Periodontology 2000, Vol.

32, 2003, 2435

Printed in Denmark. All rights reserved

Copyright # Blackwell Munksgaard 2003

PERIODONTOLOGY 2000
ISSN 0906-6713

Microorganisms as risk indicators

for periodontal disease
Paul J. Ezzo & Christopher W. Cutler

The composition of bacterial plaque has been studied

for many decades using various microbiological and
molecular techniques. These efforts have been ostensibly motivated by the dream that, once we identify
the causative agent(s) of periodontitis, we will be able
develop a rapid test (e.g. the rapid Strep test used in
physicians offices (75)) to assess microbial risk at
chairside. This information, along with that obtained
from antibiotic susceptibility testing, could be applied
for treating periodontitis like other infections in the
body administer the appropriate antibiotic and the
patient gets better. However, attempts to develop rapid
chairside microbial-risk tests and integrate them into
dental practices have not been successful for many
reasons. As the technology of bacterial detection has
evolved, from morphologic detection to cultivation to
molecular probe analysis, the sensitivity of detection
has improved logarithmically. Now over 400 bacterial
species have been identified, with surely more to come
(52). While it is in our nature to try to identify those
specific organisms most likely to cause or be associated with disease, dental practitioners are faced with
an increasingly daunting task how to make rational
decisions based on an increasingly complex database
of microbial risk factors. They must decide how to (and
indeed whether to) integrate this information into
their practices, so they can provide efficient, state-ofthe-art patient care. Moreover, while some dental
offices do use antibiotic susceptibility testing services,
the overall concept of antimicrobial therapy for treatment of periodontitis has not caught on. The simple
fact that conventional therapy (i.e. scaling and root
planing) is the standard-of- care and appears to be
effective when used alone is an important factor in
this regard. Moreover, there are apt concerns ingrained
in the dental profession about the use of antibiotics
being the driving force for antibiotic resistance.
This review will attempt to clarify the role that
microbes and their products play in periodontitis and

24

try to provide a clinical rationale for assessing microbial risk. It is hoped that this will facilitate an understanding of the pathogenesis of periodontal disease
and perhaps allow for enhanced treatment outcomes.

Plaque 101 and disease-associated

species
For a historical prospective to this topic, readers are
referred to earlier reviews that have described the nonspecific and specific plaque hypotheses (88). The plaque biofilm consists largely of microbes and host proteins that adhere to the teeth within minutes of a
dental prophylaxis. In gingival health, gram-positive
organisms like Actinomyces and streptococci dominate the plaque biofilm. In the later stages of plaque
biofilm formation (i.e. days to weeks of poor oral
hygiene) the plaque matures, resulting in a shift
towards gram-negative anaerobes and motile organisms. Some of the most common organisms associated with periodontal diseases are Porphyromonas
gingivalis, Prevotella intermedia, Bacteroides forsythus,
Campylobacter rectus, and Actinobacillus actinomycetemcomitans, as well as the treponemes. At the 1996
World Workshop on Clinical Periodontics, a group
reviewing specific periodontal pathogens as causative
agents in periodontal disease limited their findings to
three organisms: A. actinomycetemcomitans, B. forsythus, and P. gingivalis (46), presumably because
they met Socranskys modifications of Kochs postulates (70). Among other criteria, these modifications
require that, to be considered a periodontal pathogen:

the organism must occur at higher numbers in
disease-active sites than at disease-inactive sites.

elimination of the organism should arrest disease
progression.

the organism should possess virulence factors
relevant to the disease process.

Microorganisms as risk indicators for periodontal disease

the organism should elicit a humoral or cellular

immune response.

animal pathogenicity testing should infer disease
potential.
Implicit in the first two requirements is the need to
conduct longitudinal clinical studies, which has been
achieved for these three species. This review will therefore begin by focusing on each of these three organisms in turn, with the caveat that they do not exist in
isolation in vivo, but are part of a microbial community. We will briefly discuss the virulence factors they
produce and the longitudinal studies that implicate
them as risk indicators in periodontal disease. As the
term virulence could be construed as a microbial risk
factor for causing disease, we will emphasize the commonalities in virulence or pathogenesis exhibited by
these organisms. We will discuss microbial products
common to many of the so-called high-risk species
and how they might confer risk to periodontal disease.
It is important to mention in this context that, unlike
many of the other risk factors described in this volume,
we chose to refer to periodontal pathogens as risk
indicators due to the fact that the odds ratios between
the presence of these specific bacteria individually,
and periodontitis are not high enough to classify them
as risk factors, notwithstanding the essential role for
the plaque biofilm in the initiation of periodontitis.

A. actinomycetemcomitans as a risk
indicator for periodontitis
A. actinomycetemcomitans is a bacterial species
whose association with localized aggressive periodontal disease (formerly localized juvenile periodontitis) has been most clearly demonstrated (69, 87).
The subgingival prevalence of A. actinomycetemcomitans varies widely yet it typically increases with
disease (20, 82, 87). For example, 026% of healthy
children were found to exhibit A. actinomycetemcomitans subgingivally (71). On the other hand, 40
100% of subgingival sites in patients with aggressive
disease have A. actinomycetemcomitans (71). These
data suggest that A. actinomycetemcomitans is a causative agent in localized aggressive periodontal disease, but this has been difficult to prove, presumably
due to the episodic nature of disease activity. The
absence of the organism in a diseased state may be
due to the window of cultivability being missed.
A. actinomycetemcomitans has also been implicated
in some cases of chronic periodontitis (2, 50, 60).
The distribution of A. actinomycetemcomitans
serotypes within disease categories may be more

specific and indicative of A. actinomycetemcomitans

as a true pathogen or high-risk organism. For
instance, seroype c strains are more commonly
found in extraoral infections and in periodontal
health (3, 89). Furthermore, many serotype b strains
of A. actinomycetemcomitans, like JP-2, produce
increased amounts of leukotoxin, an important virulence factor, and are found most often associated
with periodontal disease (19).
In a more sensitive approach to identifying the
relatedness of A. actinomycetemcomitans isolates in
disease, DiRienzo and McKay (18) used restriction
fragment length polymorphism (RFLP) analysis to
type over 800 clinical isolates of A. actinomycetemcomitans. The identification of particular genetic variants of A. actinomycetemcomitans causing a
particular disease (or not) would suggest a high (or
low) risk organism. 13 RFLP patterns were found
using a 4.7-kb DNA probe from A. actinomycetemcomitans strain Y4. An oligonucleotide homologous to
the 5 end of the leukotoxin A gene specifically identified RFLP group II isolates that exhibited a 530-bp
deletion in the leukotoxin operon. This suggests less
genetic variability than the oral commensal P. gingivalis (36), consistent with A. actinomycetemcomitans
being a higher risk or more pathogenic organism,
especially in younger individuals. In a separate study,
nine out of 36 localized aggressive periodontal disease-susceptible individuals showed evidence of periodontal breakdown and six of these nine subjects
exhibited RFLP group II variants of A. actinomycetemcomitans (19) that were later shown to be high
leukotoxin producers (26). These studies serve as an
example of how molecular patterns characterized
within a species of bacteria can allow us to improve
the reliability of diagnostic information related to risk
assessment, thus sorting the commensals from the
true pathogens.
A. actinomycetemcomitans interacts with the host
by the production of several virulence factors. Leukotoxin, the most studied virulence factor made by
A. actinomycetemcomitans, is an RTX (Repeats in
Toxin) toxin (33) and shares sequence similarity with
the a-hemolysin from Escherichia coli, the cytolysin
from Pasteurella haemolytica and the leukotoxin
from Actinobacillus pleuropneumoniae (16). A. actinomycetemcomitans leukotoxin has been shown to
kill human and non-human primate polymorphonuclear leukocytes and peripheral blood monocytes
(4, 83), thereby evading the innate immune response
by attacking it directly. In the process, tissue-damaging granule contents from polymorphonuclear leukocytes and monocytes are released. Conversely,

25

Ezzo & Cutler

leukotoxin-mediated killing is related to the induction of apoptosis in HL-60 cells (31). Apoptosis, or
programmed cell death, is a process where host cells
undergo nuclear degeneration, triggering their clearance by phagocytes. Apoptosis, when properly regulated, is a normal part of tissue and immune cell
homeostasis and, indeed, of the immune response.
For example, apoptosis is induced by cytotoxic T
cells when clearing infected target cells. Similarly,
proteins made by A. actinomycetemcomitans (most
notably the leukotoxin) can induce apoptosis in a
variety of host immune cells. The significance of this
finding may be that, in contrast to necrosis, lysis of
the cells does not occur. Although cell lysis spills
tissue-degrading enzymes, it also releases antimicrobial peptides (e.g. defensins) that can kill bacteria,
and attract other inflammatory cells. Thus A. actinomycetemcomitans might benefit at some stage in
the disease process (e.g. when it emerges from an
infected gingival epithelial cell) by obtunding its
leukotoxin production and thereby blunting the
inflammatory response. Considering that A. actinomycetemcomitans has been shown to penetrate host
cells (40), one might presume that this organism
might benefit from eliciting apoptosis.
The A. actinomycetemcomitans lipopolysaccharide
or endotoxin also imparts virulence capabilities to
this organism (30, 63). Like the lipopolysaccharide
made by E. coli and Salmonella typhimurium,
A. actinomycetemcomitans endotoxin has the potential to modulate host responses and contribute to
tissue destruction. Most pertinent may be the ability
of the A. actinomycetemcomitans lipopolysaccharide
to stimulate macrophages to release interleukin (IL) -1,
IL-1b, and tumor necrosis factor (TNF); these cytokines, among other activities, are capable of stimulating bone resorption (63). Very little is known about
the biological activity of A. actinomycetemcomitans
lipopolysaccharide. We do not understand the type
of immune response it elicits (i.e. Th1/Th2) and how
it signals the innate immune cell response (i.e.
through CD14, Toll-like receptor [TLR] 4 or TLR 2).
In general, the structural features of A. actinomycetemcomitans lipopolysaccharide are typical and
include a surface antigen composed of various sugars,
a lipid A region deep within the outer membrane, and
an inner core polysaccharide (30). While the core polysaccharide and lipid A structure remain conserved
between the different serotypes of A. actinomycetemcomitans, there is a distinct difference between the
surface or O-antigen component (22). As an example,
the serotype b O-antigen has been defined as a repeating unit containing D-fructose, L-rhamnose, and

26

N-acetyl-D-galactosamine (53). The O-antigens of

the other serotypes of A. actinomycetemcomitans are
distinct from serotype b (22). As the serotype b strains
seem to produce more leukotoxin (19) and produce a
different O-antigen, it would seem that diagnostic
efforts to identify this high risk organism and immunotherapeutic efforts to combat its infection in vivo
should focus on serotype b strains.
Perhaps most relevant to the ability of A. actinomycetemcomitans to evade the innate defenses and survive mechanical periodontal therapy is its ability to
invade gingival tissues (8, 64) and, in particular, to
invade epithelial cells (40). It has been speculated that
once A. actinomycetemcomitans gains entry into the
connective tissue, its production of collagenase will
allow it to acquire nutrients and possibly disseminate
(59). One of the classic findings in periodontitis, collagen breakdown, could result (49). However, the direct
proteolytic activity of A. actinomycetemcomitans
enzymes are likely to be eclipsed by host metalloproteinases released upon A. actinomycetemcomitans lysis
of polymorphonuclear leukocytes and monocytes.
Many, but not all, strains of A. actinomycetemcomitans have been found to invade mammalian cell lines
(6, 40, 76). Invasion efficiency can be affected by the
mammalian cell type used and by the strain of A. actinomycetemcomitans tested (40). In a human epidermoid carcinoma cell line (KB), invasion is initiated by
the contact of A. actinomycetemcomitans with microvilli. Once in the cell, A. actinomycetemcomitans SUNY
465 can spread intracellularly by utilizing host cell
microtubules (41). Recently, A. actinomycetemcomitans, among the other periodontal pathogens discussed in this review, have been detected in human
buccal epithelial cells. This group was able to show
A. actinomycetemcomitans, B. forsythus, and P. gingivalis actively growing in these cells. Arguably, the ability of a microorganism to penetrate host cells is an
important trait common to those organisms most
likely to be associated with disease. The ability of periodontal pathogens to conceal themselves in host cells
might enable them to survive conventional periodontal
therapy and emerge to cause disease again.

A. actinomycetemcomitans as a
risk indicator for periodontitis
longitudinal studies
The presence of A. actinomycetemcomitans and the
progression or initiation of localized aggressive periodontal disease has been evaluated longitudinally.
In the 1996 World Workshop, it was acknowledged

Microorganisms as risk indicators for periodontal disease

that the presence of A. actinomycetemcomitans in

younger patients was a risk indicator for the development of periodontal disease (46). Since that time,
many studies have investigated whether the subgingival presence of A. actinomycetemcomitans in adults
coincides with periodontal disease. The majority of
these studies have been unable to show that the
presence of A. actinomycetemcomitans puts adults
at further risk of disease progression. This supports
most of the work done prior to 1996 that suggests
that it is the presence of P. gingivalis or B. forsythus
that more frequently correlates with progressive
chronic disease. Therefore, one can describe A. actinomycetemcomitans as being an initiator of aggressive disease, but not essential for it to occur. The true
mediator of disease is likely to be how the host
responds other bacterial species may play surrogate roles in initiating a similar response.
Since 1996, one group has investigated longitudinally whether the presence of A. actinomycetemcomitans puts adolescents at higher risk of aggressive
disease (80, 81). This group described three risk markers: age, presence of local factors and subgingival
A. actinomycetemcomitans. When A. actinomycetemcomitans was found subgingivally in Indonesian adolescents the odds ratio for progressive disease was
4.61. Like other studies analyzing the natural history
of disease, these subjects had no access to dental care
and therefore the prevalence of disease was high. In
another study where army recruits were analyzed for
the presence of A. actinomycetemcomitans and the
progression of disease, significant numbers of A. actinomycetemcomitans-positive samples were found in
active patients after 12 months (44). However, no
statistical difference was found at the patient level,
leading these investigators to conclude that A. actinomycetemcomitans was not a risk factor in these patients.
In general, few longitudinal studies describing
the risk of subgingival A. actinomycetemcomitans to
the progression of aggressive disease have been done
since 1996. Further studies will be needed to prove
the cause and effect relationship of A. actinomycetemcomitans and aggressive disease. However, these
studies should be approached using a systems biology approach, taking into account the genetics of the
host and of the pathogen.

Bacteroides forsythus as a risk

indicator of periodontitis
Of the three periodontal pathogens discussed in this
review, B. forsythus is the least understood, possibly

because it is the most difficult to cultivate in vitro. Its

association with periodontitis has gained more attention in recent years. B. forsythus possesses several
virulence traits including the production of a trypsin-like protease and lipopolysaccharide (43, 77) but
more recently its ability to penetrate host cells or
induce apoptosis has received attention (1, 62).
These recent findings have given rise to new ideas
for how this organism, as well as the others discussed
in this review, cause disease. As we begin to understand the mechanisms by which this organism can
cause disease, a clear view of the commonalities
between different periodontal pathogens emerges.
It is widely accepted that many of the periodontal
pathogens may attach and penetrate oral epithelial
cells. Since A. actinomycetemcomitans and P. gingivalis are perhaps the most studied periodontal
pathogens, their ability to invade cells in vitro and
in vivo has been well described. Understanding that
B. forsythus is almost always detected where P. gingivalis is present, investigators have hypothesized
that B. forsythus might also penetrate oral epithelial
cells. Recently, investigators found B. forsythus in
buccal epithelial cells first using polymerase chain
reaction methodology and later using fluorescent in
situ hybridization (FISH) (61, 62). This group has
been able to show, using this technology, that B. forsythus, in addition to P. gingivalis and A. actinomycetemcomitans, may be actively growing within these
cells. This would imply that the bacteria maintain an
intracellular reserve in areas otherwise difficult to
colonize due to their anaerobic characteristics. In
addition, it is possible that these infected cells may
act to transmit bacteria from site to site and host to
host during cellular turnover, possibly protecting
them from the harsh hypotonic conditions of saliva
(5). The ability of B. forsythus to attach and penetrate
cells may be related to its surface (S-) layer production. This protein component of B. forsythus shows
hemagglutination activity and is important in
abscess formation in animal models (34). Conversely,
other groups have not seen B. forsythus intracellularly in primary cultures of human gingival epithelial
cells (HGEC) (25). The authors went on to mention
that other invasive strains of B. forsythus may exist
and be more capable of intracellular penetration. Of
the pathogens tested in these studies, Fusobacterium
nucleatum, however, was found to be highly invasive
in HGEC and KB cells.
Perhaps the most intriguing aspect of B. forsythus
virulence is its ability to induce apoptosis. When
B. forsythus extract was added to HL-60 and other
human leukemic cells, a cytocidal activity was

27

Ezzo & Cutler

described. This effect was characterized by DNA

ladder formation and caspase-3 activation. Furthermore, the loss of mitochondrial membrane potential and membrane integrity also characterize the
apoptotic process induced by this organism (1).
The control organism in these experiments was
A. actinomycetemcomitans serotype b that, by its
production of leukotoxin, has also been shown to
induce apoptosis in these cells (31). As apoptosis of
cells results in their being recognized and engulfed
by resident (non-activated) macrophages, this raises
the provocative question of whether B. forsythus triggers a type of innate autoimmune response. In this
scenario, resident macrophages attempt to eliminate
the apoptotic epithelial cells infected by B. forsythus
through a well intentioned attempt at maintaining
tissue homeostasis.

Porphyromonas gingivalis as a risk

indicator of periodontitis
P. gingivalis is one of the best characterized of the
opportunistic oral pathogens that inhabit the oral
biofilm (1214, 24, 55, 85). P. gingivalis expresses
three major virulence factors - fimbriae, gingipains
and lipopolysaccharides. The fimbriae of P. gingivalis and other human pathogens mediate adherence
to specific receptors on host cells, such as epithelial
cells. Fimbriae also induce bacterial internalization
by activating and mobilizing the epithelial cell cytoskeleton. The fimA gene of P. gingivalis has been
cloned and sequenced (17). fimA encodes the structural unit protein (fimbrillin) of the major fimbriae of
P. gingivalis. fimA is present in a single copy in the
chromosome and is monocistronic. DNA sequence
analysis revealed that the fimbrillin of P. gingivalis
might represent a unique class of fimbrial subunit
proteins. Inactivation of the fimA gene and generation of fimbriae-deficient mutants has been invaluable
in the study of their function (39). Fimbriae-deficient
mutant strains, such as DPG-3, bind poorly to
salivary components, tooth surfaces and epithelial
cells. P. gingivalis strain DPG-3 also fails to invade
epithelial cells and does not induce periodontitis in
rats (21, 39, 65, 66, 86). Moreover, vaccination against
the fimbriae protects animals against periodontitis
(39). In addition to their role in adherence and
invasion, P. gingivalis fimbriae also modulate the production of proinflammatory cytokines such as IL-1b,
IL-6 and TNFa (47). P. gingivalis fimbriae induce T
cell activation in mice (28). Gingipains are P. gingivalis proteases whose major function is nutrient

28

acquisition through degradation of proteins into

peptides. The degradation of serum opsonins and
host tissue by gingipains contributes to phagocytosis
resistance and to the formation of spreading
abscesses in mice, respectively (24). As of 1995, at
least 39 apparently distinct proteolytic activities were
claimed to have been purified from P. gingivalis,
most being referred to as trypsin-like enzymes
(55). Most recently, three cysteine proteinases have
been purified from P. gingivalis, two capable of
hydrolyzing peptide bonds at Arg-X residues (Arggingipain or RGP) and one with Lys-X specificity
(Lys-gingipain or KGP) (54). These have since been
renamed gingipain R and gingipain K, respectively.
To date, a number of genes encoding proteinases
have been cloned and sequenced; most notably,
rgp1 and kgp, which encode a multidomain protein
of 1704 and 1723 amino-acid residues, consisting of a
prepropeptide (227 amino-acid residues), a catalytic
domain (492 and 509 amino-acid residues, respectively) and a hemagglutinin domain. The various
soluble and membrane bound forms of gingipains
are apparently derived through proteolytic processing of the preproenzymes by cleavage at Arg-X-peptide bonds (55). Several isogenic mutant strains of
P. gingivalis deficient in defined proteinases, including gingipains R and K, have become available and
have been tested on animal models (23, 45, 51). Gingipains R and K are critical to expression of virulence
by P. gingivalis. Gingipain R mediates vascular permeability through bradykinin release, enhances
binding of fimbriae to fibroblasts and destroys the
proteins of the complement system. Gingipain K also
mediates similar activities and is the most potent
fibrinogenase described to date (54).
Lipopolysaccharide is an important amphipathic
outer membrane constituent of gram-negative bacteria that enhances their structural integrity and
biological activity. The lipopolysaccharide of P. gingivalis is unique, based on the chemical structure of
its core polysaccharide and lipid A regions (48) and
in its biological activity. Unlike enteric lipopolysaccharide, P. gingivalis lipopolysaccharide induces the
symptoms of endotoxic shock in non-responder or
endotoxin-tolerant mice (C3H/HeJ) (79). These mice
have a point mutation within the region that codes
for the TLR4. Whereas TLR4 is the main transmembrane receptor for lipopolysaccharide of gram-negative bacteria, TLR2 is the key in responsiveness
to yeasts and gram-positive bacteria (i.e. PGN and
zymosan). Possible exceptions include the lipopolysaccharide of the oral pathogen P. gingivalis (27, 29,
56) and of the spirochete Leptospira interrogans (84),

Microorganisms as risk indicators for periodontal disease

both of which can apparently use TLR2. TLR2 -triggering of murine macrophages by P. gingivalis lipopolysaccharide leads to distinct patterns of inflammatory
gene expression, as compared to TLR4 (27). Moreover,
P. gingivalis lipopolysaccharide appears to stimulate a
Th2-biased response in mice (56) and human dendritic
cells (29), suggesting a mechanism for how P. gingivalis lipopolysaccharide can regulate the class of adaptive
immune response. P. gingivalis lipopolysaccharide
also binds poorly to recombinant soluble CD14 relative to E. coli lipopolysaccharide (11). Interestingly, the
serum antibody reactivity to P. gingivalis lipopolysaccharide by Western blotting analysis seems to correlate
particularly well to the clinical parameters of periodontal disease, with odds ratios equal to 40.8 (P 0.001)
(15). Thus P. gingivalis lipopolysaccharide appears
able to have a regulatory effect on the class of the
immune response, favoring a humoral response, which
might enhance its survival in vivo.

Longitudinal studies of B. forsythus/

P. gingivalis and periodontal
disease
Of the three organisms discussed in this review,
B. forsythus is by far the least well understood in
regard to its virulence. Recently, however, this organism has gained recognition as one of the most commonly found organisms associated with chronic
periodontal disease. In the consensus report from
the World Workshop in 1996, this organism was
described along with A. actinomycetemcomitans
and P. gingivalis as being the most likely candidate
organisms that could be referred to as risk indicators
(46). Since then, many longitudinal studies have
been described which continue to illustrate the
importance of this bacterium in chronic disease.
Longitudinal studies (i.e. trying to associate a particular bacterial species with disease activity over a
period of time) are extremely difficult to do when
trying to determine causality of a particular organism
to disease, for the reasons alluded to earlier. Prior to
describing the studies that associate B. forsythus with
disease, it is important to realize that P. gingivalis is
almost always found with B. forsythus. It is difficult
to discuss one organism without discussing the
other. When bacterial complexes are characterized
using technological development geared for the analysis of large numbers of species, a relationship
between these two organisms is frequently found
(72). Often, these organisms are analyzed concomitantly and there is a potential relationship between

these two organisms that allows for their mutual

colonization. Some have hypothesized that B. forsythus may in fact precede P. gingivalis in most
cases. This is emphasized in studies of conversion
of gingivitis to periodontitis. For example, numbers
of B. forsythus are elevated in gingivitis, while P. gingivalis is found in low numbers until more advanced
bone loss associated with periodontal disease is
detected (78). Similarly, when Machtei et al. (37)
followed 415 subjects with minimal disease over 5
years, the presence of B. forsythus at baseline
resulted in greater alveolar bone loss and a greater
proportion of attachment loss. Importantly, patients
harboring B. forsythus had twice the amount of tooth
loss at 5 years compared to B. forsythus-negative
patients (37). When evaluating patients with more
advanced disease, a profound increase in the numbers of P. gingivalis and B. forsythus were found (50).
In this large-scale analysis of 148 Chinese subjects,
up to 14 subgingival microbial samples from each
patient were analyzed with the Checkerboard
DNADNA hybridization technique (Socransky technique) over a 10-year period. A surprisingly high
frequency of all 18 species studied was evident. Diseased sites, however, when compared to stable tooth
sites, exhibited profound increases in P. gingivalis,
Treponema denticola and B. forsythus (50). In conclusion, recent data support the importance of B. forsythus and P. gingivalis in the initiation of chronic
periodontitis as well as the progression to advanced
periodontitis in longitudinal studies.
Further evidence suggests that B. forsythus, and
presumably P. gingivalis, are associated with disease
when patients are followed post-therapy. For example, chronic disease patients frequently show one or
both organisms in active sites detected post-therapy
(67). Perhaps the most telling examples of B. forsythus association with disease are established by
those studies where more sensitive disease detection
techniques are used to evaluate periodontal bone
loss. For example, Chaves et al. (7) used computer
assisted densitometric image analysis (CADIA) to follow periodontal bone loss following different treatment modalities. They attempted to correlate the
progression of disease with A. actinomycetemcomitans, P. gingivalis, P. intermedia, and other species.
The use of CADIA allows for the more sensitive
detection of an episode of bone loss in a patient.
The ability to detect subtle changes in bone destruction can allow for a more accurate correlation of
bone loss with the presence of specific pathogens.
In this study, P. gingivalis was commonly present in
the plaque of patients who experienced progressive

29

Ezzo & Cutler

bone loss. Positive and negative predictive values

were relatively high (84%, 85%) and an odds ratio
of 32 was calculated (7). B. forsythus was not analyzed in this study due to the nature of the detection
system used. A. actinomycetemcomitans was not
analyzed due to its infrequent presence subgingivally
in these chronic periodontitis patients. In another
study, Renvert et al. (57) attempted to relate microbiological parameters to the success of periodontal
treatment. Over 5 years, 12 patients were treated with
the aim of suppressing A. actinomycetemcomitans,
P. gingivalis, and P. intermedia and evaluated for
improvement in clinical parameters associated with
microbiological changes. A slight but significant correlation between patient clinical attachment loss and
persisting or re-established bacterial parameters was
detected. In conclusion, the studies done over the
past several years reaffirm, if not strengthen, the data
described prior to 1996. That is, the presence of
P. gingivalis and B. forsythus in a patients subgingival plaque increases their risk of developing chronic
periodontitis and reduces the chance of successful
periodontal therapy.

Other microbes associated with

periodontitis
Although we have focused on the three bacterial
species considered most likely to initiate the events
resulting in periodontitis, many other microbes have
been identified in the subgingival flora. Their roles,
if any, in the pathogenesis of disease, have yet to
be fully appreciated. The World Workshop in 1996
discussed a list of organisms that were described
as moderately associated with disease (46). These
organisms were found in the subgingival plaque
and were associated with disease. However, the
results of longitudinal assessment, if done, were inconclusive. These species include: Campylobacter
rectus, Eubacterium nodatum, Fusobacterium nucleatum, Prevotella intermedia/nigrescens, Peptostreptococcus micros, Streptococcus intermedius-complex,
and T. denticola (46).
In an effort to characterize the total bacterial diversity in the human subgingival plaque, Paster et al.
(52) used culture-independent molecular methods.
DNA was extracted from subgingival plaque and
16S ribosomal DNA was polymerase chain reaction-amplified and cloned into E. coli. The sequences
were compared with those of known species and
closest relatives. Based on the sequence data, the
subgingival flora contains 347 species or phylotypes,

30

and they estimated 68 additional unseen species were

present, for a total of 415 species in the subgingival
plaque (52). Paster et al. (52) further estimated that
total species diversity in the oral cavity, including
cheek, tongue and teeth, is about 500 species.
Human viruses have also been implicated in periodontitis. The presence of the genomes from viruses
within the Herpes family have been isolated from the
lesions of periodontitis patients when investigated in
cross sectional studies (68). Their genomes have
been found in chronic periodontitis (9), aggressive
disease (42), and periodontitis associated with systemic disease patients (68). The authors hypothesize
that environmental or systemic factors would allow
for the activation of these viruses whose inhibitory
impact on various host factors would allow for the
accumulation of periodontal pathogens and therefore disease.

The microbial community

A systems biology approach is important to understanding the role of microbial communities as risk
indicators. Systems biology approaches have been
applied to characterizing the constellation of species
(and their interrelationships) in disease and health
(72) as well as the bacterial clonotypes (at the
sub-species level) associated with different levels of
disease and health. This latter approach, called
molecular epidemiology, is useful for identifying
the following:

patterns of transmission from person to person
(35).

genetic relatedness of individual clones within
species to identify those species that behave as
pathogens vs. those that behave as commensals
(36).

changes in the bacterial genome upon growth
under different environmental conditions or in
different host cells, using microarray chip screens
(58).

the changes in the host cell genome when host
cells are presented with different bacterial species, clones or their products (38).
These approaches are necessitated by the fact that
the virulence traits of individual species in vitro
might bear little resemblance to their behavior in a
microbial community and, indeed, in vivo.
With the advent of new technology, we can gain
an appreciation for the complexity of the subgingival microbiota as further knowledge of bacterial
groupings becomes available. In 1998, Socransky et al.

Microorganisms as risk indicators for periodontal disease

(72) discussed the various complexes of bacteria

found in diseased and healthy patients. Although this
data is cross-sectional and may be difficult to apply
to the assessment of patient risk, the results are intriguing. Using Checkerboard DNADNA hybridization techniques (74), this group was able to analyze
13,261 subgingival plaque samples from 185 patients.
In this study, community ordination, a procedure
designed to relate species to each other and to look
at how complexes of bacteria relate within an ecosystem, was utilized to analyze the subgingival
microbiota. An in-depth analysis resulted in the
separation of five complexes. The red complex of
organisms includes P. gingivalis, B. forsythus and
T. denticola and is most often correlated with clinical
parameters of periodontal disease. As described in
detail earlier, in 2001 Paster et al. (52) undertook the
large task of analyzing the bacterial diversity of subgingival plaque in health and disease using a polymerase chain reaction-based rRNA analysis. In this
report, they evaluated 2,522 rRNA sequences from
cultivable and not-yet-cultivated bacterial samples
from 26 patients with various forms of periodontitis
and five healthy control patients. Even with the bias
of a smaller number of healthy controls, 29 species
(only 18 of which are named) were isolated exclusively from diseased sites. B. forsythus and P. gingivalis were among the named organisms isolated (52).
Importantly, as we grow to understand the contribution of those members of the community at the
molecular level, we can distinguish those individual
serotypes and clonotypes most associated with disease. Within a particular species of pathogenic bacteria, there exist multiple serotypes (based on the
lipopolysaccharide or capsule type) that can infect
the host. Some bacterial serotypes are associated
with acute illness or death (e.g. group B streptococci)
(75), while other serotypes may be relatively benign.
Within a particular serotype, there is the potential for
multiple clonotypes (based on the DNA sequence)
imparting their own level of virulence. This is perhaps most apparent in the genetic studies of clonality
(36), where, for example, strains of a specific organism produce an increased amount of an important
virulence trait allowing those strains to cause disease.
This would indicate that it is not the bacterial species
that is related to disease but more likely a specific
variant or clone of that species (hence its virulence
factor(s)) that is causing disease. This sub-grouping,
therefore, would likely express a specific molecular
pattern that could be more closely associated with
disease than the mere presence of the bacterial species itself.

The host genome as it modifies

bacterial risk
For many years we have been trying to associate
specific periodontal pathogens with the periodontitis
that develops in patients with higher risk systemic
factors. For the most part, subtle shifts in microbial
flora have been noted in smokers or diabetics, for
example. Over the past several years, several groups
have questioned whether genotype positive patients
that express higher levels of IL-1 and IL-1b due to
differences in their polymorphic alleles, harbor specific bacterial species that may be related to higher
risk of progressive disease. The general hypotheses
from these studies say that patients with a genetic
predisposition to an altered level of inflammatory
response may indeed be less capable of tolerating
the presence of specific organisms. This would be
perhaps the best example of how specific periodontal
pathogens may put certain patients at higher risk of
periodontitis. As with all causal relationships,
described in this article, this type of association is
difficult to prove. Although little has been done longitudinally, we have some clues that lead us to believe
that specific bacterial species or groups of bacteria
may indeed put genotype positive individuals more
at risk. These studies may also explain why results
from past studies looking at specific bacteria as risk
factors may have been skewed. For example, the
results of well-controlled longitudinal studies
describing the odds ratios associated with the presence of specific bacteria to disease may have been
higher if patients had been analyzed separately based
on genotype. As we begin to understand the contribution of the host to the pathogenesis of disease, we
may be more capable of understanding the specific
bacteria that target or evade those host defenses that
are most protective. This would therefore result in an
improvement in the selection of an appropriate antibiotic or therapeutic technique. Many of us feel that
if we could eliminate one bacterium from the oral
cavity, another would take its place and cause disease. This idea would not hold true if we could identify the causative organisms, eliminate them and
subsequently arrest disease progression.
A good example of a longitudinal study that analyzes the progression of periodontal disease and genotype status was done by Cullinan et al. (10). In this
study, clinical parameters were monitored every year
for 5 years and P. gingivalis, A. actinomycetemcomitans and P. intermedia were detected using ELISA. In
that study, IL-1 genotype positive smokers with

31

Ezzo & Cutler

advancing disease would have 70% more pocketing

than genotype negative smokers. Importantly, using
a generalized linear modeling analysis, this group
showed that when genotype positive patients exhibited
P. gingivalis, but not A. actinomycetemcomitans or
P. intermedia, subgingivally these individuals would
have 80% greater chance of increases in pocket
depth  3.5 mm (10). The Socransky study using the
checkerboard DNADNA hybridization technique to
identify subgingival taxa in severe periodontitis
patients (73) found increasing proportions of red and
orange complex bacteria in both genotype positive and
negative individuals and a tremendous increase in total
DNA probe count was found in the deeper pockets of
genotype positive patients (73). These data describe
patients at higher risk of colonization with high numbers of organisms specific to different bacterial complexes that are capable of causing disease. Taken as a
whole, both of these studies illustrate the importance
of specific bacteria as risk indicators of periodontitis.
As is true for most research that implicates specific
bacteria in disease, the greatest limitation of these
studies is time. Due to the episodic nature of periodontitis, it is difficult to assign a chronological succession of events. Therefore, it is difficult to say that the
presence of certain bacteria in IL-1 genotype positive
patients causes their disease to intensify. It may be
that these organisms only colonize in high numbers
after disease has provided a niche for growth. These
clues, however, do give us an idea that indeed certain
bacteria like P. gingivalis and B. forsythus are more
important than others when considering risk indicators of chronic periodontitis.

Conclusions
Clearly, it is difficult to ascertain the causality of specific pathogens in periodontitis. Due to the episodic
nature of periodontal disease and our lack of sensitive diagnostic clinical tests that can detect disease
activity, we cannot accurately answer the age-old
chickenegg question. Was the specific organism
present when disease was initiated, implicating that
organism, or did the organism colonize a site after the
disease occurred? Although there are other reasons
why causality or risk is difficult to relate to specific
bacterial species, recent research in microbial complexes and host genome markers elucidates other reasons why assessment of specific periodontal pathogens
as risk indicators is so complex.
In conclusion, this review has attempted to delineate recent findings in the area of microbes as risk

32

factors for periodontitis. The scope of this review was

confined to elaborating and updating the findings
described at the 1996 World Workshop in Clinical
Periodontics (46). The consensus derived from that
conference implicated three periodontal pathogens,
A. actinomycetemcomitans, P. gingivalis, and B. forsythus as risk factors in periodontitis. Several groups
have completed or extended longitudinal analyses of
these organisms. Therefore, in our review of the literature since 1996, we have described studies that
continue to support, if not further emphasize, the
importance of these pathogens in the initiation or
progression of periodontitis.
As we begin to hone in on the organisms most likely
to put a patient at risk of periodontal disease, we can
begin to assess the commonalities of these bacteria.
The three main commonalities of A. actinomycetemcomitans, P. gingivalis, and B. forsythus include:

all are gram-negative, and therefore produce lipopolysaccharide, which can modulate the local
inflammatory response in host cells that express
pattern recognition receptors (i.e. TLRs) (27, 29, 56).

all appear capable of invasion of the mucosal
barrier to infection and possibly of being sequestered inside epithelial cells. Thus they can waitout the bad times and re-emerge when conditions are permissive for their growth.

all produce factors that enable them to evade the
antibacterial functions of the innate immune
response either passively (anti-phagocytic capsule) or actively (leukotoxin, gingipains, other
proteases, induction of apoptosis).
As we study the organisms that contribute to
periodontal disease to assess their risk, it becomes
clear that we should focus more on the common
virulence traits/products shared by the organisms
(especially pathogen associated molecular patterns,
or PAMPs), and how they modulate/regulate the
responses (at the genomic level) of the host cells that
are most likely to encounter them. Resident host cells
most likely to encounter pathogenic/commensal bacteria in the gingiva include epithelial cells/keratinocytes, Langerhans cells/dendritic cells, macrophages,
and T cells. The technology now exists that will allow
us to better identify and further characterize the different bacterial clones that express PAMPs in situ and
study how these bacteria interact with these cells. We
must also begin to understand how groups of organisms (with their respective PAMPs) may co-exist in a
synergistic relationship capable of exaggerating their
potential for virulence as individual bacterial species.
We know that the host response to the pathogens we
have described is responsible for much of the tissue

Microorganisms as risk indicators for periodontal disease

destruction seen in periodontitis. Clearly, a full understanding of the host susceptibility factors in addition to
the microbial factors that put our patients at risk is
essential for the successful treatment of patients with
periodontal disease.

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