
Conclusion
References
2
3. More than 500 cultivable bacterial species have been isolated from the gingival
crevices of human beings.
But the actual species count may be thousands, since a wide proportion of bacteria
remains uncultivable or unidentified.
The sub gingival bacterial flora consists of facultatively anaerobic gram positive
species in the healthy oral cavity, but in gingivitis the proportion of gram negative
bacteria increases.
3
4. In periodontitis , only 10 to 30 species, mainly gram negative anaerobic bacteria, are
putative pathogens.
Mainly three species
Actinobacillus actinomycetemcomitans ,
Porphyromonas gingivalis and
Bacteroides forsythus
Are presently considered as primary etiological agents in periodontitis (WWP 1996 ).
4
5. A. actinomycetemcomitans was first isolated and identified in 1912 by Klinger.
It was so named because of its consistent association with Actinomyces israelii in
actinomycotic infections. (1934 Klaber ).
The genus name Actinobacillus refers to actin- star shaped bacillus- rod shaped
actinomycetum comitans- referring its close association with actinomyces israelii in
actinomycotic lesions
5
6. In 1951 – Home put forward that A.a could cause disease in humans.
Bacterium actinomycetemcomitans which was changed to Bacterium comitans by
Lieske(1921) and finally to Actinobacillius actinomycetemcomitans by Topley and
Wilson (1959)
In 1959 Heinerish suggested that it a part of normal flora.
Kilian and Schiott in 1975 were the first to demonstrate that Aggregatibacter
actinomycetemcomitans was present in dental plaque.
In 1976 , Newman, Socransky and Slots – related A.a to Juvenile periodontitis.
In 1979 , Tsai et al, discovered A.a leukotoxin.
6
7. Page et al 1991,Perry et al 1996,Kaplan et al 2001- described Six serotypes, a–f.
In 2004 , Roe et al – identified the complete A.a genome.
In 2006- Nørskov-Lauritsen and Kilian - Phylogenetic similarity of A.
actinomycetemcomitans and Haemophilus aphrophilus, H. paraphrophilus, and H.
segnis, suggesting the new genus Aggregatibacter depending upon independency of
X factor and variably dependent on V factor.
7
8. Aggregatibacter a member of family
Pasteurellaceae, has been recognized as one of
the key pathogens in periodontitis.
8
9. Gram negative
Approx. of 0.4 X 1.0µm
Non motile
Saccharolytic
Capnophilic
round ended
facultative anaerobe
9
10. 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.
10
12. Serological investigations of Aggregatibacter actinomycetemcomitans have identified
specific serotypes and bacterial antigens which may be important in the etiology of
periodontal disease.
6 serotypes- a,b,c,d,e and f. SEROTYPES (Zambon 1983)
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.
12
13. 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
13
14. 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
14
15. Bacterial culture
DNA probe hybridization checkerboard technique (Socransky SS 2004)
Specific antibody immunofluorescence (Schrago CG 2003)
Fluorescent in situ hybridization with labelled oligonucleotide probes (Iwata T 2005)
gene amplification via PCR methodology, including multiplex nested multiplex and
quantitative PCR (Tran SD 1996)
loop-mediated isothermal amplification and cloning and sequencing of 16S rRNA
libraries (Kumar PS 2004)
15
16. 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 .
16
17. The genome of the JP2 clone of A. actinomycetemcomitans (HK1651) has been
sequenced by the group lead by Bruce Roe at the University of Oklahoma.
Analysis of the A. actinomycetemcomitans genome has identified a number of genes
that could encode proteins involved in iron acquisition.
These include genes homologous to the haemoglobin- and haptoglobin–
haemoglobin-binding proteins and the ferrated transferrin-binding protein of
pathogenic Neisseria spp.
There are also three haemoglobin⁄ haptoglobin-binding proteins homologous to the
hgpABC proteins of Haemophilus influenzae
17
18. The genome also contains the genes of the Actinobacillus ferric uptake iron uptake
system afuABC first identified in Actinobacillus pleuropneumoniae.
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
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
18
19. 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.
19
20. 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.
1. Factors promoting the colonization and persistence in the oral cavity
2. Factors that interfere with the host’s defenses
3. Factors that destroy host tissues
4. Factors that inhibit host repair of tissues
20
21. :
A. Adhesins.
B. Invasins.
C. Bacteriocins.
D. Antibiotic resistance.
21
22. Adhesins
The bacterial surface components involved in adhesion to a specific substrate are
called adhesins.
Cell surface entities mediating adherence include fimbriae, extracellular
amorphous material, and extracellular vesicles.
Fimbriae carry curlin proteins and adhesins which attach them to the substratum so
that the bacteria can withstand shear forces and obtain nutrients.
22
23. Streptococcus sanguis , Actinomyces naeslundi and Streptococcus uberis
produced factors that were inhibitory to the growth of A.
actinimycetemcomitans. Hillman and co – workers ( 1982, 1985, 1987 )
The hydrogen peroxide formation by the “ beneficial species “, either directly
or via a host peroxidase system inhibits the growth of pathogen.
23
24. Vesicles
A bleb-like structure which are LPS in nature, originate from and are continuous with the
outer membrane.
Enclosed by lipid bilayer, vesicles can form naturally, for example, during endocytosis
Vesicles are involved in metabolism, transport, buoyancy control, and enzyme storage.
24
25. Aggregatibacter actinomycetemcomitans vesicles exhibit leukotoxic activity.
Aggregatibacter actinomycetemcomitans vesicles are endotoxin, bone resorption
activity, and a bacteriocin, termed actinobacillin.
Aggregatibacter actinomycetemcomitans vesicles also exhibit adhesive properties;
this observation prompted the hypothesis that vesicles function as delivery
vehicles for Aggregatibacter actinomycetemcomitans toxic materials.
25
26. Extracellular amorphous material
Associated with the surface of Aggregatibacter actinomycetemcomitans cells is
an amorphous material that frequently embeds adjacent cells in a matrix.
The material is most likely a glycoprotein, and has been shown to exhibit both
bone-resorbing activity and adhesive properties.
26
27. Invasins
Invasion of Aggregatibacter actinomycetemcomitans is a rapid mechanism
involving the formation of cell-surface “craters” or apertures with lip-like rims.
These invasins occur as indentations on the cell surface, as well as in membrane
ruffles where they appear to be entering into the epithelial cells.
Invasion is a dynamic process with bacteria appearing in the host cell cytoplasm
within 30 min.
27
28. The cells of Aggregatibacter actinomycetemcomitans bind through adhesins, to
surface receptors on gingival cells — transferrin receptor.
The epithelial cell membrane ruffles and efface and invaginations engulf the
bacteria, which then become internalized within a membrane vesicle.
The bacterial cells destroy the membrane vesicles by secretion of phospholipase C,
releasing the bacteria into the cytoplasm where they grow and divide rapidly.
Bacteria become localized at membrane protrusions through which they enter
adjoining epithelial cells in a microtubule dependent process
28
29. Bacteriocins
In Aggregatibacter actinomycetemcomitans they are usually associated with both
the bacterial cell surface and extracellular vesicles.
These toxic agents can confer a colonization advantage for the bacterium by
lessening the ecological pressures associated with competition by other organisms
for both nutrients and space.
Actinobacillin, a bacteriocin that is active against Streptococcus sanguis,
Streptococcus uberis, and Actinomyces viscosus, has been identified and purified.
29
30. Stevens et al. ( 1987 ) and Hammond et al ( 1987 )
Demonstrated the reverse antagonism.
A. a specifically inhibit the growth of S. sanguis, S. uberis, and A. naeslundii
genospecies 2 ( but not other species ) by the production of a bacteriocin.
This mutual antagonism is highly specific and its outcome may strongly influence
whether a subject or a site will exhibit disease due to A. actinomycetemcomitans.
30
31. Antibiotic resistance
Microorganism, are able to survive afterexposure to one or more antibiotics.
Approximately 30% of oral Aggregatibacter actinomycetemcomitans are resistant to
benzylpenicillin.
New or altered penicillin-binding proteins on the bacterial cell surface may account for
the nonenzymatic penicillin resistance of Aggregatibacter actinomycetemcomitans, as
has been observed among strains of Haempohilus influenza.
82% of 19 clinical isolates of Aggregatibacter actinomycetemcomitans were resistant to
tetracyclines and carried the tetB resistance determinant
31
32. A. Leukotoxin.
B. Lipopolysaccharides (LPSs).
C. Chemotactic inhibitors.
D. Cytolethal distending toxin (CDT).
E. Immunosuppressive proteins.
F. Fc-binding proteins.
32
33. Leukotoxin
It is a proteinaceous toxin secreted from the cell membrane of Aggregatibacter
actinomycetemcomitans.
Leukotoxin is a member of the RTX family of toxins that produce pore-forming
hemolysins.
The leukotoxin operon consists of four coding genes designated ltxC, ltxA, ltxB, and
ltxD and an upstream promoter gene.
In A. actinomycetemcomitans, the corresponding operon ltxCABD produces LtxA, and
a tolC homologue, tdeA, has been found to encode a protein required for the export of
LtxA. (Crosby JA 2007)
33
34. Leukotoxin is not only species specific but also cell-specific.
The toxin binds to neutrophils, monocytes, and a subset of lymphocytes; and
forms pores in the membranes of these target cells overwhelming their ability to
sustain osmotic homeostasis, resulting in cell death.
34
35. Interaction with polymorphonuclear leukocytes (PMNs) —
Leukotoxin has shown to efficiently cause death of human PMNs through extracellular
release of proteolytic enzymes from both primary and secondary granules, along with
activation and release of matrix metalloproteinase-8, which can contribute to
periodontal tissue destruction.
Interaction with lymphocytes —
The ability of leukotoxin to induce apoptosis in lymphocytes might impair the
acquired immune response of periodontal infections.
A shift in the balance between Th-1 and Th-2 subsets of T-cells is found in periodontal
inflammation, with the Th-2 cells to associate with chronic periodontitis.
35
36. Interaction with monocytes/macrophages —
Leukotoxin causes the activation of caspase-1, which is a cytosolic cysteine
proteinase that specifically induces activation and secretion of the
proinflammatory cytokines interleukin-1β and 18, which result in
monocyte/macrophage lysis by incorporation in a cytosolic multimer complex
named the inflammasome.
36
37. Lipopolysaccharides
LPSs are large molecules consisting of a lipid and a polysaccharide joined by a
covalent bond.
They are found in the outer membrane of gramnegative bacteria, act as endotoxins,
and elicit strong immune responses in animals.
Structure and composition — It comprises three parts:
1. O antigen
2. Core oligosaccharide
3. Lipid A
37
38. It exerts its toxic effects when released from multiplying cells, or when the
bacteria are lysed.
In monocytes and macrophages it results in production of interleukin-1, 6, and 8,
tumor necrosis factor-α, and platelet-activating factor (PAF); activation of the
complement; and coagulation cascade.
LPS may also contribute to destruction of periodontal connective tissue by
activating the pathways that lead to stimulation of matrix metalloproteinases and
plasminogen activator.
38
39. Chemotactic inhibitor
Disruption or inhibition of neutrophil chemotaxis is advantageous for
Aggregatibacter actinomycetemcomitans.
Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum, which
were not chemotactic by themselves, inhibited binding of chemotactic peptide
suggesting that in vitro chemotaxis inhibition was mediated by nonchemotactic
components that compete for the chemotactic factor receptor on the neutrophil.
39
40. Cytolethal distending toxin (CDT).
is a cell cycle-modulatory protein with immunosuppressive function. The toxin is
either secreted freely or associated with the membrane of the producing bacteria.
CDT is a tripartite structure encoded by a locus of three genes, cdtABC.
The toxin itself is encoded by cdtB, while cdtA and cdtC appear to encode
proteins that mediate interaction between the CDT complex and the host cell
surface.
40
41. While the role of cdtA and cdtC is less clear, both proteins possess putative mucin-like
carbohydrate-binding domains that predict interaction with the host cell surface.
CdtB is transported into the nucleus where it causes DNA damage though its DNase
activity resulting in apoptosis, through capase activation.
Aggregatibacter actinomycetemcomitans Cdt (AaCdt) disrupts macrophage function by
inhibiting phagocytic activity as well as affecting the production of interleukins 1-β, 6,
and 8
AaCdt is able to stimulate the production of receptor activator of nuclear factor-Kβ
ligand which may be involved in pathological bone resorption, characteristic of LAP.
41
42. Immunosuppressive factors
A 60-kDa protein secreted by Aggregatibacter actinomycetemcomitans has been
purified and shown to inhibit IgG and IgM synthesis by human lymphocytes.
It is believed that it affects immunoglobulin production by activating B cells that
downregulate the ability of B and T cells to respond to mitogens.
In addition, leukotoxin impairs the ability of lymphocytes to respond to mitogens
by inhibiting DNA, RNA, protein, IgG, and IgM synthesis.
42
43. Fc binding proteins
Fc binding proteins are found to be associated with the bacterial cell surface and are
released in soluble form during bacterial growth.
Tolo and Hegland demonstrated that molecules on the surface of Aggregatibacter
actinomycetemcomitans that are associated with capsular material and secreted into
the medium bind to the Fc portion of IgG; the binding inhibits the ability of
opsonizing antibodies to bind PMNs and reduces phagocytosis by 90%. It is
believed that the Fc receptors also play a role in complement activation.
43
44. A. Cytotoxins.
B. Heat shock proteins (HSPs).
C. Collagenase.
D. Bone resorption agents.
44
45. Cytotoxins
Aggregatibacter actinomycetemcomitans surface associated material produces
heat-labile cytotoxins which inhibit human fibroblast proliferation.
One toxin that is secreted into the supernatant has been isolated and identified
as a 50-kDa protein that inhibits DNA synthesis in the fibroblast.
Another surface-associated material cytotoxin, designated Gapstein, is an 8-
kDa protein.
The inhibition of fibroblast growth may be expressed as a decrease in collagen
synthesis which is manifested as a loss of collagen in certain forms of juvenile
periodontitis 45
46. Heat shock proteins (HSPs).
Certain HSPs have been found in the surface associated material that are molecular
chaperones and play a critical role in protein folding, intracellular trafficking of
proteins, and coping with proteins denatured by heat and other stresses.
Aggre g a t i b a c t e r actinomycetemcomitans strains have shown the presence of
HSPs, including GroEL-like (HSP60) and DnaK-like (HSP70) proteins.
46
47. Protein homologous to GroEL-like HSP is osteolytic. Purified native GroEL-
like HSP from Aggregatibacter actinomycetemcomitans promotes epithelial
cell proliferation at lower HSP concentrations, but has a toxic effect on
epithelial cells at higher HSP concentrations
47
48. Collagenase
Mechanism of virulence — Collagenases are zinc endopeptidases/extracellular
proteolytic enzymes secreted by bacteria that digest nearly all collagen fibers in
their insoluble triple helical form.
Collagenase activity is associated with two important periodontal pathogens,
Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, which
results in reduction in collagen density induced in periodontal disease.
48
49. Bone resorption agents
Mechanism of virulence — There is evidence that components of Aggregatibacter
actinomycetemcomitans like LPSs, proteolysissensitive factor in microvesicles,
surface associated materials, etc., inhibit osteoblast proliferation and synthetic
activity.
They cause activation of bone resorption and induction of osteoclast proliferation.
Aggregatibacter actinomycetemcomitans LPS, is a 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. 49
50. Inhibitors of fibroblast proliferation
Inhibitors of bone formation
50
52. Konig et al. 2016 demonstrated that periodontal pathogen Aggregatibacter
actinomycetemcomitans (Aa) induces changes in neutrophil function, including
hypercitrullination of host proteins, an abnormality that is also observed in the joints
of patients with rheumatoid arthritis.
The pore-forming toxin LtxA of A. actinomycetemcomitans may be a trigger of the
autoimmune disease rheumatoid arthritis due to its ability to stimulate protein
citrullination, a post-translational protein modification targeted by autoantibodies in
this disease.(Abbasi 2017)
52
53. An association between cardiovascular and periodontal disease may be due to
lipopolysaccharide (LPS) promoted release of inflammatory mediators, adverse
alterations in lipoprotein profile and an imbalance in cholesterol homeostasis.
Laura Lakio et al in 2006 studied the proatherogenic properties of LPS
preparations from serotypes b and d strains on macrophages
A. actinomycetemcomitans LPS preparations induced a time dependant release of
TNF – α and IL-1β . LPS induced foam cell formation and cholesterol ester
accumulation from native low density lipoprotein.
53
54. Commercial diagnostic kits:
1.Evalusite ( Kodak) This is a number of enzyme linked immunosorbant assays
(ELISA ) using antibodies to detect antigens for A.actinomycetemcomitans. The
reactions are carried out in a simple chair side reaction kit.
Subgingival plaque samples are reacted with the antibodies and detection substrate
in a multilevel reaction dish.
2. Omnigene (Omni Gene, Inc ) and BTD ( Biotechnica Diagnostics , Inc) These are
DNA probe systems for a number of subgingival bacteria.
A paper point sample of subgingival plaque is placed in a container provided and
mailed off to the company for assay.
54
55. Alter subgingival environment 1. Reduction in probing depth 2. Mechanical
removal or disruption of subgingival plaque biofilm 3. Application of
oxygenating and redox agents: The dyes can raise the redox potential of an
ecosystem.The dye most commonly used is methylene blue. 4. Use of
antimicrobials
Replacement therapy 1. Pre-eruptive colonization 2. Competitive replacement
55
56. Replacement therapy:-Phenomenon by which one member of the ecosystem can inhibit the
growth of another is termed as bacterial interference.
Use of antagonistic organism to control pathogens and prevent disease is termed replacement
therapy.
1. Pre-eruptive colonization: ecological niches within the Plaque are filled by a harmless or
potentially beneficial organism before the undesirable strain has had the opportunity to
colonize
2. Competitive displacement: here, a more competitive strain would displace a pre-existing
organism from plaque In health; it has been shown that H2O2 producing strains of S.sanguis
inhibit the growth of Aa, whereas the converse is true for plaque from sites with LAP.
56
57. A. 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 LAP.
Our understanding of this organism still lags behind that of enteric pathogens, largely
because methods for genetic manipulation have only just become available and the
genome sequence, while almost complete, still awaits annotation.
57
58. Socransky SS , Haffajee AD, Smith GLF, Dzink JL Difficulties encountered in the search
for the etiologic agents for destructive periodontal diseases. J Clin Periodontol 1987; 14 :
588-593.
Henderson B, Ward JM, Ready D "Aggregatibacter (Actinobacillus)
actinomycetemcomitans: a triple A* periodontopathogen?". Periodontology 2000. 2010 54
(1): 78–105
Nørskov-Lauritsen N; Kilian M . "Reclassification of Actinobacillus
actinomycetemcomitans, Haemophilus aphrophilus, Haemophilus paraphrophilus and
Haemophilus segnis as Aggregatibacter actinomycetemcomitans gen. nov., comb. nov.,
Aggregatibacter aphrophilus comb. nov. and Aggregatibacter segnis comb. nov., and
emended description of Aggregatibacter aphrophilus to include V factor-dependent and V
factor-independent isolates". Int. J. Syst. Evol. Microbiol. 2006 : 56 : 2135–46.
Maximilian F. Konig, Loreto Abusleme, Jesper Reinholdt, Robert J. Palmer, Ricardo P.
Teles, Kevon Sampson, Antony Rosen, Peter A. Nigrovic, Jeremy Sokolove, Jon T. Giles,
Niki M. Moutsopoulos, Felipe Andrade Aggregatibacter actinomycetemcomitans–induced
hypercitrullination links periodontal infection to autoimmunity in rheumatoid arthritis
Science Translational Medicine 2016 58
59. Moore WEC , Moore LVH The bacteria of periodontal diseases. Periodontol 2000
1994 ; 5 :66-67
CARRANZA 10TH, EDITION
59
Editor's Notes
These are proteinaceous structures found on the surface of the bacterial cell which interact and bind to specific receptors in saliva, on the surface of tooth, on extracellular matrix proteins, and on epithelial cells.