Endodontic Topics 2003, 6, 160–170 Copyright r Blackwell Munksgaard

Printed in Denmark. All rights reserved ENDODONTIC TOPICS 2003

Treponemes and endodontic

infections
ULF R. DAHLE, PIA TITTERUD SUNDE & LEIF TRONSTAD

The pathogenic potential of spirochetes and their significance in the development of oral diseases, as well as in
infections in other organs, have gained new interest for several reasons. First, these bacteria have the potential to be
pathogenic because of their number and frequency at infected sites, their production of tissue irritants, their ability
to invade tissues, their abundance in mixed infections, and their methods to evade host defense mechanisms.
Second, of the microbial species that are associated with oral infections, spirochetes are infectious agents that cause
severe diseases in other body sites. Third, during the past few years, new techniques have made it possible to obtain
more information about spirochetes at the genetic level. Thereby, we have gained better knowledge of these
bacteria, even though many cannot be cultured in the laboratory yet. Such knowledge includes the diversity of oral
species as well as antigenic similarities between the species commonly found in the oral cavity and known pathogens
such as the syphilis agent Treponema pallidum subsp. pallidum, Lyme disease agents Borrelia burgdorferi sensu lato
group, and the swine dysentery agent Brachyspira (Serpulina) hyodysenteria. This review covers the main areas of
what is known to date about Treponema. It focuses especially on factors related to their taxonomy, epidemiology,
and biology, with special emphasis on their presence and possible role in endodontic infections.

The issue of uncultivated organisms rings loudly in the
ears of microbial ecologists, particularly those con-
cerned with the interaction of cells in communities. It
has been estimated that 99% of all microbial species
have not yet been grown in culture, and many, in fact,
may be resistant to cultivation by available techniques
(1). As a result, there are limited opportunities of
studying the genetic, biochemical, and metabolic
capacities of the vast majority of single-celled organ-
isms. This paradox, that most of the microbes in the
world cannot be studied in fine detail in the laboratory,
is a major problem for our understanding of the
microbial world (1).
Oral biofilms offer a good demonstration of this gap
in our current knowledge. When viewed in the
microscope, it is clear that half the organisms in dental
plaque are spirochetes, with a distinct spiral-shaped
morphology and corkscrew motility, but most of the
observed cell types have not yet been grown in culture.
Many such uncultivated organisms are apparently
active in the community, and they appear to play a role
in endodontal and periodontal diseases. In many
communities, including the oral flora, it is not known
whether active but uncultivated microbes contribute to
community activity, and it remains a source of active
debate and investigation.
Spirochetes are notoriously difficult to cultivate and
study, but they are also special in that they include
many known pathogens from infections such as syphilis
(2), lyme disease (3, 4), swine dysentery (5), and
leptospirosis (6, 7). Spirochetes are also likely causative
agents of noma (8) and animal digital dermatitis (9),
and many spirochetes, including Treponema, Borrelia,
and Leptospira, are highly invasive organisms (10–12).
Spirochetes are thus associated with infections in many
sites of the body. It is not clear whether the Treponema
species commonly found in the oral cavity are specific
to the mouth. Still, for practical reasons, the terms oral
spirochetes or oral treponemes will be used throughout
this review, which covers the main areas of what is
known to date about Treponema. It focuses especially
on factors related to their taxonomy, epidemiology and

160

biology, the similarities between oral species and the
syphilis spirochete, and the pathogenic potential of
treponemes in endodontic infections (Fig. 1).
Morphology and taxonomy of oral
treponemes
Taxonomy, also known as biosystematics, gathers
organisms into defined groups, provides appropriate
nomenclature for the different groups, and is involved
in the identification of previously unknown micro-
organisms. To establish these groups, microbial typing
data are mandatory for the definition of so-called
species. The definition of a species is a primary
underlying concept; however, it is controversial and is
undergoing continuous refinement (13). When the
diverse microbial world is considered, many species of
microorganisms have been described on the basis of
phenotypic characteristics without any clues to their
phylogenetic status being available. Such is the case for
many oral spirochetes, although they were among the
first bacteria described when van Leeuwenhoek wrote
about the ‘animacules that bent their body into curves
in going forwards’ on September 17, 1683 (14).
The organisms belonging to the order Spirochaetales
are divided into three families: Brachyspiraceae, Leptos-
piraceae, and Spirochaetaceae. The first family contains
the genus Brachyspira (Serpulina). The second family
Fig. 1. Darkfield micrograph of oral spirochetes isolated
from infected root canal of human tooth. The long,
helically shaped Treponema cells can be seen in addition to
particles from the culture medium. During in vivo
microscopy these cells typically exhibit vigorous flexing
and turning movements as they swim around in the liquid
medium.
Treponemes and endodontics
contains species of the genera Leptonema and Leptos-
pira. Spirochaetaceae contains species of the genera
Borrelia, Brevinema, Cristispira, Spirochaeta, Spirone-
ma, and Treponema (15).
In terms of the genetic determinants of physiological
characteristics, the facultatively parasitic Leptospira
interrogans differs extensively from the strictly parasitic
pathogenic spirochetes, Treponema pallidum and Bor-
relia burgdorferi, although similarities exist in the genes
that govern their unique morphological features (6).
Spirochetes are a medically important and ecologi-
cally significant group of motile bacteria with a distinct
morphology. Most are helically shaped (16, 17), but
some species have a flat sinusoidal or meandering
waveform (11, 12). In addition to a typical bacterial
plasma membrane surrounded by a cell wall containing
peptidoglycan, they have an outer lipid bilayer mem-
brane, also referred to as an outer membrane sheath.
Characteristic of this outer sheath is a high-molecular-
mass oligomeric protein. Ishihara et al. (18) have
demonstrated that a PrtP (dentilisin)-deficient mutant
has decreased amounts of this protein and that
dentilisin activity plays a major role in the structural
organization of the outer sheath of T. denticola. The
loss of dentilisin activity and the following structural
change in the outer sheath also affect the pathogenicity
of T. denticola (18, 19).
Within the outer sheath is the protoplasmic cell
cylinder and subterminally attached periplasmic flagella
(10). The space between the protoplasmic cell cylinder
and the outer membrane sheath is referred to as the
periplasm (10). The spirochetes are unique, as their
periplasm contains these periplasmic flagella that are
similar in many respects to the external flagella of rod-
shaped bacteria (Fig. 2). Each periplasmic flagellum is
attached subterminally to only one end of the cell
cylinder and extends toward the opposite end. For
spirochetes, translational motility requires asymmetrical
rotation of the two internally located flagellar bundles.
Consequently, they have swimming modalities that are
more complex than the well-studied mechanisms of
other motile microorganisms (10) (Fig. 3).
There are many unknown factors with respect to
motility and chemotaxis gene regulation in spirochetes.
For example, little is known about the factors that
control periplasmic flagella length and number. Spir-
ochete species vary with respect to the size and number
of periplasmic flagella. The organisms can be quite
large: For example, Cristispira are 0.5–3 mm wide,
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Dahle et al.
Fig. 2. Schematic diagram of a spirochete illustrating
the outer membrane, the cell wall surrounding the
protoplasmic cell cylinder, and one periplasmic flagel-
lum attached at each end of the cell. The two flagella are
located inside the outer membrane, and stretch along
two-thirds of the cell length so that both are located inside
the middle third of the cell.
Fig. 3. Transmission electron micrograph of Treponema
vincentii. The cell is helically shaped and carries 5 flagella
at each end of the cell. One flagellum is detached and
protrudes outside the cell as a result of the preparation
procedures for microscopy.
30–180 mm long, and have over 100 periplasmic
flagella attached at each cell end (10). In contrast, the
Leptospiraceae (which include Leptospira and Leptone-
ma sp.) are approximately 0.1 mm in diameter, 10–
20 mm long, and have only one periplasmic flagellum at
each end.
For bacteria, a species is defined as an entity in which
members have a DNA–DNA homology value of at least
70% (20). Typing systems are used to define specific
characteristics of the object under study. The proce-
dures are specific for different phenotypic or genetic
parameters and can be general (i.e. applicable to any
microbial species) or species or genus specific. How-
ever, no clear definition exists for a genus, and oral
spirochetes are often classified as Treponema based on
their morphological appearance alone (Figs 2 and 3).
It is believed that all spirochetes found in the oral cavity
belong to the genus Treponema and more than 40
162
phylotypes have been identified in the human oral micro-
biota (21). So far, T. amylovorum, T. denticola, T. leci-
thinolyticum, T. macrodentium, T. maltophilum, T. orale,
T. parvum, T. pectinovorum, T. scoliodontum, T.
socranskii, and T. vincentii have been named (21, 22).
To demonstrate the difficulties in handling these
organisms in the laboratory, T. macrodentium, T. orale,
and T. scoliodontum have apparently been lost from
culture collections and these organisms need to be
isolated again to regain a standing in treponemal
taxonomy. T. vincentii have no deposited type strain
and is thus not fully accepted as a separate species and
often referred to as ‘T. vincentii’ (21, 22).
The agent of syphilis and type species of Treponema,
T. pallidum subsp. pallidum has never been cultured
continuously in vitro (23). This is also the case for many
other spirochetes, but the agent of syphilis demon-
strates, yet again, how difficult it is to culture these
organisms as the attempts to isolate and study this
organism have undoubtedly been many throughout
the centuries. Syphilis quickly reached epidemic pro-
portions in Europe after it was first reported in the late
1400s. It spread across the world during the early 16th
century and has been called the acquired immune
deficiency syndrome of that era (23). In 1939, Sir
Winston Churchill said ‘Syphilis is a riddle, wrapped in
a mystery, inside an enigma’. Although the syphilis
epidemic is currently under control, largely thanks to
penicillin and lack of antibiotic resistance in T. pallidum
subsp. pallidum, this quote may still be illustrative of
our understanding of this and other spirochetes.
T. pallidum subsp. pallidum is an obligate human
parasite, but the mechanisms of its pathogenesis are
poorly understood. No known virulence factors have
been identified and the outer membrane is mostly lipid
with a paucity of proteins. Thus, diagnostic tests for
syphilis are not optimal and no vaccine has yet been
developed (23). The T. pallidum subsp. pallidum
genome was among the first complete chromosomes to
be sequenced, and it offered a wealth of basic
information when it was published (23). It also
provided a foundation for the development of a culture
medium for T. pallidum subsp. pallidum, but no such
medium has yet been found, thus still postponing
further taxonomic, genetic, and virulence studies of
both T. pallidum subsp. pallidum and oral Treponema
species.
That treponemes constitute a diverse genus that has
been demonstrated through numerous studies. The

G1C ratio of oral treponeme DNA varies from 34 to
52 mol% (24). Choi et al. (25) have demonstrated that
one patient with severe destructive periodontitis may
harbor up to 81 spirochetal clones. If even the strictest
definition of a species-level cluster was set (98% or
greater sequence similarity), the clone sequences were
found to represent 23 different species (25). Such great
diversity within a genus complicates studies, as it is not
likely that all these species are pathogenic. Also, because
they are difficult to culture and isolate, spirochetes are
often referred to as one group of bacteria.
Since they are difficult to culture and analyze in the
laboratory, spirochetes have commonly been typed
based on morphologic characteristics, such as the size
and number of endoflagella. Traditionally, oral spir-
ochetes have been divided into three main categories:
small-, intermediate-, and large-sized morphotypes
(26). They range in size from 0.15 to 0.40 mm, and in
length from 5 to 20 mm (27). Even larger spirochetes
have been observed, but remain to be cultured (27).
Spirochetes are motile thanks to their internal flagella,
commonly known as periplasmic flagella. The same
number of flagella is attached at each end of the cell and
run within the cell for two-thirds of its length (Fig. 2).
The cell will thus contain twice as many flagella in the
middle third, as in the two terminal thirds of the cell.
This has been a popular way of characterizing oral
spirochetes and it has given rise to a nomenclature for
spirochetes. If a cell has one flagellum in each end, it is
thus named a 1 : 2 : 1 spirochete (Fig. 2).
Organisms as diverse with respect to size, metabo-
lism, and ecological niche as the spirochetes indicate
that their existence could be the result of convergent
evolution. Alternatively, the spirochetes could have
evolved divergently from a common ancestral spir-
ochete. To address these hypotheses, the 16S rRNA
genes of several pathogenic, parasitic, and free-living
species have been sequenced. The results indicate that
the spirochetes are an ancient group of bacteria, and
that they evolve from one common ancestral proto-
spirochete (15). In fact, they are one of the few phyla of
bacteria that can be identified solely from morphology
(15, 28). Recently, the entire genomes of T. pallidum
subsp. pallidum, B. burgdorferi, and T. denticola have
been sequenced (24, 29, 30). The genome sequence is
the blueprint of all the working parts of a bacterium,
and its completion is the first step toward determining
how all the parts function and interact. The DNA
sequence reveals not only the open reading frame
Treponemes and endodontics
(ORF) encoded within a gene but also associated
promoter elements, and thus information necessary to
study how expression of a gene is regulated (31). The
results of this analysis further support the conclusion
that the spirochetes are evolutionarily closely related
(10).
Methods to study bacterial epidemiology need to not
only separate the species (identification) but also to
separate the different strains within a species (typing).
In addition, the bacterial characteristic that is studied
must be stable enough not to separate all isolates of a
strain. Typing of bacterial species is important (1) for
surveillance of transmission between patients, (2) to
determine risk factors for infection, (3) to identify and
decipher outbreaks, (4) to differentiate re-infection and
new infection, as well as (5) to evaluate prophylactic
measures that have been employed. Since spirochetes
are such diverse bacteria, it has been difficult to find
methods applicable to all strains within a species.
It has been demonstrated that fresh clinical isolates
and laboratory reference strains of Treponema species
represent genomic groups, irrespective of previous
species assignment, and that characterized cultures may
contain more than one species (32). Thus, when
scientists study spirochetes from the oral cavity, they
cannot always trust that they are dealing with a
pathogenic organism. Neither can they trust that the
culture they are working with contains only one species.
This may in part explain why microbiologists often find
it difficult to identify spirochetal cultures. Problems
related to culturing and isolation of the organisms are
therefore only the first obstacle to solve in order to
conclude on the pathogenicity of spirochetes in oral
infections. These problems will be followed up by
developing methods to identify and to type the strains
before the much-needed pathogenicity studies com-
mence.
Virulence factors of oral treponemes
Genome analysis of microbial pathogens has provided
unique insights into their virulence, host adaptation,
and evolution. The advent of post-genomic approaches
and the sequencing of the human genome will enable
scientists to investigate the complex and dynamic
interplay between host and pathogen. In the last 8
years, more than 130 bacterial genomes have been
sequenced and more than 120 similar projects are
163
Dahle et al.
currently being conducted. This information is already
being used in search for new antibiotics, vaccines, and
for markers of pathogenicity. In June 2003, The
Institute of Genomic research (TIGR) in Maryland
and Stanford University in California announced that
they were starting up projects to sequence the genomes
of all microorganisms (metagenomics) present in the
oral cavity, gastrointestinal tract, and the vagina of
human beings (33). This is expected to generate a
wealth of information that will catalyze the develop-
ment of new intervention strategies to reduce the
burden of microbial-related disease also in the oral
cavity. However, until such results are available, the
information on Treponema virulence will remain
piecemeal since only three of the many species have
yet been fully sequenced.
In order to succeed as a pathogenic parasite, a
microorganism must be able to transmit between
carriers, adhere to a host surface, evade the host
defense mechanisms, and/or produce products that
cause an unwanted reaction in the host. It is also
important that the pathogen does not kill or cause life-
long immunity in a host before it has been transmitted
to another host. There is little doubt that treponemes
have ample opportunities to spread by salivary transfer
since oral infections (where they are present) are long-
lasting, even life-long infectious diseases. It has also
been demonstrated that oral treponemes can adhere to
human surfaces, evade the host defense mechanisms,
and produce tissue-destructive enzymes (22). It is not
completely understood, however, which of the oral
Treponema species hold these characteristics of patho-
genic organisms. Research has focused on T. denticola
that is often considered a representative species of the
oral treponemes.
Among the anaerobic oral spirochetes, the species T.
denticola can be cultivated in the laboratory and is
studied not only in its own right as an oral pathogen but
also as a relative of the syphilis spirochete and
representative of other oral spirochetes (34). As the
gingival epithelium is a primary colonization site for
human oral Treponema species, interactions of bacteria
with gingival cells are highly relevant to understanding
disease progression. The binding of T. denticola to
human gingival fibroblast (HGF) cells is sensitive to
proteinase K but not to trypsin, suggesting the
involvement of a trypsin-resistant proteinaceous adhe-
sin. Adhesion is also inhibited by mannose and
galactose. This implies a role for lectin-like adhesins
164
on the bacterial cell surface. Treatment of HGFs with
periodate (which oxidizes saccharides) also inhibits
bacterial adhesion (35). Thus, attachment of T.
denticola to HGFs probably involves protein–protein
and protein–carbohydrate interactions. Although Tre-
ponema adhere to a range of host proteins, the
interactions of T. denticola and T. pallidum subsp.
pallidum with fibronectin are the most fully examined.
Fibronectin-mediated adhesion has been implicated in
many important bacterial infections caused by a range
of Gram-positive and Gram-negative organisms, and in
some instances invasion of host tissues is fibronectin
dependent (10). Fibronectin is a 220-kDa glycopro-
tein, present as two polypeptide chains that are
important for cell adhesion, migration, repair, and
blood clotting. Fibronectin also seems to be important
for T. denticola adhesion to HGFs, as adhesion is
inhibited by anti-fibronectin antibodies (36). Binding
of T. denticola cells to HGFs results in profound
morphological alterations of the cells because of
cytoskeletal rearrangements, and then leads to HGF
cell detachment from the substratum. These processes
are independent of each other and detachment may be
related to degradation of fibronectin by T. denticola
proteases (37).
The component in T. denticola responsible for
inducing cytoskeletal rearrangements has been identi-
fied as the major surface protein (Msp) (38–40). This
protein is one of several surface proteins identified in T.
denticola and mediates binding to host cells and
extracellular matrix and function as porins. It is a
trypsin-resistant protein that perturbs calcium signal-
ing by uncoupling store-operated channels, whereby
Ca21-release from endoplasmic reticulum stores is
retarded and subsequent calcium influx is inhibited
(41). Msp binds both putative epithelial cell surface
receptors and cytoplasmic proteins, and the Msp
complex forms ion channels in the cytoplasmic
membrane of epithelial cells (40). These properties
can contribute to the cytopathic effects of T. denticola
on host epithelial cells.
Many spirochetes, including Treponema, Borrelia,
and Leptospira, are highly invasive pathogens. Motility
is likely to play a major role in many disease processes,
including some in the oral cavity. These organisms can
swim in highly viscous, gel-like media, such as
methylcellulose, that slows down or stops most
externally flagellated bacteria (10). This attribute may
allow T. denticola and other spirochetes to penetrate,

invade, and adapt to specific ecological niches that
exclude other bacterial species (16). Studies of infec-
tions such as acute necrotizing and ulcerating gingivitis
(ANUG) and noma have demonstrated that spirochete
species in such tissue are diverse (42). Spirochetes have
also been observed to invade surrounding tissues of
ANUG sites (42), and they are major candidates to
cause noma (8).
A chymotrypsin-like protease complex, which is
composed of the 72-kDa PrtP protein and two auxiliary
proteins with molecular masses of approximately 40 and
30 kDa, is also involved in localization and oligomeriza-
tion of the T. denticola Msp (39, 41–45). This protein
complex has been named dentilisin. It is implicated in
degradation of host cell molecules as it degrades
fibronectin and host cell protease inhibitors (39, 43–
46). Dentisilin also contributes to tissue invasion and
probably plays an important role in the ability of
spirochetes to invade host tissues and spread infectious
disease within the host. Outer membrane (OM) extracts
of a PrtP-deficient mutant of T. denticola has dimin-
ished stress fiber-disrupting activity in HGF than the
OM extracts of the parent strain. The OM extract of the
parent strain completely disrupts epithelial resistance,
while that of the mutant has negligible effects (47).
Interestingly, isogenic msp mutants of T. denticola that
are inactivated at a centrally located site of the msp gene
produce increased amounts of dentisilin. In contrast, if
mutations are constructed near the 3 0 end of the msp
gene, T. denticola produces a truncated Msp and no
dentisilin (45). Thus, important interactions between
Msp and dentisilin transport or assembly of the outer
membrane complex exist.
Spirochetes that may be suspected to be associated
with the pathogenesis of a disease are motile bacteria
that can be found in the most advanced regions of
infected tissue (48). Previous studies have shown that
the motility of spirochetes is a key virulence factor, since
spirochete motility mutants fail to infect their host (49,
50). While it is evident that pathogenic spirochetes do
move within the tissues of their respective hosts, it is
still unclear whether these cellular movements are
random or directed by chemotaxis systems. Genome
sequence analyses of Borrelia burgdorferi, Treponema
pallidum subsp. pallidum, and T. denticola have
revealed that these spirochetes not only have complete
flagellum-based motility systems but also possess the
genes necessary for chemotaxis that may direct the
flagellar movement (23, 29, 51–54).
Treponemes and endodontics
The migration of T. denticola through epithelial
tissue is dependent upon motility and an intact
chemotaxis system. T. denticola penetrates to a greater
degree than T. socranskii or T. vincentii. Additionally,
spirochetes isolated from fresh dental plaque seem to
penetrate the endothelium to a greater extent and/or
in greater numbers than reference strains (54). As
opposed to intercellular penetration, which is the mode
of penetration used by T. pallidum subsp. pallidum to
pass through endothelium, it seems that oral spiro-
chetes may utilize intracellular passage as well. Both
inter- and intracellular penetration occurs with B.
burgdorferi (55, 56).
Recently, T. denticola mutants (in flgE, cheA, dmcA,
and dmcB genes) were found to be deficient in the
penetration of gingival epithelial cell layers (57, 58).
These results suggest that both motility and chemotaxis
are involved in tissue penetration. Motility-deficient T.
denticola mutants, lacking the central kinase of the
chemotaxis pathway, are unable to penetrate epithelial
tissues (57). T. denticola cells demonstrate high affinities
for binding to type I and type IV collagen as well as to
gelatin (59). T. vincentii adheres to collagen type I, III,
IV, and V. Collagen adhesion has been attributed to the
activity of Msp (60). These interactions of Treponema
with tissue matrix components such as collagen and
hyaluronan (61) probably facilitate penetration of host
cell layers. It thus seems that for the oral bacterium T.
denticola, the epithelium of the gingival tissue consti-
tutes a natural site of entry into the host (57, 58).
Spirochetes have been identified in dental plaque
using monoclonal antibodies to putative T. pallidum
subsp. pallidum-specific proteins. Also, serum from
subjects with necrotizing ulcerative gingivitis has been
shown to contain immunoglobulin G to molecules
considered to be restricted to T. pallidum subsp.
pallidum (62). Serum from subjects with periodontitis
may contain IgA, IgG, and IgM antibodies that cross-
react with 47-, 39-, and 15-kDa molecules from T.
pallidum subsp. pallidum. However, there is no
association of cross-reactive immunity to T. pallidum
subsp. pallidum with the detection of these oral
spirochetes in subgingival plaque. This means that oral
treponemes harbor antigenic similarities to those of T.
pallidum subsp. pallidum. Such findings help indicate
that oral treponemes may serve as models for the
specific syphilis pathogen.
In the absence of antibiotic therapy, T. pallidum
subsp. pallidum establish lifelong persistent infection
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Dahle et al.
in a host, despite humoral and cellular immune
responses. The mechanisms by which these organisms
evade the immune response to persist for decades are
not yet fully known. However, outer surface molecules
of many bacterial pathogens undergo antigenic varia-
tion. This allows organisms to evade the host immune
system and establish a chronic infection, since immune
response mechanisms may not recognize the infectious
agent. This also appears to be the case for some
spirochetes. The large number of serotypes of the
relapsing fever Borrelia spirochete, Borrelia hermsii, is
in part due to gene conversion occurring in the vsp and
vlp genes (63, 64). The Lyme disease agent B.
burgdorferi also undergoes antigenic variation to
produce a high level of diversity of the vlsE gene (65–
67). Changes in the vlsE variable regions have been
shown to alter VlsE antigenicity during infection with
B. burgdorferi (68). Recently, LaFond et al. (69) have
demonstrated that tprK sequences in T. pallidum
subsp. pallidum obtained directly from syphilis patients
are heterogeneous. Also, tprK sequences from a rabbit-
propagated isolate reveal that individual variable
regions have different levels of diversity. The expression
of new TprK proteins may, in part, explain how T.
pallidum evades the intense immune responses in an
infected host, as well as being able to re-infect
previously exposed individuals. The tpr family of genes
is believed to be central to pathogenesis and immunity
during syphilis infection and some of these genes in
T. pallidum have homology to the msp genes of
T. denticola (38).
Treponema in endodontic infections
Root canal infection
More than a hundred years ago, Miller (70) reported
the occurrence of spirochetes in endodontic infections
and suggested that these bacteria were involved in the
pathogenesis of pulpitis and apical periodontitis. It is
assumed that the pulp will not be infected as long as it is
vital. Rather, it is bacterial products that initially cause
pulpal inflammation, either through a direct cytotoxic
effect or indirectly by their antigenic properties. The
classic opinion is that not until the pulp injury is so
severe that a localized area of necrosis has developed,
will it be possible for bacteria to enter the pulp cavity
and establish colonies in the necrotic tissue. However,
166
presently it is well known that invasiveness is an
important aspect of the virulence of many oral
pathogens. Thus, during the spread of inflammation in
the pulp, bacteria may well be present both in the part of
the pulp that has become necrotic and in the super-
ficial layers of the subjacent vital tissue. The front of
infection, in other words, will be the transition zone be-
tween the necrotic and vital (inflamed) pulp tissue (71).
The invasive capacities of spirochetes are well
established (72, 57), but their possible role in the
development of pulpal inflammation is not really
known. However, spirochetes are commonly present
in the necrotic pulp of non-vital teeth as evidenced by
both anaerobic culture (27, 32, 73, 74) and scanning
electron microscopic findings (27, 75). Using a novel
method for recovering spirochetes, Dahle et al. (73)
have reported the findings of previously unidentified
oral species. One spirochete from the root canal of a
tooth with apical periodontitis was characterized
morphologically (27). This spirochete was seven times
longer (140 mm) and five times thicker (2 mm) than
previously observed spirochetes (26). The bacterial cell
had a structured surface with small scales similar to
those described for several Treponema species (76).
Protruding processes from the cell gave an impression
of feet-like appendages with an average length of
1.5 mm in several areas. Based on the morphology,
motility, and site of isolation, it was assumed that this
organism belonged to the genus Treponema.
Recently, molecular genetic analyses have confirmed
the presence of oral treponemes in endodontic infec-
tions, and T. denticola, T. socranskii, T. vincentii, T.
maltophilum, T. pectinovorum, T. amylovorum, T.
medium, and T. lecithinolyticum have all been detected
in infected root canals (77–82). With the checkerboard
DNA–DNA hybridization technique, T. denticola and
T. socranskii have been identified in as many as 40% and
60% of root canals from teeth with necrotic pulps (77).
With PCR-based identification, T. denticola was
detected in 78%, T. socranskii in 41%, T. maltophilum
in 39%, and T. lecithinolyticum in 26% of necrotic pulps
(80, 81). The findings of the genetic analyses are of
special interest in the light of the studies on margi-
nal periodontitis where the ‘red cluster’ bacteria
T. denticola, P. gingivalis, and P. forsythus have been
found to have a decisive influence on the progression of
disease in patients with active marginal periodontitis.
These findings, and the fact that a significant relation-
ship has been observed between treponemes and

P. gingivalis also in root canals of teeth with apical
periodontitis (79–82), seem to suggest that trepo-
nemes are involved in the development of disease also
in the pulp.
Extraradicular infection
As in the root canal, the infection of the periapical
lesion is characterized by a wide variety of combinations
of bacteria (83–85). In situ hybridization studies of
periapical granulomas, using a universal bacterial
probe, have demonstrated several spirochete-like or-
ganisms of different sizes (84, 86, 87). The spirochetes
were seen in biofilm-like settings, coaggregating and
forming microcolonies with bacteria of different
morphologies, and also as single cells or in groups,
free in the granulation tissue. In scanning electron
microscopic studies, spirochetes are seen interspersed
with other bacteria in bacterial aggregates that have the
form of granules of up to 3–4 mm in diameter (‘sulfur
granules’) (86).
Using the checkerboard DNA–DNA hybridization
technique, T. denticola and T. socranskii were identified
in about 60% of periapical endodontic lesions (83, 87).
With fluorescence in situ hybridization, T. vincentii was
detected directly inside the periapical granuloma (84).
T. vincentii and T. vincentii-related organisms, which
are Group 1 treponemes, have been associated with
periodontal diseases (25, 42, 48, 88). Since T. vincentii
has been recovered by molecular methods both from
the root canal (79), the periapical lesion (84), and from
pus of endodontic abscesses (80), this group of
organisms may play a role in endodontic infections as
well. In addition, a number of spirochete-like organ-
isms observed in the periapical tissue by means of the in
situ hybridization technique could not be identified by
means of treponeme-specific probes, suggesting a
considerable genetic diversity in this group of organ-
isms (25, 42) also in endodontic infections.
Concluding remarks
Deciphering the roles of spirochetes in endodontic
infections is at its early stages (89). Many researchers
have long believed that motility and chemotaxis are
major virulence factors of such organisms. This belief is
supported by the fact that 5–6% of the genomes of T.
pallidum and B. burgdorferi are putative chemotaxis
and motility genes (10). In addition, several spirochete
Treponemes and endodontics
species, including those found in infected root canals,
are known to be highly invasive to host tissues (57, 58).
Spirochetes isolated from infected root canals of teeth
and from periodontal pockets of patients with marginal
periodontitis have been shown to carry several putative
virulence factors identical or similar to invasive organ-
isms such as T. pallidum (38, 62). Also, when genes in
these putative endodontic pathogens are mutated, the
bacteria lose their ability to penetrate tissue (57, 58).
Chemotaxis and motility may be essential for the
spirochetes to migrate from infected sites into adjacent
tissues and thus contribute to the progression of the
infection.
Our understanding of treponemes and other micro-
organisms, pathogenic or otherwise, certainly will be
altered by the increasing availability of whole genome
sequences. Virulence studies using targeted mutants
are currently being performed in oral spirochetes, and
now that genetic tools have been and are being
developed, such studies are expected to provide
important new insights into their biology. It will be
exciting in the near future to relate specific gene
functions, including the genes involved with chemo-
taxis and motility, to virulence and possibly to
pathogenesis of endodontic infections.
Acknowledgement
We thank Dr Ellen Namork for taking the electron micro-
graph of the negatively stained ‘T. vincentii’.
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