Aggregatibacter actinomycetemcomitans

AGGREGATIBACTER
ACTINOMYCETEMCOMITANS
Dr. PRADNYA
WAGH

CONTENTS
1. Introduction
2. Taxonomy
3. Historical perspective and ethymology.
4. Morphological characteristics of Aggregatibacter
actinomycetemcomitans.
5. Biochemical properties of Aa
6. Transmission of A.a

7.Virulence factors of A. actinomycetemcomitans
8.Cellular and humoral response of A.a in periodontal
disease
9.Factors influencing the growth and viability of aa
10.Diagnostic methods for A.a

11.Localized aggressive periodontitis and A.a
12.Effect of periodontal treatment on subgingival A.
13.Dissemination of A.a causing non oral infections.
14.Conclusion

 In 1902, Lignieres & Spitz isolated a non-motile,
non-branching, gram-negative bacillus from
lesions in cattle suffering from a disease that
resembled actinomycosis.
 The organism was called actinobacillus, and the
disease to which it gave rise, actinobacillosis.

 The genus actinobacillus includes species
isolated from human beings and other
mammals.
 The only species routinely isolated from the
human oral cavity is Aggregatibacter
actinomycetemcomitans which has been
implicated as a major periodontal pathogen.

Four lines of evidence to regard Aa as a prime
organism associated with AgP (Socransky and
Haffajee in 1992)
 Association studies:
 Aa is isolated from > 90% of LAP patients and
much less frequently in healthy patients.
 It was possible to demonstrate Aa is the sites
showing evidence of recent or ongoing destruction.
 Demonstration of virulance factors:
 Aa produces several pathogenic substances including
leukotoxin and is capable of causing disease in
experimental animals. (Zambon et al 1988)

 Findings of immune responces:
 Significantly elevated levels of serum antibodies to
Aa reported in LAP patients. (Listgarten 1981)
 Such patients also produce local antibodies against
these organisms at diseased sites.(Slots and Rosling
1983)
 Treatment outcomes and levels of Aa:
 Treatment seems to reduce the levels of Aa
 Unsuccesful treatment associated with failure in the
reduction of the microorganism (Aa) levels.

Taxonomy
 FAMILY- Pasteurellaceae ( consists of large
group of gram-negative chemoorganotrophic,
facultative anaerobic and fermentative bacteria).
 GENERA- Pasteurella (Trevisan 1887),
Actinobacillus (Brumpt 1910) and
Haemophilus (Winslow et al. 1917).
Aggregatibacter (Niels Nørskov
2006)

Aggregatibacter actinomycetemcomitans

1) The morphological and cultural characteristics
of A. actinomycetemcomitans were first
described by Klinger in 1912.
 The first isolates of this gram-negative,
facultatively anaerobic rod, recovered from
cervicofacial actinomycosis, were given the
name Bacterium actinomycetem comitans.

2) This name was changed twice, first by Lieske
in 1921 to Bacterium comitans and later by
Topley &Wilson in 1929 to Actinobacillus
 The specific epithethelium means “with
actinomycetes”, referring and reflects its initial
isolation in association with an actinomyces
species.

ETYMOLOGY
 Greek words,
Actes, meaning ray, because of the star
on the top of the agar colony and,
Mycetes, meaning fungus, because
Actinomyces was originally thought to be a
fungus.
 The Latin word, comitans, meaning in
common with, or accompanying Actinomycetes
spp., reflects the association of Actinobacillus
with Actinomycetes.

3) King & Tatum (1962) described the close
phenotypic similarity of Actinobacillus
actinomycetemcomitans with H. aphrophilus,
and Actinobacillus actinomycetemcomitans
was subsequently reassigned to the genus
Haemophilus (Potts et al., 1985).

 This transfer did not give rise to a satisfactory
classification.
 Actinobacillus actinomycetemcomitans is
unrelated to Haemophilus influenzae, the type
species of the genus Haemophilus, and the
reclassification of Actinobacillus
actinomycetemcomitans was not favoured by
the ICSB Subcommittee on Pasteurellaceae and
related organisms (Frederiksen, 1987).

5) Members of the family Pasteurellaceae are
incapable of synthesizing nicotinamide adenine
dinucleotide (NAD) de novo and acquire this
essential nutrient from their environment as
either NAD or a limited number of precursors.

 Actinobacillus actinomycetemcomitans,
Haemophilus aphrophilus, Haemophilus
paraphrophilus and Haemophilus segnis
exhibited the nicotinamide
phosphoribosyltransferase gene (nadV)
conferring V factor- {nicotinamide adenine
dinucleotide NAD nicotinamide
mononucleotide (NMN) nicotinamide riboside
(NR)} independent growth.

 This led to the transfer of Actinobacillus
actinomycetemcomitans, Haemophilus
aphrophilus and Haemophilus segnis to a new
genus Aggregatibacter gen. nov. as
Aggregatibacter actinomycetemcomitans comb.
nov. (Niels Nørskov-Lauritsen and Mogens
Kilian 2006)

 Kilian and Schiott were the first to demonstrate
that A. actinomycetemcomitans was present in
dental plaque.

MORPHOLOGICAL
CHARACTERISTICS OF A.

 A. actinomycetemcomitans is a gram-negative
coccobacillus approximately 0.4±0.1 to1.0 ± 0.4
ųm in size.
 A. actinomycetemcomitans is capnophilic,
requiring an atmosphere containing 5-10% CO2
for good growth.

 It is microaerophilic and a facultative anaerobe
and can grow under anaerobic conditions.
 A. actinomycetemcomitans is nonsporulating,
nonmotile, non-hemolytic, and oxidase and
catalase positive.
 Upon primary isolation, A.a forms small
colonies approximately 0.5-1.0 mm in diameter.

 The translucent (or transparent) colonies with
irregular edges appear smooth, circular and
convex.
 The colonial morphology of fresh isolates is
distinctive with the internal star-shaped or
crossed cigar morphology form embedding in
the agar that gives A.a its name.

Colonies of Actinobacillus actinomycetemcomitans on TSBV
Tryptonesoy-serum-bacitracin-vancomycin (TSBV) agar. R-colonies to the
left, S-colonies in the middle and to the right.
Colonies of Actinobacillus
actinomycetemcomitan with
protrusions into TSBV agar.
Gram-stain of Actinobacillus
actinomycetemcomitans cells

Surface ultrastructure of
A . actinomycetemcomitans
 A significant feature of A.
actinomycetemcomitans is its surface
ultrastructure which includes
1. Fimbriae,
2. Vesicles, and
3. Extracellular amorphous material.

Fimbriae
 Aa may exhibit fimbriae, small filamentous cell
surface appendages associated with bacterial
colonization of host tissues.
 Aa fimbriae may be more than 2 mm in length
and 5 mm in diameter and often occur in
bundles.
 Freshly isolated strains are fimbriated, but in
vitro subculture results in organisms that lack
fimbriae.

 Fimbriated strains produce colonies with a star-
shaped interior structure, designated star
positive, colonies produced by nonfimbriated
strains lack a structured interior and thus are
designated star negative.
 A number of studies indicate that colonial
variation and fimbriation are associated with A.a
adhesion.

 The colony surface of fimbriated A.a will be
rough whereas non fimbriated will be smooth
which exhibit poor adherence and poor biofilm
forming capacity.
 However some studies have shown the presence
of integrin binding sites and other adhesion
molecules which help in adhesion.

 Fimbria associated protein, an attachment factor
of Aa is expressed in fimbriated genes, but not
in non-fimbriated ones.
 Thus, a positive correlation between Aa
fimbriation and adhesion exists.

Vesicles
 A prominent feature of the surface of A.a is
vesicles (blebs).
 These structures, which are lipopolysaccharide in
nature, originate from and are continuous with the
outer membrane.
 Vesicles are also released into the external
environment in large numbers.

 The surface of highly leukotoxic A.a strains has
an abundance of extracellular membranous
vesicles, in contrast to minimally or
nonleukotoxic strains, which have few or no
vesicles.
 Furthermore, vesicles exhibit leukotoxic activity.

 Other biologically active components of A.
actinomycetemcomitans vesicles are endotoxin,
bone resorption activity and a bacteriocin
termed actinobacillin.
 A. actinomycetemcomitans vesicles also exhibit
adhesive properties, this observation prompted the
hypothesis that vesicles function as delivery
vehicles for A. actinomycetemcomitans toxic
materials.

Extracellular amorphous material
 Associated with the surface of certain A.a cells is
an amorphous material that frequently embeds
adjacent cells in a matrix.
 The material is a protein, most likely a
glycoprotein, and has been shown to exhibit
both bone-resorbing activity and adhesive
properties.

Slots (1982) –130 strains of A. a.
 All of the isolates were small, non-motile,
capnophilic, G –ve rods that did not require X or
V factor for growth.
 All decomposed H2O2, were oxidase negative and
benzidine positive.
 Reduced nitrate to nitrite.
 Produced strong alkaline and acid phosphatases.
 Fermented fructose, glucose and mannose.
 Some strains ferments galactose.

Strain is a group of organisms within a species or
variety, characterized by some particular quality,
as rough or smooth strains of bacteria.
R strain
S strain
Wild type strain

Biotypes of Aa
Biotypes
A group of variant strains of a bacterial species,
differing in identifiable physiologic
characteristics.
First described in A.a by King and Tatum (1962)
by fermentation reactions with
 Galactose, Mannitol and Xylose  8 biotypes
 Dextrin, Maltose, Mannitol and Xylose  10
biotypes
Biotyping based on fermentation of galactose was
given Pulverer and Ko (1970).

 Biochemical properties of Aa in blood agar
colonies are small gray to white, translucent,
smooth and nonhemolytic.
 Growth is stimulated by the addition to CO2 and
it grows well when agar cultures are incubated at
5-10% CO2.

 TryptoneSoy-serum-bacitracin-vancomycin
(TSBV) agar yields white translucent colonies
with star shaped, internal structure that are
adherent to the agar.
 It ferments a range of sugars including glucose
and fructose but not sucrose or lactose.

 Acid end products include lactate, succinate,
acetate, and propionate.
 Eight to ten biotypes on the basis of
fermentative ability of A.a strains to utilize
galactose, dextrin, maltose, mannitol and xylose
permits the biotyping of this organism into
several biotypes and serves to distinguish this
organism from other members of the oral flora.

 They do not require factor X (hemin) and V
(NAD) for growth.
 They decompose hydrogen peroxide.
 They are oxidase positive, benzidine positive,
reduce nitrate, produce strong alkaline and acid
phosphatases and ferment fructose, glucose and
mannose.
 Some isolates also produce small amount of gas,
but hydrogen sulphide is not generated.

Serotypes
Serotyping
Serotyping is a taxonomic subdivision of bacteria based
on the kinds and combinations of constituent antigens
present in the cell, or a formula expressing the antigenic
analysis.
■ The number of recognized serotypes in A.
actinomycetemcomitans are five: a, b, c (Zambon et al),
d and e (Asikainen S et al 1991; Saarela M et al 1992).
■ Recently another serotype- f has been classified. (Niels
Nørskov-Lauritsen 2006)

■ Clinical isolates predominantly belong to
serotype b. Healthy subjects frequently carry
serotype c strains.
■ The serological specificity is defined by six
structurally and antigenically distinct O-
polysaccharide components of their respective
lipopolysaccharide molecules and also by
molecular weight. (Page et al., 1991; Perry et al.,
1996a, b; Kaplan et al., 2001).

Serogroup
 Serogroup is a group of bacteria containing a
common antigen, possibly including more than
one serotype, species, or genus or a group of
intimately related microorganisms distinguished
by a common set of antigens.

• Pulverer and Ko (1972) 24 groups of A.a and
1-6 agglutinating antigens on each strain.
• King and Tatum (1962) three serotypes
based on a heat stable component among non-
oral A.a.
• Taichman et al (1982) 4 serogroups based on
surface antigens and proteinaceous leukotoxin.

Three serotypes a, b & c
 Serotype a & b are common in oral cavity.
 Serotype c - 10% of oral cavity, extraoral
infections.
 Serotype b – LJP (Slots, 1991)
 Serotype c – Health (Slots, 1991)
 Serotype b and c- severe and EOP in blacks
 Serotype a - severe and EOP in whites (Solley et al,
1991)
Zambon et al (1983)

Later serotypes raised to five
– a, b, c, d & e.
 Single serotype.
 Serotype stability.
 Serotype b - multiple serotype antibodies.
 Serotype b LPS Ab protective in G-EOP.
 Intrafamilial Transmission.

Serotype antigens of A.a have
 high molecular weight
 are heat stable and
 have primary carbohydrate moieties.
Serotype antigens can be detected by
immunofluorescent techniques on whole cells
as they are part of outer cell surface, possibly
the microcapsule.
Chemical composition

 Isolates belonging to the same serotype were
genetically identical in the same individual, but
non-identical if they belonged to different
serotypes.
 Isolates of the same or different serotypes are
genetically non-identical in different individuals.
 Arbitrarily primed PCR (AP-PCR) to fingerprint
clinical A.a isolates showed 15 different AP-PCR
genotypes among 93 A.a isolates (Asi Kainen et
al, 1995).
Genetic Dissimilarity of serotypes

 RFLP analysis has identified 5 groups of genetic
variants of Aa
 Of these, group II and IV are associated within
aggressive periodontitis, with group II showing
the strongest correlation of seroconversion from
a healthy to a diseased periodontal status.

 Each of serotype a, b, c, d and e comprises
genetically isolated subpopulations.
 Non-serotypable strains of A.a may be serotype-
deficient variants originating from strains of
known serotypes.
 Serotype b and c strains may contain
transmittable DNA sequences not found in
strains of the other serotypes.

• Genetic analysis by multilocus enzyme
electrophoresis of an A.a population of 88 clinically
well characterized isolates divided the 5 serotypes
into two phylogenetic lineages: -
• Serotypes b & c
• Serotypes a, d & e
• Recently, in 6 Serotypes (a-f) based on
O-polysaccharide component of LPS i.e.,
Omp 100, 64, 39, 29, 18 & 16 has been described
by Komatsuzawa et al, 2002.

1) Vertical transmission
2) Horizontal transmission- between siblings and
spouses (Von Troil-Linden B 1995)
3) Route of infection from person to person-
Since there is no evidence that periodontal
pathogens would be disseminated in aerosols
as, for example, respiratory pathogens, it is
likely that the person- to-person transmission
occurs via salivary and mucosal contact or an
inanimate object.

 The role of saliva as a transport vehicle has been
supported by the findings that A.
actinomycetemcomitans and P gingivalis can be
cultured from salivary samples.

VIRULENCE FACTORS OF A.

 Factors that promote colonization and persistence in
the oral cavity
• Adhesins
• Invasins
• Bacteriocins
• Antibiotic resistance
 Factors that interfere with the 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

1) Adhesion of Aa
 The bacterial surface components involved in
adhesion are adhesins.
 In most cases, adhesins are proteinaceous
structures found on the surface of the bacterial
cell. They interact and bind to very specific
receptors in saliva, on the surface of the tooth,
on extracellular matrix proteins and on
epithelial cells.

Epithelial cells
 Most A. actinomycetemcomitans strains that
have been tested adhere strongly to epithelial
cells. Binding occurs very rapidly, reaching
saturation levels within 1 hour after infection.
 Cell surface entities that mediate adherence
include fimbriae, extracellular amorphous
material and extracellular vesicles.

2) Extracellular matrix proteins.
 In order to initiate disease in extraoral sites
(such as endocarditis and osteomyelitis), A.
actinomycetemcomitans must bind to the
extracellular matrix, the complex network of
proteins and polysaccharides that underlies
epithelial and endothelial cells and surrounds
connective tissue.

 Autotransporters constitute the largest known
family of bacterial extracellular proteins. Recent
studies have shown that A
actinomycetemcomitans produces three
autotransporter proteins:
 Aae;
 EmA; and
 ApiA (also known as Omp100)

 The major component of the extracellular
matrix is collagen.
 It has recently been demonstrated that A.
actinomycetemcomitans binds to immobilized
collagen types I, II, III and V but not to type IV
collagen.

3) Antibiotic resistance
 Tetracyclines, as an adjunct to mechanical
debridement, are antibiotics frequently
employed in treating infections of localized
juvenile periodontitis.
 In a recent study, 82% of 19 clinical isolates of
A. actinomycetemcomitans were resistant to
tetracyclines.

4) Bacteriocins
 Bacteriocins are proteins produced by bacteria
that are lethal for other strains and species of
bacteria.
 The mode of action is to increase the
permeability of the cell membranes of target
bacteria, which leads to leakage of DNA, RNA
and macromolecules essential for growth.

5) Bone resorption
A characteristic feature of periodontal disease is
the loss of bone supporting the teeth. A.
actinomycetemcomitans has been shown to
stimulate bone resorption by several different
mechanisms:
a) Lipopolysaccharide,
b) Proteolysis-sensitive factor in microvesicles
c) Surface-associated material.

6) Collagenase
 A major feature of periodontal disease is a
marked reduction in gingival collagen fiber
density. Collagenase activity is associated with
two important periodontal pathogens, A.
actinomycetemcomitans and P gingivalis.
 Some of the reduction in collagen density may
also be due to tissue collagenases induced in
periodontal disease.

7) Cytotoxins
 Many oral bacteria express toxins that inhibit
human fibroblast proliferation,But the heat-
labile cytotoxin produced by A.
actinomycetemcomitans is especially cytotoxic.
 The toxin is considered a virulence factor due
to its impact on fibroblast viability. 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.

 A. actinomycetemcomitans surface-associated
material at very low concentrations has also
been shown to inhibit fibroblast proliferation.
The active component of surface-associated
material, designated Gapstein, is an 8-kDa
protein.

8) Extracellular membranous vesicles
 Almost all strains of A.a examined extrude
membrane vesicles from their surface. These
vesicles often contain leukotoxin, endotoxin,
bone resorption activity and a bacteriocin.
Scanning electron micrograph
of A. actinomycetemcomitans
Strain revealing the presence
of large amounts of fibrillar
membranous vesicles (arrows)
on the cell surface.

 A. actinomycetemcomitans vesicles must also
contain adhesins, since their addition to weakly
adherent or nonadherent strains significantly
increases the ability of those strains to attach to
epithelial cells. It is not clear what role vesicles
play in the pathogenesis of A.a.

9) 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 polymorphonuclear leukocytes,
binding of the antibody may be inhibited and,
thereby, inhibit phagocytosis. Fc binding
proteins have been associated with A.

 Tolo & Hegland demonstrated that molecules
on the surface of A. actinomycetemcomitans
that are associated with capsular material and
secreted into the medium bind to the Fc portion
of immunoglobulin G (IgG), the binding
inhibits the ability of opsonizing antibodies to
bind polymorphonuclear leukocytes and reduces
phagocytosis by 90%.

10) Leukotoxin
 One of the most studied virulence factors of A.
actinomycetemcomitans is leukotoxin.
 Leukotoxin is a member of the RTX (repeat in
toxins) family of toxins that produce pore-forming
hemolysins.
 They have a varying no. of glycine rich calcium
binding tandem repeats in the N- terminal fo the
structural molecule, hence referred as repeat in
toxins.

 The leukotoxin gene (ltxA) resides in an operon
consisting of four genes, C, A, B and D.
 ltxB and ltxD code for proteins that are involved
in transporting the toxin to the surface of the
cell, while ltxC post-translationally activates the
toxin.
 Gene A encodes the ltx itself and is produced in
an inactive protoxin state.

 The C gene product is required to activate
protoxin
 B and D genes are involved in secretion of the
leukotoxin from the bacterial cell.

 It is secreted outside the periplasmic space, but
remains adherent to the nucleic acids that coat
the outer surface of Aa cells.
 The RTX have a membrane spanning domain in
the C terminus end of the structural toxin
molecule that can insert into the PMLN-
membrane phospholipids, resulting in the
formation of a pore that allows influx of Ca and
efflux or K and macromolecules such as ATP.

Molecular basis of variability in leukotoxin
production:
 This is attributed to the variation in the DNA
sequene of leukotoxin promoter region.
 Bacterial promoters consist of specific segments
of DNA that provide a recognition and binding
site for the enzyme RNA polymerase which is
responsible for RNA synthesis.

 Investigation of the DNA sequence upstream of
the ltxC gene revealed that the highly toxic
strains have a deletion of 530 base pairs of DNA
as compared with the minimally toxic strains.

Immunosupressive factors:
 A protein which blocks cell cycle progression in
G2 by a unique mechanism of action and has a
potent proinflammatory cytokine stimulating
activity, with extremely potent induction of IL-6
and IL-8 synthesis by monocytes and
fibroblasts.
 PGE2 is suggessted to be involved in the
mechanism of formation of osteoclast like cells
mediated by Aa

MECHANISM OF LEUKOTOXIN ACTION
 Ltx A mediated mechanisms of cell death is
through necrosis or apoptosis.
 Necrosis - exposure of neutrophils and
macrophages to strains that produce large amount
of Ltx A results in killing in a short period of time.
 this cell death is thought to result from the ability
of Ltx A to form pores in the membrane of target
cells, leading to osmotic lysis caused by water influx
into the cell.

 Apoptosis – prolonged exposure of lymphocytes
and NK cells to Ltx A results in the induction
of apoptosis.
 Lower concentrations of Ltx A result in
apoptosis whereas higher concentrations result
in necrosis.

11) Lipopolysaccharide
 Lipopolysaccharides (endotoxins) have a high
potential for causing destruction of an array of
host cells and tissues.
 Tissue destruction is a key feature of periodontal
diseases; thus, the lipopolysaccharide of A.
actinomycetemcomitans has been extensively
characterized.

 It causes skin necrosis(Schwartzmann reaction),
bone resorption and platelet aggregation, and it
activates macrophages.
 Low concentrations of A. actinomycetemcomitans
lipopolysaccharide stimulate macrophages to
produce interleukins (IL- lα, IL-lβ) and TNF,
cytokines involved in tissue inflammation and
bone resorption.

 These data suggest that macrophages that
migrate to gingival sites of A.
actinomycetemcomitans infection will be
stimulated to produce these cytokines, which
may then be involved in gingival inflammation
and alveolar bone resorption.

12) Immunosuppressive factors
 A. actinomycetemcomitans has been shown to
elaborate many factors capable of suppressing
these host defense mechanisms.
 The organism produces a protein that inhibits
DNA, RNA and protein synthesis in mitogen-
activated human T cells.

 A 60-kDa protein secreted by A.
actinomycetemcomitans has been purified and
shown to inhibit IgG and IgM synthesis by
human lymphocytes.

 Many strains of periodontal pathogens including
every strain of Aa are resistant to the bactericidal
action of serum.
 The only way a host can clear an infection by a
serum-resistant organism necessarily involves
phagocytes.

 Polymorphonuclear leukocytes and their intact
function are absolutely required to regulate the
levels of serum resistant bacteria external to the
epithelium.

HUMORAL IMMUNE RESPONSE
FOR Aa IN PERIODONTAL
DISEASE

FACTORS INFLUENCING
THE GROWTH AND
VIABILITY OF Aa

1.Appropriate culture
medium
• selective media
• defined media
2. Effects of
supplements
• Yeast extract
• cystine
• hormones
• iron
• Ph
• Salt

Selective media
 Selective media enable one to determine the
organism’s role in disease by assessing its
incidence and numbers in disease and healthy
sites.
1. MGB, a selective medium that utilizes a
trypticase soy broth with malachite green and
bacitracin
2. Trypticase soy agar and serum with bacitracin
and vancomycin added as the selective agents
(TSBV)

Defined media
 Precise nutritional requirements of a
microorganism are determined using chemically
defined media because the exact composition of
each medium is known.
 For Aa , the defined medium is tissue medium
ex: RPMI 1640
Dulbecco’s modified eagle medium

 Effect of supplements
 Yeast extract.
■ Yeast extract has been consistently used to
supplement media for the growth of A.
actinomycetemcomitans and many other
periodontal bacteria.
■ The addition of increasing amounts of yeast
extract to trypticase soy broth enhances the
growth of most strains of
A.actinomycetemcomitans.

Cystine
 Cystine and thiamine were two components
identified from a number of vitamins and
aminoacids tested that promoted the growth of
A.actinomycetemcomitans.
 The addition of cystine, an essential amino acid,
significantly increased the growth of all strains of
A. actinomycetemcomitans.

Hormones
 Steroid hormones including estrogen,
progesterone and testosterone are capable of
enhancing the growth of Aa
Iron
 Aa expresses iron binding proteins and has
hemin binding activity.
 Furthermore, Aa down regulates expression of a
70 KDa membrane protein in iron limited
conditions

pH
 The pH of the gingival crevice varies from below
7.0 during health to above 8.0 during inflammatory
disease.
 A. actinomycetemcomitans demonstrates good
growth between pH 7.0-8.0, with optimal growth at
pH 7.5

Salt concentration
 The concentration of sodium in the gingival
fluid ranges from 90 mEq/l in health to 136
mEq/l following inflammation.
 A. actinomycetemcomitans demonstrates
optimal growth between 85.1 mEq/l and 170
mEq/l.

DIAGNOSTIC METHODS FOR
ACTINOBACILLUS
ACTINOMYCETEMCOMITAM

 Detection methods for A.
actinomycetemcomitans and P gingivalis be
divided into four general categories:
1. Culture,
2. Immunodiagnostic,
3. Nucleic acid probe and
4. PCR
5. Gene expression profile dileniation

Bacterial culturing
 Culture has been used for decades in the
detection of A. actinomycetemcomitans and P
gingivalis and is frequently used as the reference
method.
 Most significantly, culture can provide antibiotic
susceptibility of A. actinomycetemcomitans and
P gingivalis and total microbial content,
information that is not obtainable by other
current microbial detection methods.

IMMUNODIAGNOSTIC METHODS
 Employs antibodies that recognize specific
bacterial antigens to detect target micro
organisms.
 This reaction can be revealed using a variety
of procedures, including
1. Direct and indirect immuno fluorescent
microscopy assays (IFA)
2. Flow cytometry
3. ELISA

4. Membrane assay
5. latex agglutination
6. microarray
 Immunodiagnostic methods have the
advantage of being fast and inexpensive.
However, cross-reactivity with non-target
organisms may occur.
 Also, immunodiagnostic methods generally
provide poorer detection limit for A.
actinomycetemcomitans and p gingivalis than
culture, nucleic acid probe or PCR assays.

 NUCLEIC ACID PROBES that hybridize to
species-specific regions of the genome may
show a good detection limit and no cross-
reactivity with other oral bacterial under optimal
conditions.
 PCR demonstrates an excellent detection limit
and is highly specific for A. actinomycetemcomitans
and P gingiualis under optimized amplification
conditions.

GENE EXPRESSION PROFILE
DILENIATION:
■ IVIAT- in vivo induced antigen technology- can
identify microbial antigens that are expressed
during infection in a host, using pooled sera from
infected individuals.
■ IVET- in vivo expression technology- is a
genetic system designed to identify virulence
genes that are induced during infection.

LOCALIZED AGGRESSIVE
PERIODONTITIS AND A.a

 Kochs stringent criteria cannot or can only with
great difficulty be fulfilled for organisms that
cannot be grown in pure culture (which includes
A a), that demonstrate a very long incubation
period or often occur in an asymptomatic carrier
state (most suspected periodontal pathogens),
that express pathogenicity first after another
infectious agent has weakened the host immune
response -

(superinfecting organisms in HIV and
herpesviruses infections) and that exhibit a host
range that is restricted to humans or to animal
species in which the human disease cannot be
reproduced (P gingivalis does not usually colonize
animals).
 Another set of limitations in fulfilling Koch’s
postulates is apparent for periodontal disease.

 The actual state of periodontal disease
progression can be difficult to determine, the
same clinical signs and symptoms may be
produced by several organisms and the same
pathogenic organism may give rise to a variety of
clinical disease features.

 Koch’s postulates have been modified by
various authors to designate the microorganisms
as responsible for a particular diseaese.
 The one accepted for A a is given by Socransky
(Socransky SS, Haffajee AD: The bacterial
etiology of destructive periodontal disease:
Current concepts. j Periodontol 1992; 63:322.)

Criterion A actinomycetemcomltans
Association •Increased in localized aggressive
periodontitis (LAP) lesions
•Increased in some chronic
periodontitis lesions
•Detected in the tissues of LAP
lesions

Elimination •Suppressed or eliminated in
successful therapy
•Found in recurrent lesions
Host response Increased serum and local
antibody levels in LAP
Animal studies Capable of inducing disease in
gnotobiotic rats

Virulence factors •Host tissue cell invasion,
leukotoxin, collagenase,
endotoxin
•(LPS), epitheliotoxin,
fibroblast inhibiting
•factor, bone resorption-
inducing factor

 The following possible reasons for the
limitation of periodontal destruction to certain
teeth have been suggested:
1) After this initial colonization, adequate
immune defenses are stimulated to produce
opsonic antibodies to enhance the clearance
and phagocytosis of invading bacteria

2) Bacteria antagonistic to A. actinomycetemcomitans
may colonize the periodontal tissues and inhibit A.
actinomycetemcomitans,
3) A. actinomycetemcomitans may lose its leukotoxin
producing ability for unknown reasons,
4) The possibility that a defect in cementum formation
may be responsible for the localization of the lesions
has been suggested.

 Several lines of clinical evidence that support the
association of A. Actinomycetemcomitans with
localized aggressive periodontitis.
1. The organism is found more frequently in
samples obtained from subjects with localized
aggressive periodontitis compared with samples
obtained from periodontally healthy subjects or
subjects with gingivitis or other forms of
periodontal disease.

2. Subjects with localized aggressive periodontitis
were consistently found to have elevated serum
and locally produced antibody titers to A.
Actinomycetemcomitans,
3. Several studies indicated that the treatment of
subjects with LAP with the intention of
reducing A. actinomycetemcomitans to
undetectable levels resulted in marked clinical
improvement,

while a lack of clinical improvement was found
to correlate with a failure to significantly reduce
the level of A. Actinomycetemcomitans.
4. Furthermore, disease exacerbation was shown
to relate to continued growth of A.

 Difficulty removing A. actinomycetemcomitans
from the subgingival environment by rigorous
debridement procedures was attributed to the
ability of A. Actinomycetemcomitans to
repopulate or re-infect subgingival sites from
other locations in the oral cavity.

Temporal aspects of disease
 The first step in the infectious process occurs
when the organism in question is transmitted
from a carrier to a susceptible individual.
 Once the pathogenic organism has colonized its
preferred target tissue site (the junctional or
pocket epithelium) a series of complicated
interactions take place in this local environment
over a defined period of time.

 The time prior to a tissue-damaging infection
varies and relates to the virulence of the
organism and the susceptibility of the host.
1. Incubation period- Time required for the
organism to reach the appropriate
concentration so that it can overwhelm the
host at the epithelial tissue surface interface.

 For A. actinomycetemcomitans, it is estimated
that the minimal concentration required for
disease is 1 × 106/ml.
 It would be logical to assume that it might
require a minimum of 2–3 weeks to achieve this
minimal infective dose.

2. The prodromal period- Infecting organism
produces a subclinical infection only detectable
by sophisticated clinical tools.
3. The period of disease- frank tissue-destructive
activity takes place and the host is
compromised.

4. The 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 which counteracts the
forces of destruction.
 The disease becomes quiescent.

Attachment of Aa
 For A. actinomycetemcomitans to act as the
driving pathogenic force in disease initiation, A.
Actinomycetemcomitans would be required to
1. attach (via A. Actinomycetemcomitans
adhesins and other cell-associated proteins),
2. evade the host defense(via A.a leukotoxin and
cytolethal distending toxin),
3. penetrate its target tissue (possibly through A.a
cdt, api A, aphA) and then cause tissue
destruction

 A. actinomycetemcomitans has been designated
as a tertiary colonizer, an organism that attaches
poorly and binds to bacteria that have already
colonized tooth surfaces.

 A. Actinomycetemcomitans attachment is
carbohydrate dependent, a fact which has been
exploited and has led to the discovery of an
enzyme, hexaminidase, that attacks N-
acetylglucosamine residues that play a role in A.
actinomycetemcomitans aggregation.

Colonization
 Recent literature indicates that A.
Actinomycetemcomitans can colonize
predentate children.
 This implies that the oral mucosa may, in fact,
be the initial colonizing site in the oral cavity.
 Rudney et al described that buccal epithelial
cells could be thought of as a protected reservoir
for A. actinomycetemcomitans.

 Once the initial colonizing organisms take hold,
they provide a new set of conditions for the
succeeding populations of microorganisms.
 In this manner, the pioneer community sets up
the environment for the pattern of succession.

 This phenomenon has been studied by
examining co-aggregating species, leading to
scenarios which suggest that there are pioneers,
secondary and then tertiary colonizers.
 This scenario suggests an exquisite order to
developing dental plaque biofilm communities.

 A. Actinomycetemcomitans has also been
isolated from freshly cleaned tooth surfaces of
humans after 2–6 h, and from macaque monkeys
3–5 h after thorough cleaning, suggesting that A.
actinomycetemcomitans has the potential to be
an early colonizer of teeth.

 High prevalence of LAP is seen in African
Americans.
 Hubek et al in 1996 discovered JP2 clonal type
of Aa characterized by 530 base pair deletion in
the leukotoxin gene operon leading to increase
in production of leukotoxin.

 A.a is also isolated from chronic periodontitis
lesions but less frequently and in lower numbers
than LAP. (Rodenburg et al 1990, Slots et al
1990).

Prevention and control of periodontitis caused
by Aa
1. Alter subgingival environment
 reduction in probing depth
 mechanical removal or disruption of subgingival plaque
biofilm
 application of oxygenating and redox agents
2. replacement therapy
 pre-eruptive colonization
 competitive replacement

Reduction in probing depth
Surgical or non-surgical has been successful in the
treatment of periodontal disease, achieving an
immediate ecological change that favors a facultative
anaerobic gingival microflora and depriving the
subgingival microflora of its anaerobic environment at
the base of the deep pockets which is mandatory for
the growth of Aa .

Mechanical removal or disruption of
subgingival biofilm
Mechanical removal of biofilm changes the ecology and
the remaining micro-organisms become accessible to
both host factors and Antimicrobial agents

Application of oxygenating and redox agents
• Although the use of redox agents do not release
oxygen, the dyes can raise the redox potential of an
ecosystem.
• The dye most commonly used is methylene blue.

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.
• 2 main approaches to the use of replacement therapy to
prevent periodontal disease are:

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
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

EFFECT OF PERIODONTAL
TREATMENT ON SUBGINGIVAL
A. ACTINOMYCETEMCOMITANS

 In 1983, Slots & Rosling showed that scaling
and root planing alone was unable to remove A.
actinomycetemcomitans from localized juvenile
periodontitis lesions.
 The study also showed that nonsurgical therapy
had the least effect on A.
actinomycetemcomitans counts in heavily
infected periodontal lesions.

 The failure of nonsurgical therapy to effectively
control A.a from subgingival sites may be due
the ability of the organism to invade gingival
tissue and thereby evade the effect of
mechanical debridement and periodontal
healing.
 A. actinomycetemcomitans cells in gingiva may
constitute a reservoir for repopulating
periodontal pockets. Saglie FR).

 Periodontal surgery also often fails to control
effectively subgingival A. actinomycetemcomitans.
 Modified Widman flap surgery may suppress A.
actinomycetemcomitans to below detectable levels
in about 50% of localized juvenile periodontitis
lesions and may be even less effective in adult
periodontitis lesions. (Slots J, Rosling BG. 1983).

 Tuan et al. 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 A. actinomycetemcomitans.

 Other studies have also indicated that apically
positioned flap surgery or gingivectomy is
capable of controlling subgingival A.
 Resective types of periodontal surgery are more
effective than access flap surgery in combating
subgingival A. actinomycetemcomitans.

 The superior performance of resective
periodontal surgery may be due to the excision
of A. actinomycetemcomitans-infected gingival
tissue and to pocket depth reduction to levels
permitting adequate cleaning by tooth brushing,
flossing or other oral hygiene measures.

 Systemic antibiotic therapy has the potential to
eradicate A. actinomycetemcomituns residing in
periodontal pockets and gingival tissue.
 Tetracyclines (250mg qid for 7 days) were the
first antibiotics to be used against periodontal A.
actinomycetemcomitans infections. Tetracycline
combined with scaling or root planning or with
periodontal surgery may markedly suppress or
eliminate subgingival A. actinomycetemcomitans
in localized juvenile periodontitis lesions. ( Slots
J, & Rosling ).

 Systemic tetracycline has been ineffective in
suppressing A. actinomycetemcomituns in some
localized juvenile periodontitis lesions and in
other types of periodontal disease. ( Muller HP
& Lange DE).
 Systemic metronidazole has demonstrated good
anti-A. actinomycetemcomitans activity in
localized juvenile periodontitis patients but not
in adult periodontitis patients.( Saxen &
Asikainen S)

 Systemic use of amoxicillin-metronidazole has
shown striking clinical results in the treatment of
A. actinomycetemcomitans-associated localized
juvenile periodontitis, adult periodontitis and
refractory periodontitis, even in the absence of
other periodontal therapy (Van Winkelhoff et al
1996-recommended regimen 250 mg of
metronidazole and 375mg of amoxycillin tid for
7 days).

 However, this is not always guarenteed, as
shown in a recent report on patients with
periodontitis who revealed subgingival A.
actinomycetemcomituns after the amoxicillin-
metronidazole combination therapy ( Fleming et
al).

Replacement therapy
 S. sanguis produces H2O2 which either directly
or by host enzyme ampification kills A.a.
 Dongary and Miyasaki in 1991 showed the
bactericidal effect of H2O2 on strain of A.a may
involve intracellular formation of OH which can
directly induce DNA strand scission.

A.a & PERIODONTAL
REGENERATION

 The regenerative periodontal devices are placed in
the potentially highly infected periodontal
environment, and their successful application
depends upon the prior removal or effective
suppression of periodontal pathogens at treated
site(s).
 A. actinomycetemcomitans and P gingivalis can
attach to barrier membranes and P gingivalis can
penetrate porous barrier membranes from one
side to the other.

 Both organisms have been implicated in failing
regenerative periodontal therapy.
 Machtei et al. detected A.a in periodontal sites
exhibiting suboptimal regeneration.

A. actinomycetemcomitans in ailing or
failing dental implants and in
periodontal regeneration
 Mengel et al. studied 35 healthy implant sites in
five partially edentulous patients who had been
treated for severe periodontitis. None of the 136
implants in the two studies yielded A.
actinomycetemcomitans or P gingivalis.

 In 37 healthy implant sites in 18 edentulous
patients and one partially edentulous patient,
Ong et al. detected A.
actinomycetemcomitans in one implant site and
no P gingivalis in any site.
 George et al. recovered A.
actinomycetemcomitans from 14 of 98 implants
in edentulous and partially edentulous patients.

 A. actinomycetemcomitans and especially P
gingivalis comprise major pathogens in
infectious implant failure.

Other oral infections
 A. actinomycetem-comitans, in concert with
cytomegalovirus or Epstein-Barr type 1 virus,
plays an important role in the development of
Papillon-Lefevre syndrome periodontitis.
 The organism may also act as a pathogen in
some types of advanced adult periodontitis,
particularly in lesions recalcitrant to mechanical
therapy. (Jogren Slots 1999)

Pathways of oral microbial
dissemination and Non oral infections
 The tissue-invasive property of A.
actinomycetemcomitans makes this organism a likely
candidate for dental focal infection.
 Healthy individuals exhibit a rapid clearing of
bacteria in the bloodstream.
 However, damaged heart valves, prosthetic
devices or other locus minoris resistentiae can
provide a niche for the survival of oral bacteria.

 Aspiration of oropharyngeal secretions, dental
plaque and suppurating periodontal lesions can
lead to oral bacteria gaining access to the lower
respiratory tract and causing pleuropulmonary
infection.
 In 1911, Hunter suggested that swallowed
bacteria, from periodontitis lesions could cause
gastric ulcers.

 Even though Hunter’s notion of a microbial
cause for gastic ulcer proved correct, the
causative bacterium H. pylori is not a resident
member of the subgingival microbiota, even
though the organism may occasionally occur in
other sites of the mouth.
 Cyanotic congenital heart disease constitues a
risk factor for intracranial infection.

 A right-to-left intracardiac shunt allows oral
microorganisms and other microemboli to
bypass the normal filtration of the pulmonary
circulation and enter the cerebral circulation.
 Oral organisms may subsequently adhere to and
grow in microinfarcts of the brain, occurring as
a result of other pathological events, and give
rise to intracranial infection.

 Endocarditis represents the most frequent
nonoral A. actinomycetemcomitans infection.
 Bacteria from infective endocarditis lesions can
be disseminated via the blood vessels and
colonize other body sites.
 The event can take place unnoticed or be
associated with septicaemia.

LAP and caries
Daniel H Fine et al 2006
 The contrast between the exaggeratedly
aggressive proximal periodontal disease seen in
children with localized aggressive periodontitis
and the reduced proximal decay seen in these
same children suggests that local environmental
regulatory mechanisms could influence these
two distinctly different infections.

 As such, in localized aggressive periodontitis
the growth and colonization of the gram
negative (disease-provoking) microflora appears
to occur at the expense of the gram-positive
caries-provoking flora.

 Unlike caries, where the bacteria colonize the
tooth, ingest carbohydrates and produce acid
that causes tooth demineralization, localized
aggressive periodontitis is an infectious disease
that appears to require bacterial colonization of
atleast three separate habitats or domains. Each
of these three domains has different
environmental specifications.

 Thus, in the case of localized aggressive
periodontitis, A. actinomycetemcomitans
appears to be endowed with the machinery that
allows it to colonize and survive on the oral
mucosa, the tooth surface and in the subgingival
domain.

Aa and orthodontics
 The presence of orthodontic appliances
produces a steadily increased rate of subgingival
colonization by Aa among individuals presenting
with an initially healthy periodontium.
Paolantonio M et al 1997.

CONCLUSION
 There is significant evidence implicating A.
actinomycetemcomitans as a microorganism that
is highly associated with an aggressive form of
periodontal disease found in young adults.

 New genetic methods may have to be used to
account for the horizontal transfer of genetic
material from one organism to another in the
plaque matrix and how this transfer may account
for new phenotypes that are critical for disease
pathogenesis.

A. actinomycetemcomitans possesses traits that
enable it to colonize, invade, avoid the host-defensive
strategies and cause tissue destruction .All of this evidence
would lead one to believe that there is a cause and effect
relationship between A. a and localized aggressive
periodontitis. Nevertheless, the exact mechanism of A.
a interaction with its fellow community inhabitants, and
the collective or individual influence of these associations
on host cell signalling, are just at the earliest stages of
investigation and future studies are needed to get deeper
insight into this small creature.

REFERENCES
 Slots and Taubman- Contemporary oral
microbiology and immunology.
 Per Axelsson- diagnosis and risk prediction of
periodontal diseases, vol 3.
 Daniel H. Fine et al. How we got attached to
Actinobacillus actinomycetemcomitans: a model
for infectious diseases. Periodontology 2000,
Vol. 42, 2006, 114–157.

 Jorgen Slots & Miriam Ting. Actinobacillus
actinomycetumcomitans and porphyromonas
gingivalis,perio 2000, 1999, 20, 82-121.
 Microbiological findings after periodontal therapy
using curettes, Er:YAG laser, sonic, and ultrasonic
scalers. J Clin Periodontol 2007; 34: 588–598.
 Microbiology of periodontal diseases: selected
pathogens and treatment. Perio 2000, vol 42.2006.
 Caranza clinical periodontology 10th edition.

 Niels Nørskov-Lauritsen and Mogens Kilian.
International journal of systematic and
evolutionary microbiology 2006 56 2135-2146
 Ingar OlsenTaxonomv and biochemical
characteristics of Actinobacillus
actinomycetemcomitans and Porphyromonas
gingivalis. Periodontology 2000, Vol. 20, 1999,
14-52

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