2. Aggregatibacter actinomycetemcomitans (AAC) is a Gram-negative, facultative anaerobe, nonmotile
bacterium that is often found in association with localized aggressive periodontitis, a severe infection
of the periodontium. It is also suspected to be involved in chronic periodontitis.
Less frequently, it is associated with nonoral infections such as endocarditis. Its role in
aggressive periodontitis was first discovered by Danish-born periodontist Jørgen Slots, a professor
of dentistry and microbiology at the University of Southern California School of Dentistry.
'Bacterium actinomycetem comitans' was first described by Klinger (1912) as coccobacillary bacteria
isolated with Actinomyces from actinomycotic lesions in humans.
It was reclassified as Actinobacillus actinomycetemcomitans by Topley & Wilson (1929) and
as Haemophilus actinomycetemcomitans by Potts et al. (1985). The species has attracted attention
because of its association with localized aggressive periodontitis
3. AGGREGATIBACTER ACTINOMYCETEMCOMITANS
Scientific classification
Domain: Bacteria
Phylum: Proteobacteria
Class: Gammaproteobacteria
Order: Pasteurellales
Family: Pasteurellaceae
Genus: Aggregatibacter
Species: A. actinomycetemcomitans
Binomial name
AGGREGATIBACTER
ACTINOMYCETEMCOMITANS
(Klinger 1912) nørskov-lauritsen and
kilian 2006
Synonyms
•Actinobacillus actinomycetemcomitans (Klinger
1912) Topley and Wilson 1929 (Approved Lists 1980)
•"Bacterium actinomycetemcomitans" Klinger 1912
•Haemophilus actinomycetemcomitans (Klinger 1912)
Potts et al. 1985
4. NOMENCLATURE:
Recent studies have shown a phylogenetic similarity of A.
actinomycetemcomitans and Haemophilus aphrophilus, H. paraphrophilus, and H.
segnis, suggesting the new genus Aggregatibacter for them.
IMPORTANCE
It is one of the bacteria that might be implicated in destructive periodontal disease.
Although it has been found more frequently in localized aggressive
periodontitis,prevalence in any population is rather high. It has also been isolated
from actinomycotic lesions (mixed infection with certain Actinomyces species, in
particular A. israelii).
It possesses certain virulence factors that enable it to invade tissues, such as the
pore-forming toxin leukotoxin A. It has also been isolated from women
with bacterial vaginosis and as an etiologic agent in endocarditis.
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
5. VIRULENCE FACTORS
• Leukotoxin A: kills granulocytes, monocytes, and other white blood cells expressing integrin
beta-2 (CD18)
• Cytolethal distending toxin
• Immunosuppression factors that inhibit blastogenesis, antibody production, and activate T-
suppressor cells
• Inhibition of granulocyte functions
• Resistant to complement-mediated killing
• Lipopolysaccharides
• Surface antigens
• Heat shock proteins
• Antimicrobial resistance
6. A. actinomycetemcomitans is a non-motile, gram-negative, capnophilic, fermentative coccobacillus
which closely resembles several Haemophilus species but which does not require X or V growth
factors
The organism has been categorized into 10 biotypes based on the variable fermentation of dextrin,
maltose, mannitol, and xylose and into 3 serotypes on the basis of heat stable, cell surface antigens.
A. actinomycetemcomitans primary human ecologic niche in the oral cavity.
It is found in dental plaque, in periodontal pockets, and buccal mucosa in up to 36% of the normal
population.
The organism can apparently seed from these sites to cause severe infections throughout the human
body such as brain abscesses and endocarditis.
7. There is a large body of evidence which implicates A. actinomycetemcomitans as an important
micro- organism in the etiology of localized juvenile periodontitis including:
(1) an increased prevalence of the organism in almost all localized juvenile periodontitis patients
and their families compared to other patient groups;
(2) the observation that localized juvenile periodontitis patients exhibit elevated antibody levels to
A. actinomycetemcomitent in serum, saliva and gingival crevicular fluid;
(3) the finding that localized juvenile periodontitis can be successfully treated by eliminating
A.actinomycetemcomitans from periodontal pockets;
(4) Histopathologic investigations showing that A actinomycetemcomitans invades the gingival
connective tissue in localized juvenile periodontitis lesions;
8. The demonstration of several pathogenic products from A.actinomycetemcomitans including
factors which may:
(a) facilitate its adherence to mucosal surfaces such as capsular polysaccharides;
(b) inhibit host defense mechanisms including leukotoxin, a polymorphonuclear leukocyte
chemotaxis inhibiting factor, and a lymphocyte suppressing factor
(c) cause tissue destruction such as lipopolysaccharide endotoxin, a bone resorption- inducing
toxin, acid and alkaline phosphatases, collagenase, a fibroblast inhibiting factor and an
spitheliotoxin
Although A.actinomycetemcomitant is an apparently important micro-organism in the etiology
of localized juvenile periodontitis, additional research is needed to pinpoint subjects at risk of
contracting this disease, to develop means of preventing localized juvenile periodontitis, and to
examine the role of this organism in other forms of periodontal disease.
9. Human periodontal disease includes a number of clinically discrete entities, some of which may
be associated with specific bacteria (Socransky 1977, Slots 1979). Perhaps the clearest such
relationship yet described is that between localized juvenile periodontitis and Actinobacillus
actinomycetemcomitans.
A number of recent studies incriminate this bacterium in the etiology of localized juvenile
periodontitis as well as in serious medical infections such as bacterial endocarditis.
10. Bacteria inhibit the oral cavity from birth to death. They colonize the soft tissues, including the gingiva, cheeks and
tongue and, when teeth are present, bacteria colonize them both above and below gingival margin.
While non-specific accumulations of dental plaque are associated with gingival inflammation, accumulated data
indicate that a small group of bacteria are important in periodontal disease exhibiting loss of connective tissue
attachment and alveolar bone.
Prominent among these mainly Gram-negative species are Actinobacillus actinomycetemcomitans, Bacteroides
forsythus, Campylobacter rectus, Fusobacterium nucleatum, Prevotella intermedia, Peptostreptococcus micros, and
Streptococcus intermedius,Porphyromonas gingivalis.
Periodontitis is the inflammatory response in gingival and connective tissue elicited by bacteria which accumulate on
adjoining teeth. In terms of quality of plaque, three specific bacteria have been identified as etiologic agents for
periodontitis: Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Bacteroides forsythus.
Actinobacillus actinomycetemcomitans is strongly implicated as an etiologic agent of this destructive disease.
INTRODUCTION
11. Actinobacillus actinomycetemcomitans was first recognized as a periodontal pathogen by its increased
frequency of detection and higher numbers in lesions of localized juvenile periodontitis when compared with
numbers in plaque samples from other clinical conditions including periodontitis, gingivitis and health.
Actinobacillus actinomycetemcomitans is major putative periodontopathic bacteria.
Actinobacillus actinomycetemcomitans has been closely associated with periodontitis in young individuals and
with cases of refractory adult periodontitis. Clinical, microbiological and immunological studies all provide
evidence of a strong correlation between Actinobacillus actinomycetemcomitans and periodontal disease.
12. TAXONOMY AND BIOCHEMICAL CHARACTERISTICS OF ACTINOBACILLUS
ACTINOMYCETEMCOMITANS:
Actinobacillus actinomycetemcomitans is a member of the genus Actinobacillus that belongs to the family
Pasteurellaceae. This family was constructed to accommodate a large group of gram-negative chemoorganotrophic,
facultative anaerobic and fermentative bacteria consisting of the genera Pasteurella, Actinobacillus and Haemophilus,
and several other groups of organisms showing complex phenotypic and genotypic relationships with the
aforementioned genera. Most members of this family cause disease in mammals including humans, birds, and/or
reptiles.
CELLULAR LIPIDS AND CARBOHYDRATES OF PASTEURELLACEAE
The distribution of cellular lipids and carbohydrates in the family Pasteurellaceae was reviewed by Mutters et al. The
cellular fatty acid composition was uniform with minor variations. Also the distribution of phospholipids was uniform,
while the lipoquinone content was useful for discrimination of groups within the family. Cellular carbohydrates showed
a common pattern within all members but with profiles that could distinguish between groups, often at the species level.
POLYAMINE PATTERNS OF PASTEURELLACEAE
Moller Analysis of polyamines was promising for discriminating members of the family Pasteurellaceae. The strains
examined included the type strains of species belonging to the genera Actinobacillus, Haemophilus and Pasteurella and
additional strains of selected 4 Dew species, as well as numerous unnamed strains. Members of the genus Actinobacillus
contained 1, 3-diaminopropane as the predominant compound.
13. Genus Actinobacillus
In 1902 Lingnieres 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. Brumpt in 1910 was the first to use a binominal for this bacterium. In the ninth edition of Bergeys
manual of determinative bacteriology the genus Actinobacillus contained 11 species.
Genetic studies
DNA-DNA hybridization studies changed the taxonomic picture of Actinobacillus. New species were transferred to the
genus due to homologies above 55%. These included one of the recognized species of Pasteurella, P. ureae, which was
reclassified as A. ureae.Another new species was Actinobacillus pleuropneumoniae, which was transferred from
Haemophilus by Pohl et al in 1983." Actinobacillus muris was created by Bisgaard and Actinobacillus rossii and
Actinobacillus seminis by Sneath & Stevens, Actinobacillus delphinicola by Foster et al and Actinobacillus minor,
Actinobacillus porcinus and Actinobacillus indolicus by Moller et al. Based upon DNA- DNA homology studies.
Bisgaard suggested that the genus Actinobacillus should also include Bisgaard's taxa. True actinobacilli were included in
rRNA cluster 4A of Dewhirst et al, which in addition to A. lignieresii. A pleuropneumoniae, Bisgaard's taxon, A. ureae,
A. equuli and A. suis, included the type strain of Haemophilus parahaemolyticus. Additional sequencing work included
A. hominis.
14. HISTORICAL BACKGROUND
A. actinomycetemcomitans was first described by the German microbiologist Klinger, who
isolated the organism from cervicofacial actinomycosis.
It was originally named Bacterium actinomycetem comitants (Klinger 1912) which was changed
to Bacterium comitans (Lieske 1921) and finally to Actinobacillus actinomycetemcomitans
(Topley & Wilson 1929), like many terms in microbiology, the name is descriptive.
"Actinobacillie" refers to the internal star-shaped morphology of its bacterial colonies on solid
media (Colebrook 1920) and to the short rod or bacillary shape of individual cells (Linke 1921,
Slots 1982); actinomycetemcomitans reflects its close association with Actinomyces israelli in
actinomycotic lesions So little has been known about A. actinomycetemcomitans that it is listed
in the 8th Edition of Bergey's Manual of Determinative Bacteriology (Buchanan & Gibbons
1974).
15. Biochemical properties of A. actinomycetemcomitans
Slots studied 135 biochemical characters in 6 reference strains and 130 strains of A. actinomycetemcomitans freshly
isolated from the oral cavity. Up to then biochemical studies had been few and somewhat conflicting. All the isolates
were small, nonmotile capnophilic gram-negative rods that did not require X or V factor for growth.
They all decomposed hydrogen peroxide, were oxidase negative and benzidine-positive, reduced nitrate, produced
strong alkaline and acid phosphatases and fermented fructose, glucose and mannose. Variable fermentation results were
obtained with dextrin, maltose, mannitol and xylose.
With the exception of Bergey's manual of determinative bacteriology, eighth edition, A. actinomycetemcomitans had
been invariably reported to reduce nitrate to nitrite. Various results had been achieved with sugar fermentation. Several
studies found some strains to be galactose-positive.
Pulverer & Ko used fermentation of galactose as a differential character in describing biotypes of A.
actinomycetemcomitans. Variable results had also been listed with dulcitol, lactose and sucrose. Furthermore, positive
reactions had been recorded for arabinose, glycogen, inulin, raffinose and starch.
16. In Slots study the biochemical reaction pattern of A. actinomycetemcomitans was remarkably uniform. Characters useful
for distinguishing A. actinomycetemcomitans from the closely related H. aphrophilus included the catalase reaction,
fermentation of lactose, starch, sucrose, and sodium fluoride resistance, as well as the ability of H. aphrophilus to produce
ẞ-glucosidase and B-galactosidase.
A.actinomycetemcomitans was distinguished from Haemophilus species requiring X and V factors by its ability to grow in
absence of these factors.
Tanner et al found that none of their A. actinomycetemcomitans isolates from advanced periodontal pockets fermented
galactose, lactose, sucrose or trehalose.
Most H. aphrophilus and H. paraphrophilus strains fermented lactose, sucrose and trehalose but not mannitol or xylose.
Since not all A. actinomycetemcomitans strains decomposed hydrogen peroxide and isolates of H. aphrophilus occasionally
are catalase positive, the presence or absence of this feature was considered less useful for separating A.
actinomycetemcomitans from H. aphrophilus.
The ability of A. actinomycetemcomitans to ferment lactose can be tested for by using a ẞ galactosidase or MUG reaction.
17. Twenty out of 33 A. actinomycetemcomitans strains formed hemolytic colonies on horse blood agar plates under
anaerobic condition. Human erythrocytes were most susceptible.Presence of various biotypes in A. actinomycetem
comitans was first described by King & Tatum.
Fermentation reactions with galactose, mannitol and xylose permitted definition of 8 biotypes, and fermentation
based on dextrin, maltose, mannitol and xylose gave 10 biotypes.
18. Serotypes of A. actinomycetemcomitans
Pulverer & Ko, using tube agglutination assays, detected 24 groups within A. actinomycetemcomitans and 1 to 6
agglutinating antigens on each strain.
These serogroups, based on a heat stable component, were distinguished among normal A. acti- nomycetemcomitans by
King & Tatum. Taichman et al described four serogroups based on surface antigens and proteinaceous leukotoxin. Three
serotypes, a, b and c, were defined by Zambon et al.
They were similar to those reported by King & Tatum. Serotypes a and b were most common in the oral cavity, while
serotype e constituted 10% of the human oral isolates.
Serotype c seems to be particularly important in extra oral infection. A relationship was found of serotype b to localized
juvenile periodontitis and serotype c to periodontal health in adults.
In a study by Gunsolley et al, severe periodontitis and early-onset periodontitis were associated with higher levels of
serotype b and c in black subjects, whereas in white subjects serotype a was associated with severe periodontitis.
19. The number of recognized serotypes in A. actinomycetemcomitans was later extended to five: a, b, c, d and e. Most
subjects are infected with only one serotype, and A. actinomycetemcomitans infections seem to be relatively serotype-
stable. Variation in serotype determination may occur due to the bacterial growth and preparation procedures used.
A. actinomycetemcomitans-infected individuals with periodontal disease exhibiting elevated antibodies to multiple
serotypes were most consistently colonized with serotype b.
Furthermore, antibody reactive with A. actinomycetemcomitans serotype b lipopolysaccharide was protective in
generalized early-onset periodontitis.
Selective culture combined with an immunodiffusion assay was used to determine the serotype distribution of A.
actinomycetemcomitans in families.
The parents harbored the same serotype as the child in 22 of 23 A.actinomycetemcomitans-positive families suggesting
intrafamilial transmission of A. actinomycetemcomitans.
20. SEROTYPE-SPECIFIC ANTIGENS CHEMICAL COMPOSITION
According to Zambon, the serotype antigens of A. actinomycetemcomitans have a high molecular weight, are heat-
stable and have primarily carbohydrate moieties.
The serotype antigens can easily be detected by immunofluorescent techniques on whole cells indicating that they are
part of the outer cell surface, possibly the microcapsule.A. actinomycetemcomitans possesses at least one common
antigen with other Actinobacillus species and one antigen shared with H. aphrophilus and H. parainfluenzae.
Serotype-specific polysaccharide antigens from A. actinomycetemcomitans ATCC 29523 (serotype a) and NCTC 9710
(serotype c) have been reported to be new polysaccharide antigens.
The serotype a antigen consisted solely of 6-deoxy-D-talose, whereas the serotype c antigen was composed of 6-
deoxy-L-talose. Both these antigens contained closely related repeating units.
They had one acetyl group/2 sugar residues. Recently, the serotype b antigen of A. actinomycetemcomitans was
suggested to be defined by a trisaccharide repeating unit in the O-polysaccharide of lipopolysaccharide consisting of L-
Rha and D-GalNac, and that this structure is unrelated to that of the O-antigens of other serotypes.
21. SEROTYPES - GENETIC DISSIMILARITY
There is a major genetic dissimilarity between A. actinomycetemcomitans serotypes. Isolates belonging to the same serotype
were genetically identical in the same individual, but non-identical if they belonged to different serotypes. Furthermore, iso-
lates of the same or different serotypes were genetically non-identical in different individuals.
Serotypes of A. actinomycetemcomitans differed as much as many species in the phylogenetic tree of Pasteurellaceae
established after 16S rRNA sequencing, Poulsen et al found that each of the serotype a, b, c, d and e comprises generically
isolated subpopulation.
Successful horizontal transfer of genomic DNA between different serotypes of A. actinomycetemcomitans appears to be
extremely rare in vivo.
22. On the other hand, recombination between strains of the same serotype may take place. Thus non-serotype able
strains of A. actinomycetemcomitans may be serotype-deficient variants originating from strains of known
serotypes, while serotype b and c strains may contain transmittable DNA sequences not found in strains of the
other serotypes.
Among enzymes representative of the pentose phosphate pathway/hexose monophosphate shunt, glucose-6-
phosphate dehydrogenase and malate dehydrogenase were the most readily detected and stable enzymes in cell-
free extracts from the five serotypes of A. actinomycetemcomitans.
In these serotypes, three groups were delineated after enzyme electrophoresis with glucose-6-phosphate
dehydrogenase and malate dehydrogenase.
Genetic analysis by multilocus enzyme electrophoresis of an A. actinomycetemcomitans population of 88
clinically well characterized isolates divided the five serotypes into two phylogenetic lineages, one comprising of
serotypes a, d and e.
23. The association of A. actinomycetemcomitans with various forms of periodontal disease such as juvenile periodontitis,
rapidly progressive periodontitis and refractory periodontitis is strongly implicated by a great deal of evidence.
However, this organism can also be detected in non-diseased sites.
Socransky & Haffajee proposed that a small number of clones within species of periodontal pathogens might actually
be responsible for attachment loss in periodontitis. Recent studies have increasingly evaluated the clonality of oral
pathogens.
The past decade has seen extensive efforts to exploit the potential of clinical microbiology in the diagnosis and
treatment of periodontal disease. Clinicians are primarily interested in microbial diagnosis to
(i) Evaluate current periodontal disease activity.
(ii) Help to select an effective treatment modality and
(iii) Predict future periodontal disease status that is. Prognosis
24. SENSITIVITY, SPECIFICITY, DETECTION LIMIT AND CROSS-REACTIVITY
Sensitivity denotes the percentage of positive test results of a detection method employed to identify different strains
of the target microbial species, and specificity is the percentage of negative results when the detection method is
applied to a variety of non-target bacterial species.
The ideal detection method displays sensitivity and a specificity of 100% "cross-reactivity" connects reactivity with
non-targeted organisms.
Sensitivity may have the same meaning as "detection limit", denoting the minimum bacterial level for detecting the
target microorganism.
Sensitivity and specificity may indicate the ability of a diagnostic test to identify a disease condition.
25. CULTURE METHODS
Culture has a long history in the detection of A actinomycetemcomitans is subsequently used as the reference method when
determining the performance of a new detection method
Culture is the only current method capable of determining the in vitro antimicrobial susceptibility of periodontal pathogens.
Culture can also provide a quantitative measurement of all major viable microorganisms in the specimen
Culture identifies only the bacteria immediately after therapy, it may be important to determine the levels of viable
microorganisms and not antigens or bacterial DNA from possible dead bacteria unable to sustain a pathogenic infection.
Immunodiagnostic methods or nucleic acid-based detection methods do not discriminate between viable and nonviable
bacteria. However culture requires sophisticated equipment and experienced personnel and is relatively time-consuming and
expensive.
TSBV agar is an excellent primary selective medium for A. actinomycetemcomitans that can detect the microorganism in
levels as low as 20 viable cells per ml. A. actinomycetemcomitans on TSBV(Tryptone soya bacitracin vancomycin) agar
plates is identified on the basis of an adherent colony and positive catalase reaction.
26. IMMUNODIAGNOSTIC METHODS
Immunodiagnostic methods employ antibodies that recognize specific bacterial antigens to detect target microorganism.
The methods do not require viable bacteria and are less susceptible to variations in sample processing.
Immunodiagnostic methods are generally less time-consuming and easier to perform than culture. However, the accuracy
of immunodiagnostic tests depends greatly on the quality of the reagents used. In addition Immunodiagnostic methods
generally show poorer detection limits than nucleic acid probe or PCR assay.
Bonta et al, described an indirect immunofluorescence identification method of A. actinomycetemcomitans with a detection
limit of 500 cells/ ml, a sensitivity of 82- 100% and a specificity of 88-92% compared with selective and non-selective
culture
Evalusite Test (Eastman Kodak Company. Rochester, NY) is a commercially developed antibody based sandwich enzyme-
linked immunosorbent assay for the detection of A. actinomycetemcomitans.
27. NUCLEIC ACID PROBE
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. Whole genomic probes are more likely to cross-react
with non-target organisms due to the presence of homologous sequences between different bacterial species. Specific
genes such as 16S rRNA genes, contain signature sequences limited to organisms of the same species.
Oligonucleotide probe based on species-specific sequences may display limited or no cross-reactivity with non- target
organisms.
The A. actinomycetemcomitans cloned DNA probe showed cross-reactivity with Haemophilus aphrophilus, Haemophilus
influenzae, Haemophilus parahaemolyticus and Haemophilus parainfluenzae".
Socransky et al developed a checkerboard DNA- DNA hybridization assay for the detection of oral bacteria.
28. POLYMERASE CHAIN REACTION (PCR)
PCR involves a 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. Among
various detection methods PCR displays the best detection limits, identifying as few as 3-5 cells and shows no cross
reactivity under optimized amplification conditions.
Compared with culture of clinical samples, the PCR method demonstrated 45% sensitivity and 79% specificity for A.
actinomycetemcomitans and 100% sensitivity.
The low sensitivity for A. actinomycetemcomitans is due to the very low detection limit of the microorganism in
selective culture.
PCR detection of A. actinomycetemcomitans leukotoxin gene demonstrated a detection limit of 1000 cells/ml or 15
cells per 15µl of samples in a PCR reaction mixture
29. Thus, It is necessary to carefully consider the purpose of periodontal microbiology testing in order to choose a suitable
detection method. Hence Detection methods for A. actinomycetemcomitans divided into four general categories: culture,
immunodiagnostic nucleic acid probe and PCR.
Culture has been used for decades in the detection of A. actinomycetemcomitans and is frequently used as the reference
method. Most significantly, culture can provide antibiotic susceptibility of A. actinomycetemcomitans total microbial
content, information that is not obtainable by other current microbial detection methods.
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 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. In contrast, whole genomic probes may react with
phylogenetically related species and are less useful for the detection of A. actinomycetemcomitans
PCR demonstrates an excellent detection limit and is highly specific for A. actinomycetemcomitans under optimized
amplification conditions.Future availability of quantitative PCR assays may further improve the utility of PCR based
detection of A. actinomycetemcomitans.
30. Virtually all types of human periodontal disease are the result of bacterial infections and possibly herpes virus-bacterial co-
infections. Unfortunately destructive periodontal disease represents one of the most difficult challenges facing scientists
interested in infectious diseases.
A variety of very different infectious agents seem capable of producing periodontitis. Moreover, periodontal pathogens
reside in the deeply anaerobic environment of the periodontal pocket, which may compromise important antimicrobial
mechanisms of polymorphonuclear leukocytes and other protective host cells, also several putative periodontal pathogens
evolved devastating strategies to avoid various host Defense and to cause tissue breakdown.
31. SUBGINGIVAL PREVALENCE OF A. ACTINOMYCETEMCOMITANS
Several difficulties are associated with determining the occurrence of a periodontal organism in a population. Studies
examining the percentage of bacterium- positive sites may not provide a good estimate of the prevalence of a specific
organism in a population.
Study shown by Christersson et.al, the detection of periodontal A.actinomycetemcomitans may require the sampling of
a minimum of 25 random sites. Relationship to oral and systemic disease three lines of evidence have led to
conclusions that associate and implicate A. actinomycetemcomitans in oral and systemic diseases:
Clinical and microbiological data linking A. actinomycetemcomitans to the initiation, progression and recurrence of
disease in localized aggressive periodontitis.
Genetic and experimental data linking reputed virulence factors possessed by A. actinomycetemcomitans to
pathogenic events known to occur in localized aggressive periodontitis.
Reports indicating that organisms of the HACEK group (Haemophilus spp., A. actinomycetemcomitans,
Cardiobacterium hominis, Eikenella corrodens and Kingella kingae), and A. actinomycetemcomitans, in particular, are
associated with systemic diseases distant from the oral cavity.
32. RELATIONSHIP TO PERIODONTAL DISEASE
Periodontally healthy children below 11 years of age exhibit an occurrence of A. actinomycetemcomitans from 0% to
26%.
Destructive periodontal disease in children is frequently associated with A. actinomycetemcomitans. Prepubertal
periodontitis and other types of early onset periodontitis yield the organism in prevalence rates of 40-100%. The close
relationship between A. actinomycetemcomitans and early-onset periodontitis incriminates the organism in the
development of many cases of the disease.
Localized juvenile periodontitis is the most notorious disease associated with A. actinomycetemcomitans. Despite
uncertainty about clinical diagnosis and prior periodontal therapy; studies have isolated A. actinomycetemcomitans
from 75-100% localized juvenile periodontitis lesions.
The factors responsible for the establishment and overgrowth of A. actinomycetemcomitans in localized juvenile
periodontitis and other types of periodontitis need to be delineated. Undoubtedly, the dynamics of subgingival A.
actinomycetemcomitans populations is the result of a complex bacterium host inter- relationship". New findings point
to the possible roles of the cytomegalovirus and Epstein-Barr type 1 virus in the development of localized juvenile
periodontitis.
33. A. actinomycetemcomitans is also associated with periodontitis lesions of Papillon-Lefevre syndrome patients.
Velazco et al reported on an 11-year-old girl with Papillon-Lefevre syndrome who yielded subgingival A.
actinomycetemcomitans as well Epstein-Barr type I virus. Papillon-Lefevre patients exhibit decreased function of
monocytes, neutrophils, and lymphocytes, which in part may be due to cytomegalovirus infection.
A. actinomycetemcomitans seems to be a particularly frequent organism in refractory periodontitis lesions, possibly due
to the organism's ability to invade gingival tissue and thereby evade the cleaning efforts of the dentist and the patient.
Most periodontally healthy adults do not show detectable levels of subgingival A. actinomycetemcomitans. In contrast,
30-40% and higher proportions of adult periodontitis patients exhibit the organism. In addition, the proportion of the
subgingival microbiota comprising A. actinomycetemcomitans increases considerably with increasing periodontal
pocket depth.
Also, A. actinomycetemcomitans has been detected four times as frequently in periodontal lesions with angular than
with horizontal alveolar bone loss
A. actinomycetemcomitans strains differ in ability to produce leukotoxin. A. actinomycetemcomitans strains vary in
their capability of invading epithelial cells. However most strains of the periodontopathic A. actinomycetemcomitans
RFLP group II show poor intracellular epithelial invasiveness suggesting that virulence factors other than epithelial cell
invasiveness are also important determinants of disease development.
34. SURFACE ULTRASTRUCTURE OF A. ACTINOMYCETEMCOMITANS
BASIC CONSIDERATION
A significant feature of A. actinomycetemcomitans is its surface ultrastructure which includes fimbriae vesicles, and
extracellular amorphous material. The expression of all of these entities appears to be a function of the strain per se,
well as culture conditions, such as broth agar, aerobicity, anaerobicity or nutrients Fimbriae
Like many other gram-negative bacteria. A. actinomycetemcomitans may exhibit, fimbriae, small filamentous cell
surface appendages associated with bacterial colonization of host tissues. A actinomycetemcomitans fimbriae occur
in peritrichous arrays, may be more than 2 um in length and 5 nm in diameter and often occur ion bundles. Freshly
isolated strains are fimbriated but in duo 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 arid fimbriation are associated with A. actinomycetemcomitans adhesion.
VESICLES
A prominent feature of the surface of A. actinomycetemcomitans 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. actinomycetemcomitans strains
has an abundance of extracellular membranous vesicles.
35. In contrast to minimally or nonleukotoxic strains, which have few or no vesicles.Furthermore vesicles per section 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. actinomycetemcomitans cells is an amorphous material that frequently embeds
adjacent cells in a matrix. In an early study it was reported that cells growth in liquid lacked the amorphous material.
However, others did observe this material on cells grown in liquid culture, but not on all strains. Production of the
material may be in part associated with growth in a tryptone-based medium.
Thus, like fimbriae and vesicles, the expression of the amorphous material is determined by culture conditions. The
material is a protein, most likely a glycoprotein, and has been shown to exhibit both bone-resorbing activity and adhesive
properties.
The material is easily removed by washing cells with phosphate-buffered saline. Bacteria from which the amorphous
material has been removed exhibit reduced adhesion to epithelial cells. Furthermore A. actinomycetemcomitans strains,
which normally exhibit low levels of adhesion exhibit increased levels of adhesion when suspended in extracellular
amorphous material, a phenomenon termed conveyed adhesion.
36. ORAL INFECTIONS
BASIC CONSIDERATION
A. actinomycetemcomitans is an important pathogen in severe and recurrent forms of periodontitis. The prevalence of A.
actinomycetemcomitans is nearly 90% in localized juvenile periodontitis and 30-50% in severe adult periodontitis. It is
frequently associated with rapidly progressive periodontitis. Among the five currently recognized serotypes of A.
actinomycetemcomitans, serotype b strains often predominate in periodontal lesions of localized juvenile periodontitis
patients although there is considerable overlapping in the serotype distribution. Localized juvenile periodontitis:
Localized juvenile periodontitis constitutes a periodontal condition in adolescents that exhibits rapid destruction of
periodontal tissue, which slows with time, Periodontal tissue around the incisors and first molars is usually affected.
Clinically, periodontal bone loss resembles a "mirror image" on each side of the dental arch. Large numbers of A.
actinomycetemcomitans are frequently isolated from patients, although smaller amounts of capnocytophaga spp., E.
corrodens, Fusobacterium nucleatum, Bacteroides cappillus and Eubacterium brachy are sometimes isolated as well. A.
actinomycetemcomitans was first implicated as the cause of juvenile periodontitis in 1976 by Newman et al. and by
Slots6667,. This disease is now called localized aggressive periodontitis.
37. These and other studies implicating A. actinomycetemcomitans were cross-sectional observations that documented the
association of A. actinomycetemcomitans with disease where bone loss had already occurred.
There are also a few longitudinal studies that documented the relationship of A. actinomycetemcomitans with disease
progression where stable diseased sites had converted to active disease sites.
In these studies, subjects with diseased pocket sites harboring A. actinomycetemcomitans showed progressing disease
activity over a short observation period, while pockets without A. actinomycetemcomitans remained stable during the
observation period Other studies have demonstrated that disease recurs with the reappearance of A.
actinomycetemcomitans, even after thorough debridement and removal of A. actinomycetemcomitans had been
theoretically accomplished.
Thus, there are several lines of clinical evidence that support the association of A. actinomycetemcomitans with localized
aggressive periodontitis.
38. First, as described above, 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.
Second, subjects with localized aggressive periodontitis were consistently found to have elevated serum and locally
produced antibody titers to A. actinomycetemcomitans.
Third, several studies indicated that the treatment of subjects with localized aggressive periodontitis 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.
Many studies have implicated A. actinomycetemcomitans as the agent responsible for the pathogenesis of localized
juvenile periodontitis. Microbiological,clinical and immunological evidence as described below, have been used to
implicate this agent.
39. Large numbers of A. actinomycetemcomitans are routinely isolated from localized juvenile periodontitis lesions. Whereas, the
isolation of the bacterium from healthy sires is low. A. actinomycetemcomitans is isolated from 97% of localized juvenile
periodontitis cases and the numbers of A. actinomycetemcomitans are six times as many as those found in healthy sites.
The eradication of A. actinomycetemcomitans from diseased sites is usually correlated with recovery from clinical symptoms
of disease.
The presence of large numbers of A. actinomycetemcomitans in the periodontal pocket is correlated with a significant humoral
immune response.
A. actinomycetemcomitans produces a wide array of potent cell bound and secreted virulence factors that haw been
implicated in the pathogenesis of disease
RAPIDLY PROGRESSING PERIODONTITIS
A severe or rapid loss of bone is hallmark of rapidly progressing periodontitis, a periodontal condition that affects young
adults 25-30 years of age. The isolation of A. actinomycetemcomitans, P. gingivalis, B. capillus, P. intermedia, E. corrodens
and Campylobacter rectus from disease sites has been reported either singly or in combination. In addition A.
actinomycetemcomitans has been associated with active periodontal lesions that are refractory to standard therapy
40. EXTRAORAL INFECTIONS
BASIC CONSIDERATION
A. actinomycetemcomitans has been reported to cause serious infections at several sites outside the mouth.
Extraoral infections by A. actinomycetemcomitans have been reported from the brain, meninges,
septicemia, urinary tract infections, vertebral osteomyelitis and abscesses of the abdomen, brain, face, hand
and thyroid gland.
Among the reports of extraoral infections, endocarditis and soft tissue abscesses remain the most common
infections with the latter syndrome generally occurring in association with Actinomyces. Endocarditis
remains the most commonly reported extra oral infection by A. actinomycetemcomitans.
41. Virulence factors of
A. actinomycetemcomitans
Factors that promote
colonization and persistence
in the oral cavity
Adhesins
Invasins
Bacteriocins
Factors that interfere
with the host's defenses
Leukotoxin
Chemotactic inhibitors
Immunosuppressive
proteins
Fe-binding proteins
Factors that inhibit host
repair of tissues
Inhibitors of fibroblast
proliferation
Inhibitors of bone
formation
Factors that destroy host
tissues
Cytotoxins
Collagenase
Bone resorption agents
Stimulators of
inflammatory mediators
42. VIRULENCE FACTORS OF A. ACTINOMYCETEMCOMITANS
Virulence factors are attributes of a microorganism that enable it to colonize a particular niche in its host overcome the
host defenses and initiate a disease process. These factors frequently involve the ability to be transmitted to susceptible
hosts.
Organisms with many virulence factors are more pathogenic. Numerous studies have focused on the virulence of A.
actinomycetemcomitans because of its strong association with periodontal diseases and related extraoral infections.
The colonization and growth of specific bacteria in a particular habitat occurs because the colonizing environment offers
special ecological advantages. It is logical to expect that the mother or caretaker is the source for transmission of the
majority of contaminating microbes that are found in the first several years of the infants life.
Contamination occurs on a daily basis, and most contaminating organisms do not survive and thus appear in the oral
cavity for a fleeting period of time. Only those organisms that can attach and hide are sustained in their specific
ecological niche, and only those oral domains that can support the colonization and growth of these microbes over time,
will provide the long-term residence for the contaminating organisms.
43. It must be remembered that a proposed disease-initiating species, such as A. actinomycetemcomitans, exists in a dense
polymicrobial matrix and that the ensuing infection occurs in a time- and host-dependent manner. These complexities
can be studied for dental diseases because the oral cavity provides an ideal environment in which these complex
microbiological interactions can be monitored.
Microbial material in the form of dental plaque can be obtained in a noninvasive manner so that dental plaque can be
removed at one point in time, or over time, in groups of preselected study subjects.
Because the plaquerelated diseases, caries and periodontal diseases progress at a relatively slow rate, intervention can
be instituted at the first sign of disease without serious consequences for the patient. Treatment can reverse disease and
provide patients with long-term follow up that will prevent any permanent damage.
44. Work by Socransky and Haffajee illustrates these points" and provides a detailed and innovative approach to human microbial
ecology. In vitro, A. actinomycetemcomitans exhibits numerous phenotypes that may contribute to its ability to colonize the oral
cavity. These phenotypes include attachment to human epithelial cells, attachment to hydroxyapatite, invasion of nonphagocytic
human cells, co-aggregation with fusobacteria, auto-aggregation and biofilm formation
In addition, A. actinomycetemcomitans cells secrete two cytolethal toxins - leukotoxin and cytolethal distending toxin- that may
facilitate evasion of the host immune system during the colonization process and may also contribute to pathogenesis.
As colonization is a vital and necessary prerequisite in the process of infection, this section of the review will focus on recent
studies aimed at identifying and characterizing A. actinomycetemcomitans surface proteins and other surface-associated
components, such as pili, fimbriae and extracellular polysaccharides, which may play a role in the colonization process.
A. actinomycetemcomitans has been shown to possess a myriad of virulence factors that enhance its survival in the oral cavity
and enable it to circumvent the host's protective strategies. Many of these virulence factors may be involved in the pathogenesis
of periodontitis. They include the ability to attach to extracellular matrix proteins and epithelial cells; antibiotic resistance, a
bacteriocin; bone resorption by either endotoxin or surface-associated material; a chemotactic inhibitor; a collagenase; cytotoxin;
Fc-binding proteins; a leukotoxin; immunosuppressive factors: and the ability to invade epithelial cells and tissues.
45. A. actinomycetemcomitans has clearly adapted well to its environs: its armamentarium of virulence factors ensures its
survival in the oral cavity and enables it to promote disease.
Factors that promote A. actinomycetemcomitans colonization and persistence in the oral cavity include adhesins.
bacteriocins invasins and antibiotic resistance. It can interact with and adhere to all components of the oral cavity (the
tooth surface. other oral bacteria, epithelial cells or the extra-cellular matrix).
The adherence is mediated by a number of distinct adhesins that are elements of the cell surface (outer membrane proteins.
vesicles, fimbriae or amorphous material).
A.actinomycetemcomitans enhances its chance of colonization by producing actinobacillin an antibiotic that is active
against both streptococci and Actinomyces primary colonizers of the tooth surface. The fact that A. actinomycetem
comitans resistance to tetracyclines, a drug often used in the treatment of periodontal disease is on the rise is an added
weapon.
Periodontal pathogens or their pathogenic products must be able to pass through the epithelial cell barrier in order to reach
and cause destruction to underlying tissues (the gingiva, cementum, periodontal ligament and alveolar bone).
46. A. actinomycetemcomitans is able to elicit its own uptake into epithelial cells and its spread to adjacent cells by
demaging normal epithelial cell function. It may utilize these remarkable mechanisms for host cell infection and
migration to deeper tissues.
A. actinomycetemcomitans also orchestrates its own survival by elaborating factors that interfere with the host’s
defense system (such as factors that kill phagocytes and impair lymphocyte activity inhibit phagocytosis and phagocyte
chemotaxis or interfere with antibody production).
Once the organisms are firmly established in the gingiva, the host responds to the bacterial onslaught, especially the
bacterial lipopolysaccharide by a marked and continual inflammatory response, which results in the destruction of the
periodontal tissues.
A. actinomycetemcomitans has at least three individual factors that cause bone resorption (lipopolysaccharide,
proteolysis-sensitive factor, as well as a number of activities (collagenase, fibroblast cytotoxin, etc.) that elicit
detrimental effects on connective tissue and the extra cellular matrix.) It is of considerable interest to know that A.
actinomycetemcomitans possess so many virulence factors but unfortunate that only a few have been extensively
studied.
47. The Relationship between Gingivitis and Colonization by Porphyromonas gingivalis and Actinobacillus
actinomycetemcomitans in Children was evaluated in a study. Plaque and sera were obtained from 40 healthy children, 2
to 18 years old. Gingival health was assessed by the periodontal disease index (PDI), papillary bleeding score (BS) and
the modified total papillary margin attachment index (M-PMA). P. gingivalis and A. actinomycetemcomitans in plaque
samples were detected by slot immunoblotting (SIB). Serum antibody levels against these microorganisms were evaluated
using ELISA. The findings showed that P. gingivalis and A. actinomycetemcomitans were readily detected as early as 3
years of age and that their presence is associated with the onset and severity of gingivitis.
REVIEW OF LITERATURE
Morinushi T, Lopatin DE, Poperin NV, Ueda Y. The Relationship between Gingivitis and Colonization by
Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans in Children. J Periodontol 2000;71:403-
409.
48. A longitudinal study was conducted to evaluate A. actinomycetemcomitans as a diagnostic indicator for periodontal
disease in treated and periodontally maintained patients. Following comprehensive mechanical/surgical and
supportive amoxicillin plus metronidazole therapy in 13 subjects with A. actinomycetemcomitans-associated
destructive periodontal disease, they monitored subgingival A. actinomycetemcomitans at 4 individual sites in each
patient up to 3 years post-therapy. The periodontal status was determined, and A. actinomycetemcomitans levels
were quantitatively enumerated on TSBV agar in CFU/ml. Six patients with a persistence of subgingival A.
actinomycetemcomitans at each re examination within 3 years post-therapy were selected to be at risk for minor
periodontal treatment outcomes and further recurrence of periodontal disease (test group). Seven subjects with a
complete suppression of 4. actinomycetemcomitans at each post-therapy visit served as controls. It concluded that
although in advanced periodontal disease, comprehensive mechanical and antimicrobial treatment is an appropriate
regimen for sustained improvement of periodontal health, long-term control of subgingival infection with A.
actinomycetemcomitans could not be achieved. In the maintenance care of destructive periodontitis, the persistence
of A. actinomycetemcomitans is not a diagnostic parameter for periodontal disease.
Buchmann R, Müller RF, Heinecke A. Lange DE. Actinobacillus actinomycetemcomitans in Destructive
Periodontal Disease. Three-Year Follow- Up Results. J Periodontol 2000;71:444-453.
49. In a study, the distribution, clonality, and intrafamilial transmission of highly leukotoxic A. actinomycetemcomitans were
examined in order to determine the importance of leukotoxin in the pathogenesis of periodontitis. The polymerase chain
reaction (PCR) was used to differentiate highly leukotoxic from minimally leukotoxic strains in examining 1,023 fresh
A. actinomycetemcomitans isolates and strains from our culture collection. These were obtained from 146 subjects
including 71 with localized juvenile periodontitis (LJP), 4 with early-onset periodontitis, 11 with post-localized juvenile
periodontitis, 41 with adult periodontitis, and 19 periodontally normal subjects. The arbitrarily primed polymerase chain
reaction (AP-PCR) analysis of 30 oral isolates from each of 25 subjects was used to determine the intraoral distribution
of A. actinomycetemcomitans clones. AP-PCR was also used to examine the transmission of A. actinomycetemcomitans
in 30 members of 6 families. The clonality of 41 highly leukotoxic A. actinomycetemcomitans strains was evaluated by
both AP-PCR and ribotyping. This study suggested that localized juvenile and other forms of Actinobacillus-associated
periodontitis are primarily associated with the highly leukotoxic clone of A. actinomycetemcomitans.
Hariharan G, Tinoco EMB, Cortelli JR, Lally ET, Davis E, Zambon JJ. Evidence for the Role of Highly Leukotoxic
Actinobacillus actinomycetemcomitans in the Pathogenesis of Localized Juvenile and Other Forms of Early-Onset
Periodontitis. J Periodontol 2000;71:912-922.
50. The occurrence of A. actinomycetemcomitans, P. gingivalis, and P. intermedia was determined using a DNA
probe in progressive adult periodontitis in Chileans in a study. Sixty patients (mean age 43.6+ 8 years) who had
not previously received any type
of periodontal therapy were selected. Bleeding on probing, probing depth, and clinical attachment level
measurements were made with an automated probe. Patients were monitored at 2-month intervals until at least 2
sites exhibited 22 mm attachment loss. Two subgingival plaque samples from active sites were taken in 56
subjects and matched with 2 plaque samples from inactive sites in the same individuals. It concluded that a high
prevalence of P. gingivalis and P. intermedia was found in adult periodontitis, and the occurrence of these
bacteria appears to be higher in Chileans than in other populations. No apparent association exists between A.
actinomycetemcomitans and progressive adult periodontitis in Chileans.
López NJ. Occurrence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and
Prevotella intermedia in Progressive Adult Periodontitis. J Periodontol 2000;71:948-954.
51. The associations of periodontal status with general health conditions and with serum antibody titers for
Porphyromonas gingivalis (Pg) and Actinobacillus actinomycetemcomitans (Aa) in a Japanese population was
evaluated in a study. Subjects (1,314) aged 40 years or older were recruited for the study. Their periodontal status was
evaluated using the community periodontal index of treatment needs (CPITN). Records from general health
examinations were used for the present analyses, including data on age, gender, body mass index, systolic and
diastolic blood pressure (SBP/DBP); levels of serum alkaline phosphatase, total cholesterol, high-density lipoprotein
cholesterol, triglyceride, fasting blood glucose, and C-reactive protein (CRP); counts of white blood cells (WBC) and
platelets; smoking habit; and electrocardiogram recordings. Serum IgG. antibody titers for Pg fimbrial antigens
(IgG.Pg.Fim.), Pg whole-cell antigens (IgG.Pg.whol.), and Aa whole-cell antigens (IgG.Aa.whol.) were determined
using enzymelinked immunosorbent assay. The subjects were divided, according to their maximum CPITN codes, into
four periodontal groups: max.CPITN-0/1/2, -3, -4, or –MS (missing sextant). It concluded that significant associations
between periodontal status and several health conditions were found in the adult population examined, including
gender, smoking habit, diastolic blood pressure, white blood cell counts, C-reactive protein, and serum IgG antibodies
to P. gingivalis fimbriae, IgG P. gingivalis whole cell, and IgG A. actinomycetemcomitans whole cell titers.
Namariyama Y, Yotsumoto Y, Hino Y, Mishige Y, Inoue M, Izumi Y. Associations of Periodontal Status with General
Health Conditions and Serum Antibody Titers for Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans
Periodontol 2003;74:1491-1497.
52. In a study the frequencies of the five serotypes of A. actinomycetemcomitans in A. actinomycetemcomitans isolates
from various forms of periodontitis were characterized using both old and new diagnostic classifications and the
relationships between serotype and age and clinical diagnosis were determined. A total of 345 A.
actinomycetemcomitans isolates from 115 A. actinomycetemcomitans culture-positive subjects (mean age 38.0 18.3
years, 59% female) were collected. Based on the new classifications, 33 subjects had aggressive periodontitis and 82
chronic periodontitis. According to old classifications, there were six prepubertal periodontitis (PPP), 12 localized
juvenile periodontitis (LJP), 15 post-localized juvenile periodontitis (PLJP), 28 refractory periodontitis (Ref-P), and 54
adult periodontitis (AP) cases. Serotypes of A. actinomycetemcomitans were determined by an indirect
immunofluorescence assay using serotype-specific polyclonal antisera to A. actinomycetemcomitans strains ATCC
29523, ATCC 43728, ATCC 33384, IDH 781 and IDH 1705 (serotype a, b, c, d, and e, respectively). Proportions of
serotype b were examined between different diagnostic and age groups with a Z-test for proportions. The results of this
study showed that proportions of serotype b of A. actinomycetemcomitans were significantly greater in culture-positive
patients with aggressive periodontitis than those with chronic periodontitis.
Yang WH, Asikainen S, Doğan B, Suda R, Lai CH. Relationship of Actinobacillus actinomycetemcomitans
Serotype b to Aggressive Periodontitis: Frequency in Pure Cultured Isolates. J Periodontol 2004;75:592-599.
53. A study was conducted to evaluate the use of unstimulated saliva in detecting A. actinomycetemcomitans and to
compare the subgingival and extracrevicular occurrence of this pathogen in Brazilian subjects with chronic
periodontitis. Sixty-six patients (mean age 38.01 9.28 years) with advanced generalized chronic periodontitis were
sampled. Subgingival plaque samples were collected from eight sites per patient representing the two deepest sites
of each quadrant. Samples of the mucous surfaces, including dorsal surface of the tongue and cheek, were collected
with a sterile swab and placed in a microtube containing a reduced solution. Samples of unstimulated saliva were
also collected in sterile tubes and 0.1 ml of whole saliva was diluted in 1 ml of reduced solution. The presence of
A. actionomycetemcomitans was established using bacterial culture in trypticase soy bacitracin vancomycin
selective media. Polymerase chain reaction (PCR) was used to differentiate highly from minimally leukotoxic
strains in patients who presented A. actinomycetemcomitans in at least two sampled sites. The results suggested
that in advanced periodontitis, unstimulated saliva is representative of pooled subgingival plaque samples and its
use is appropriate in the oral detection of A.actinomycetemcomitans
Cortelli SC, FeresM, Rodrigues AAB, Aquino DR, Shibli JA, Cortelli JR. Detection of Actinobacillus
actinomycetemcomitans in Unstimulated Saliva of Patients With Chronic Periodontitis. J Periodontol
2005;76: 204-209.83
54. The effects of the toxin cytolethal distending toxin (CDT) of Actinobacillus actinomycetemcomitans on primary cultures
of human periodontal ligament fibroblasts (HPLF) was examined as a virulence factor in periodontal diseases in a study.
HPLF and an immortalized human gingival epithelial cell line, GMSM-K, were exposed to recombinant A.
actinomycetemcomitans CDT. Effects of the toxin on cell proliferation and cell cycle were assessed by a cell viability
assay and flow cytometry, respectively. Double-strand DNA damage was detected by pulsed field gel electrophoresis.
Binding of the toxin and its individual subunits to HPLF was examined by immunofluorescence microscopy. The results
demonstrated that HPLF are resistant to the cytotoxic effects of the A. actinomycetemcomitans CDT. The mechanism of
resistance was not known but may be related to the inability of the toxin to cause DNA damage. The difference in
sensitivities of HPLF and oral epithelial cells to the CDT has important implications for the role of this putative
microbial virulence factor in periodontal pathogenesis.
Di Rienzo J M, Korostoff J, Volgina A, Kanno F. Resistance of Human Periodontal Ligament Fibroblasts to the
Cytolethal Distending Toxin of Actinobacillus actinomycetemcomitans J Periodontol 2005;76:1189-1201.
55. A longitudinal design was used to examine subgingival colonization of Actinobacillus actinomycetemcomitans (Aa)
and alkaline phosphatase (ALP) and aspartate aminotransferase (AST) activities in gingival crevicular fluid (GCF)
in order to assess whether these parameters have potential as biomarkers of tissue responses to orthodontic tooth
movement in humans. Twenty-one patients (ages: 11.2-22.5; mean 17.1+3.3 years) participated in the study. An
upper canine from each patient undergoing treatment for distal movement served as the test tooth (DC), and its
contralateral (CC) and antagonist (AC) canines were used as controls. The CC was included in the orthodontic
appliance, but was not subjected to the orthodontic force; the AC was free from any orthodontic appliance. The
subgingival plaque and GCF around the experimental teeth was harvested from both mesial and distal tooth sites
immediately before appliance activation and on day 28. Clinical gingival condition was evaluated at the baseline
and at the end of the experimental period. Aa colonization was determined by culture methods, while ALP and AST
activities were evaluated spectrophotometrically. The results of this study suggested that Aa subgingival
colonization, and ALP and AST activities in GCF reflected the tissue responses that occurred in the periodontium
during orthodontic treatment.
Perinetti G, Paolantonio M, Serra E, D'Archivio D, D'Ercole S, Festa F, Spoto G: Longitudinal monitoring of
subgingival colonization by Actinobacillus actinomycetemcomitans, and crevicular alkaline phosphatase and
aspartate aminotransferase activities around orthodontically treated teeth. J Clin Periodontol 2004; 31, 60-67
56. A study was conducted to compare patterns of attachment loss among EOP- patients with or without JP2-type of A.
actinomycetemcomitans in dental plaque. Among 45 Moroccan adolescents with EOP (i.e. one or more teeth with
attachment loss >= 3 mm) 39 had cultivable plaque samples. Fifteen (38.5%) were culture-positive for A.
actinomycetemcomitans of the JP2-type as determined by PCR, and 24 (61.5%) were not (mean age 16.5 years in both
groups). EOP-patients culture-positive for A. actinomycetemcomitans of the JP2-type had significantly more teeth with
attachment loss (mean 5.1, median 4.0) than EOP-patients not culture-positive for A. actinomycetemcomitans of the JP2-
type (mean 2.8 teeth, median 1.0) (p = 0.02), and higher attachment loss (mean 4.3 mm vs. 3.4 mm; median 4.0 mm vs.
3.0 mm) (p = 0.01). No major differences could be detected between the two groups in the pattern of affected teeth in the
dentition. The study demonstrated an increased periodontal destruction among EOP-patients culture-positive for A.
actinomycetemcomitans of the JP2-type compared with EOP-patients without the JP2-clone.
Poulsen S,Haubek D, Ennibi O-K, Abdellaoui L, Benzarti N, Attachment loss in Moroccan early onset periodontitis
patients and infection with the JP2-type of Actinobacillus actinomycetemcomitans. J Clin Periodontol 2002;29:657-
660.86.
57. In a study, (i) the natural distribution of the three putative periodontopathogens Porphyromonas gingivalis, Prevotella
intermedia and Actinobacillus actinomycetemcomitans in an Australian population and (ii) the relationship between
these organisms, pocket depths and supragingival plaque scores were assessed. Subgingival plaque was collected
from the shallowest and deepest probing site in each sextant of the dentition. In total, 6030 subgingival plaque
samples were collected from 504 subjects. An ELISA utilising pathogen-specific monoclonal antibodies was used to
quantitate bacterial numbers. A. actinomycetemcomitans was the most frequently detected organism (22.8% of
subjects) followed by P. gingivalis and P. intermedia (14.7% and 9.5% of subjects respectively). The majority of
infected subjects (83%) were colonised by a single species of organism. A. actinomycetemcomitans presence was
over- represented in the youngest age group but under-represented in the older age groups. Conversely, P. gingivalis
and P. intermedia presence was under-represented in the youngest age group but over-represented in the older age
groups. Bacterial presence was strongly associated with pocket depth for both A. actinomycetemcomitans and P.
gingivalis. This cross-sectional study in a volunteer Australian population, demonstrated recognized periodontal
pathogens occur as part of the flora of the subgingival plaque
Hamlet SM, Cullinan MP, Westerman B, Lindeman M, Bird PS, Palmer J, Seymour GJ. Distribution of Actinobacillus
actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia in an Australian population. J Clin
Periodontol 2001;28:1163-1171
58. A study was conducted to determine the prevalence and the structure of the leukotoxin promoter region of
Actinobacillus actinomycetemcomitans in an ethnic Chinese population. Subgingival plaque samples were collected
from 42 patients with moderate to advanced periodontitis and 50 periodontally healthy patients. A.
actinomycetemcomitans was detected directly from the crude subgingival plaque by PCR using leukotoxin gene
specific primers. The presence of A. actinomycetemcomitans was determined by a single 285 bp PCR amplicon. It
concluded that high prevalence of A. actinomycetemcomitans, regardless of whether the subgingival samples were
analysed from patients with healthy or diseased periodontium suggested that this bacterial species was part of the
normal oral flora of ethnic Chinese. It also suggested that subjects who harboured the mildly toxic strain of A.
actinomycetemcomitans were potentially susceptible to aggressive forms of periodontitis.
Tan KS, Woo CH, Ong G, Song KP. Prevalence of Actinobacillus actinomycetemcomitans in an ethnic
adult Chinese population. J Clin Periodontol 2001;28:886-890
59. A study aimed to determine whether the presence of bacterial antigens for Porphyromonas gingivalis (Pg). Prevotella
intermedia (Pi), and Actinobacillus actinomycetemcomitans (Aa) in subgingival plaque of periodontitis patients after
periodontal treatment was associated with progressive alveolar bone loss. 39 subjects in good general health
previously diagnosed with adult periodontitis within the last 2 years, and still presenting with probing depth >5 mm
in 2 to 6 teeth, were studied. All subjects were treated with scaling and root planing. Half of the subjects were
randomly assigned to receive adjunctive systemic doxycycline (200 mg the 1st day, then 100 mg per day for 21 days).
Subgingival plaque samples were taken at baseline, 1, 3 and 6 months after therapy. A modified ELISA test (Evalusite
TM, Periodontal Test Kit, Eastman Kodak Co., Rochester, NY) was used to test for plaque antigens associated with P.
gingivalis, P. intermedia and A. actinomycetemcomitans. Progressive alveolar bone loss was determined using digital
subtraction radiography with standardized radiographs taken at baseline and 6 months after treatment. The presence
of P. gingivalis in plaque after treatment was significantly associated with progressive bone loss (positive predictive
value 84%, negative predictive value 85%, odds ratio 31.9, p<0.0001). In contrast, the presence of P. intermedia in
plaque after treatment was not indicative of progressive loss (positive predictive value 39%, negative predictive value
82%). Too few sites had evidence of A. actinomycetemcomitans to be amenable to statistical analysis. No significant
difference in bone loss was attributable to the systemic antibiotic therapy. The data indicated that, in that population,
the presence of P. gingivalis in plaque after treatment might be indicative of progressive alveolar bone loss.
Chaves ES, Jeffcoat MK, Ryerson CC, Snyder B. Persistent bacterial colonization of Porphyromonas gingivalis, Prevotella
intermedia, and Actinobacillus actinomycetemcomitans in periodontitis and its association with alveolar bone loss after 6
months of therapy. J Clin Periodontol 2000;27:897- 903.
60. Okada M, Hayashi F, Nagasaka N. Detection of Actinobacillus actinomycetemcomitans and Porphyromonas
gingivalis in dental plaque samples from children 2 to 12 years of age. J Clin Periodontol 2000;27:763-768.
A study was conducted to detect the presence of Actinobacillus actinomycetemcomitans and Porphyromonas
gingivalis in plaque samples from 104 children, collected from their toothbrushes using a polymerase chain reaction
(PCR). The age range of all subjects was 2-12 years. 21, 73 and 10 children with healthy gingiva, gingivitis and
periodontitis respectively were selected. Plaque samples were collected from all erupted teeth sites using a sterile
toothbrush. The mean concentration of DNA recovered from brushing plaque samples was approximately 660
mug/ml, which was sufficient for performing a PCR-based survey. Both A. actinomycetemcomitans and P. gingivalis
were detected in the primary and mixed dentition. The prevalence of A. actinomycetemcomitans in healthy subjects
was 4.8%, and those with gingivitis and periodontitis was 6.8% and 20.0% respectively, while the prevalence of P.
gingivalis was 4.8% in healthy subjects, and 9.6% and 20.0% in those with gingivitis and periodontitis, respectively.
This survey, using a toothbrush, indicated that A. actinomycetemcomitans and P. gingivalis are rarely present in oral
cavities of healthy children.
61. The oral cavity is the ecological niche for A. actinomycetemcomitans. Approximately 90% of patients with localized
juvenile periodontitis (LJP) and 50% of adult periodontitis patients harbor A. actinomycetemcomitans in the oral cavity,
and this organism may be isolated from some periodontally healthy subjects as well.
However, A. actinomycetemcomitans has not been detected in edentulous infants. A. actinomycetemcomitans was first
isolated and identified to species level in human periodontal lesions in 1979 from a patient with juvenile periodontitis.
Nearly two decades later, in 1996, it was classified as a periodontal pathogen (World Workshop in Periodontics 1996). This
organism fulfills the criteria of an etiological agent of periodontitis. A. actinomycetemcomitans is present in high numbers
in periodontal lesions, especially in LJP, and absent or in low proportions in healthy periodontium.
The elimination of the organism results in improvement of the clinical periodontal condition.
The host with a periodontal infection exhibits elevated serum and salivary antibody levels against this pathogen. A.
actinomycetemcomitans possesses virulence factors associated with the pathogenesis and the progression of the disease.
Additionally, animal models link the organism to periodontal tissue destruction.
SUMMARY AND CONCLUSION
62. 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.
Much of the enthusiasm for studies of the role of A. actinomycetemcomitans in localized aggressive periodontitis has
diminished over the last 10 years for reasons that are inexplicable and inconsistent with the progress that has been
made in efforts to understand the molecular biology of this important organism and its association with disease
pathogenesis.
Comparative genomics has allowed for in silico identification of the aae gene in A. actinomycetemcomitans, a gene
that is similar to the hap (i.e. Haemophilus adhesive protein) gene described for H. influenzae and the gene in A.
actinomycetemcomitans, a gene with similarity to the yadA (Yersinia adhesion) gene of Y. pestis.
In a similar manner, A. actinomycetemcomitans biologists have been able to enlighten microbiologists in other
disciplines to the importance of genes and operons expressed in A. actinomycetemcomitans that could have broad
significance in the world of general microbiology.
63. While A. actinomycetemcomitans does not present itself as a clear exogenous pathogen in the mold of Treponema pallidum,
Mycobacterium tuberculosis or Clostridium botulinum, A. actinomycetemcomitans does provide an interesting perspective
on pathogenic events seen in chronic infections caused by members of the resident flora.
The evidence is incomplete but it does seem that in the case of A. actinomycetemcomitans, disease takes place by means of
direct expression of A. actinomycetemcomitans virulence traits in the earliest stages of disease, and by means of the reaction
of the host defense system to traits expressed by A. actinomycetemcomitans in the later, tissue-destructive stages.
These two distinctly different modes of pathogenesis are more than likely not unique to A. actinomycetemcomitans. The
evidence is based on clinical, microbiological and immunological studies that demonstrate a robust association between A.
actinomycetemcomitans and localized aggressive periodontitis.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. actinomycetemcomitans and localized aggressive
periodontitis.
This logic is further complemented by studies which suggest that A. actinomycetemcomitans is related to disease initiation
and by studies which show that failure to remove A. actinomycetemcomitans leads to disease progression. Moreover,
repopulation of diseased sites by A. actinomycetemcomitans leads to disease recurrence.
Nevertheless, the exact mechanism of A. actinomycetemcomitans interaction with its fellow community inhabitants, and the
collective or individual influence of these associations on host cell signaling, are just at the earliest stages of investigation
