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Endodontic Topics 2012, 22, 3349 2012 John Wiley & Sons A/S
All rights reserved ENDODONTIC TOPICS 2012
1601-1538

Biofilms in endodontic infection

JOS F. SIQUEIRA JR, ISABELA N. RAS & DOMENICO RICUCCI

Bacterial biofilms are very prevalent in the apical root canals of teeth with primary and post-treatment apical
periodontitis. These bacterial endodontic communities are often found adhered to or at least associated with the
dentinal canal walls, with bacterial cells encased in an extracellular amorphous matrix and often facing host
inflammatory cells. This pattern of bacterial community arrangement in the root canal system is consistent with
the acceptable criteria for including apical periodontitis in the set of biofilm-mediated diseases. Whereas
intraradicular biofilms are common in teeth with apical periodontitis lesions, extraradicular biofilms are found
much less frequently and usually in association with symptoms. Morphological studies reveal that the structure of
endodontic biofilms can vary from case to case and a unique pattern has not been established. Bacterial biofilms
are expected to be even more prevalent in the root canals of teeth associated with long-standing pathologic
processes, including large apical radiolucencies and cysts. The very high frequency of biofilms in the root canals
of treated teeth with post-treatment disease may be interpreted as indirect evidence that, depending on location
and possibly species composition, biofilms can be a challenge for proper root canal disinfection.

Received 19 May 2011; accepted 13 November 2011.

Introduction tive in reaching and eliminating as many bacterial cells

The infectious etiology of apical periodontitis has been
well established over the last four decades and a myriad
of studies, first using advanced anaerobic culture tech-
niques and then sophisticated molecular microbiology
methods, has contributed to define the microbial
species associated with the disease (1). These studies
have demonstrated that endodontic infections are
invariably caused by a mixed community whose diver-
sity may vary according to the type of infection and
clinical manifestation of the disease (2). Some infected
root canals, especially those associated with large apical
periodontitis lesions, have been shown to harbor bac-
terial population densities in the same order of mag-
nitude as human populations inhabiting large
countries (37). Thus, bacterial diversity and density
in root canals have been a matter of continuous study
as this knowledge may have an impact on the under-
standing of the disease pathogenesis and response to
treatment. Also in this regard, knowledge concerning
the patterns of microbial colonization in the infected
site is of paramount importance, not only for a better
understanding of the disease process, but also for the
establishment of therapeutic strategies that are effec-
as possible from the root canal system.
Morphological studies of the endodontic micro-
biota in untreated (primary infection) and treated
(persistent/secondary infection) teeth have demon-
strated that the dominant pattern of bacterial coloni-
zation of the root canal system resembles that of
typical biofilms (812). These structures represent one
of the most successful strategies for microorganisms
to adapt to their environment and to resist environ-
mental stresses and threats. The recognition of bio-
films as the main form in which bacteria are found in
endodontic infections permits apical periodontitis to
be included in the set of biofilm-induced oral diseases,
along with caries and marginal periodontitis. In some
aspects, mixed (multi-species) biofilm communities
can act as a multi-cellular organism, with a resultant
collective pathogenic effect on the host. This
community-as-pathogen concept may well be applied
to the etiology of apical periodontitis (13).
This review paper focuses on the concept of apical
periodontitis as an infectious disease caused by bacte-
rial communities primarily organized in intraradicular
biofilms. Several aspects of the biofilm lifestyle, devel-
oping dynamics, collective pathogenicity, prevalence,

33
Siqueira et al.
and morphology are discussed as they relate to apical
periodontitis.
The biofilm lifestyle: an overview
It is now widely recognized that the vast majority
of microorganisms in nature invariably grow and
behave as members of metabolically integrated
communitiesbiofilms (1415). The study of micro-
bial biofilms assumes a great importance in different
sectors of industrial, environmental, and medical
microbiology. For instance, microbial biofilms are
involved in the corrosion of pipes and the degradation
of submerged structural components in off-shore oil
platforms and boats, and can threaten the safety of
drinking water by growing in water distribution pipes
(16). The importance in medical microbiology arises
from the fact that estimates indicate that biofilm infec-
tions are responsible for 6580% of human infections
in the developed world (17). In the oral cavity, caries,
gingivitis, and marginal periodontitis are typical
examples of diseases caused by bacterial biofilms in the
form of supragingival or subgingival dental plaque
(18). Recent evidence tends to include apical
periodontitis in this set of biofilm-induced oral
diseases (9).
Biofilm can be defined as a sessile multi-cellular
microbial community characterized by cells that are
firmly attached to a surface and enmeshed in a self-
produced matrix of extracellular polymeric substances
(EPS) (14,19) (Figure 1). In bacterial biofilms, indi-
vidual cells grow and aggregate to form micro-
colonies (populations) that are embedded and non-
randomly distributed in the EPS matrix and separated
by water channels (1922). In most biofilms, while the
bacterial populations account for less than 10% of the
dry mass, the EPS matrix can account for over 90%
(23). Dental biofilms can exhibit up to 300 or more
cell layers of thickness (22).
The EPS matrix confers unique characteristics to the
biofilm community and is essential to biofilm physiol-
ogy, output, and existence. EPS are hydrated biopoly-
mers (usually polysaccharides, but also proteins,
nucleic acids, and lipids) secreted by biofilm cells (14).
The EPS matrix serves the following important func-
tions in the microbial community (23):
(i) it mediates biofilm adhesion to surfaces, very
often acting as a biological glue;
(ii) it provides mechanical stability to the biofilm;
34
Fig. 1. Endodontic bacterial biofilm. Section taken at
the apical third of the mesial root of a mandibular molar
with necrotic pulp and apical periodontitis lesion. This
is typically a sessile multi-cellular bacterial community
characterized by cells attached to the dentin surface and
enmeshed in a self-produced matrix of extracellular poly-
meric substances (Taylors modified Brown & Brenn,
original magnification 1,000 ). Reproduced with per-
mission from Ricucci D. Patologia e Clinica Endodontica.
Bologna, Italy: Edizioni Martina, 2009.
(iii) it allows for extracellular enzymes to accumulate
and exert important activities, which include
nutrient acquisition and co-operative degrada-
tion of complex macromolecules;
(iv) it keeps biofilm cells in close proximity, thus
allowing for interactions including quorum
sensing, genetic exchanges, and pathogenic
synergism;
(v) in periods of nutrient deprivation, it can serve as
a nutrient source, although some components
of the matrix may be only slowly or partially
degradable;
(vi) it retains water and maintains a highly hydrated
microenvironment surrounding the biofilm
populations;
(vii) it plays a protective role against host defense
cells and molecules as well as antimicrobial
agents.
The biofilm community represents a complex bio-
logical system that is structurally and dynamically or-
ganized. Populations of cells are strategically positioned
for optimal metabolic interaction and the resultant
architecture favors the physiology and ecological role of
the community. The properties displayed by a multi-
species biofilm community are mostly dictated by the
interactions between populations, which create novel

physiological functions that cannot be observed with
individual components. Consequently, in some aspects
the biofilm community behaves in a complex way
that is somewhat similar to multi-cellular organisms
(2425).
It is also noteworthy that bacteria living in biofilms
present a different pattern of gene expression. It has
been demonstrated by proteomic techniques or DNA
arrays that genes expressed by cells organized in bio-
films differed by 2070% from those expressed by the
same cells growing in planktonic cultures (2628).
Consequently, biofilm cells exhibit a different pheno-
type that has been found to be more resistant to
antimicrobial agents, stress, and host defenses when
compared to their counterparts living in a planktonic
state. This contributes to biofilms being a great
problem with respect to treatment susceptibility.
Several aspects of the biofilm, such as encasement
and immobilization of the populations in close prox-
imity, favor co-operation and communication between
the community members. Co-operation is of utmost
importance in making nutrients available and may also
contribute to provide resistance to environmental
stresses. Communication is especially important to
virulence and survival (29). One of the most studied
communication mechanisms is that of quorum sensing
(3032). By this mechanism, bacteria are able to esti-
mate the total number of individuals in their local
community and act accordingly. For instance, by regu-
lating the growth of the overall community, nutrients
can be conserved during periods of starvation (32). As
for virulence, quorum sensing can delay the produc-
tion of virulence factors until the population reaches a
sufficient level or quorum to cause damage and resist
the host defenses (31).
To summarize, a number of advantages have been
recognized for bacteria living in biofilm communities.
They are as follows (1820,22,25,3335):
(i) creation of a broader habitat range for the growth
of a more diverse microbiota;
(ii) increased metabolic diversity and efficiency
due to food chains/webs and enzymatic
co-operation to degrade complex nutrients;
(iii) protection from competing microorganisms,
host defenses, antimicrobial agents, and environ-
mental stresses;
(iv) facilitated cellcell communication (quorum
sensing), which can influence survival and
virulence;
Biofilms and apical periodontitis
(v) facilitated genetic exchanges, which may include
genes encoding antibiotic resistance and virulence
factors;
(vi) enhanced pathogenicity, especially due to syner-
gism between different bacterial species in a
mixed community. This results in a net patho-
genic effect (36). In fact, even the ability to
form biofilms has been regarded as a virulence
factor (25).
The community-as-pathogen
concept
Since the pioneering observations of Robert Koch,
studies of the etiology of infectious diseases have tra-
ditionally been conducted under the aegis of the
single-species etiology concept. This is certainly
valid for many classic diseases caused by exogenous
and true pathogens. However, this concept may not be
successfully applicable to most human endogenous
infections, for which no single pathogen but a set of
species usually organized in mixed biofilm communi-
ties has been associated with disease causation. These
diseases have very often been regarded as having a
polymicrobial etiology.
There is a current trend to accept a more holistic
approach regarding the etiology of endogenous infec-
tions in that the microbial community as a whole is
indeed the unit of pathogenicity (13,3738). Accord-
ing to this concept, the whole can very often be
greater than the simple sum of its parts and any com-
ponent cannot be thoroughly understood except in
relation to the whole (38). The concept of the com-
munity as a pathogen is based on the principle that
teamwork is what eventually counts. The behavior
of the community and the outcome of the host
bacterial community interaction will depend upon the
species comprising the community and how the
myriad of associations that can occur within the com-
munity affect and modulate the virulence of the
species. The virulence ability of a given species is alleg-
edly different when it is in pure culture, in pairs, or as
part of a large bacterial society (community) (13,
3842).
Conceivably, the pathogenesis of apical periodontitis
requires the concerted action of bacteria in a multi-
species community. Bacterial virulence factors involved
in the pathogenesis of apical periodontitis consist of
a summation of structural cellular components,
35
Siqueira et al.
antigens, and secreted substances that accumulate in
the biofilm (43). The concentration and virulence of
this bacterial soup will depend upon the population
density, species composition, and bacterial interactions
in the community. Once the biofilm forms in the apical
canal, this soup of antigens and virulence factors
becomes in constant and direct contact with the per-
iradicular tissues to cause damage and stimulate/
modulate the host immune responses (1).
The study of bacterial communities
in apical periodontitis
It has been demonstrated that, whereas associations of
any specific species with any form of apical periodon-
titis are seldom (if ever) observed, the bacterial com-
munity profiles seem to follow patterns related to the
different clinical presentations of apical periodontitis
(13). Community profiles are essentially determined
by species richness and abundance. Many molecular
microbiology techniques used to profile communities
in different environments have been applied to the
study of the microbial communities associated with
human healthy and diseased sites. Some of these tech-
niques have been used in endodontic microbiology
research and the results of community profiling analy-
ses of the endodontic microbiota can be summarized
below.
(i) The different types of endodontic infections
were confirmed as being composed of multi-
species bacterial communities (4446). This also
applies to persistent/secondary infections associ-
ated with treated teeth (3,4651), which had
been traditionally considered as being composed
of only one or two species based on culture
studies.
(ii) Some underrepresented as-yet-uncultivated bac-
teria can be commonly found in infected root
canals as part of endodontic communities
(5253).
(iii) There is a great individual-to-individual variability
in the diversity of endodontic bacterial commu-
nities associated with the same clinical disease
(4546,51). In other words, each individual
harbors a unique endodontic microbiota in terms
of species richness and abundance. The fact that
the composition of the endodontic microbiota
differs consistently between individuals suffering
from the same disease (4445,47) indicates a
36
heterogeneous etiology for apical periodontitis,
where multiple species combinations can lead to
similar disease outcomes.
(iv) Bacterial communities seem to follow a specific
pattern according to the clinical condition
(asymptomatic apical periodontitis, acute apical
abscesses, and post-treatment apical periodonti-
tis) (45,47,54). Therefore, disease severity (inten-
sity of signs and symptoms) or response to
treatment may be related to the bacterial commu-
nity composition. In other words, from the per-
spective of the single-pathogen concept, apical
periodontitis can be considered as of no specific
microbial etiology. However, based on the
community-as-pathogen concept, it is possible
at this point to infer that some bacterial commu-
nities are more related to certain forms of the
disease than others (45,54).
(v) There seems to be a geography-related pattern in
community profiles. Inter-individual variability is
lower amongst individuals residing in the same
geographical location when compared to that
observed for individuals living in distant countries
(4445,47,55).
(vi) The apical portion of the root canal harbors a
microbiota that is significantly different in com-
position than that occurring in the more coronal
aspects of the root canal (5657). The apical bac-
terial communities are as diverse as those occur-
ring at the middle/coronal thirds. A high
variability is observed for both inter-individual
(samples from the same root canal region but
from different patients) and intra-individual
(samples from different root canal regions of the
same tooth) comparisons (56).
Biofilm and apical periodontitis
The evidence that apical periodontitis is a biofilm-
induced disease comes from in situ investigations
using optical and/or electron microscopy (8,1012,
5859). These studies have allowed observations of
bacteria colonizing the root canal system in primary
or persistent/secondary infections as sessile biofilm-
like structures usually covering the dentinal root canal
walls (Fig. 2). Apical ramifications, lateral canals, and
isthmuses connecting main root canals have all been
shown to harbor bacterial cells frequently organized
in biofilms (6062) (Fig. 3). Moreover, biofilms

Fig. 2. Apical intraradicular biofilm. Mandibular second
premolar subjected to endodontic surgery and extracted
three months later due to recurrence of symptoms and
sinus tract. (a) Section cut on a bucco-lingual plane. The
overview shows that the surgical approach was inappro-
priate, leaving the lingual canal (on the right) unde-
tected and untreated (Taylors modified Brown &
Brenn, original magnification 16 ). (b) Magnification
of the area of the lingual canal demarcated by the rec-
tangle in (a). A dense biofilm is layering both walls
(original magnification 100 ). (c) Higher magnification
of the rectangular area in (b). Note the abundance of
extracellular matrix. Some polymorphonuclear neutro-
phils are attracted to the biofilm surface (original mag-
nification 400 ).
adhered to the apical root surface (extraradicular bio-
films) (Fig. 4) have been reported and regarded as a
possible cause of post-treatment apical periodontitis
(6365).
Although the concept of apical periodontitis as a
biofilm-induced disease has been built upon these
observations, the prevalence of biofilms and their asso-
ciation with diverse presentations of apical periodon-
titis were only very recently investigated. Ricucci &
Siqueira (9) evaluated the prevalence of biofilms in
Biofilms and apical periodontitis
Fig. 3. Biofilm extending to a lateral canal. (a) Man-
dibular second premolar with necrotic pulp. There have
been several pain episodes with severe swelling and
the tooth was clinically deemed as non-restorable. The
radiograph shows a large radiolucency, located on the
mesial aspect of the root. (b) Longitudinal section
passing through the main canal, encompassing a lateral
canal at the transition between the apical and middle
thirds. A bacterial biofilm fills half of the lateral canal
and is faced by a severe concentration of inflammatory
cells (Taylors modified Brown & Brenn, original mag-
nification 50 ). (c) High power view of the area indi-
cated by the arrow in (b). Note the biofilm adhered to
the dentinal walls. The lateral canal content is character-
ized by amorphous necrotic debris heavily colonized by
bacterial flocs (original magnification 400 ).
untreated teeth (primary infections) and treated teeth
(persistent/secondary infections) with apical peri-
odontitis and looked for associations between
endodontic biofilms and clinical/histopathological
conditions. Some of the most important findings of
this study can be summarized as follows:
37
Siqueira et al.

Fig. 4. Extraradicular biofilm. (ab) Untreated mandibular canine with a destructive carious process. After extraction,
calculus can be seen exclusively around the root apex. (c) Section passing approximately at the center of the root canal.
Calculus seems to fill the foramen (Taylors modified Brown & Brenn, original magnification 25 ). (de) Higher
magnifications of the left and right calculus in (c) showing different bacterial concentrations. Inset shows a dense
biofilm with predominantly filamentous forms at the outer layer of calculus (original magnification 100 ; original
magnification of the inset 400 ).

(i) Intraradicular biofilm arrangements were in
general observed in the apical segment of 77%
of the root canals of teeth with apical periodon-
titis (80% in untreated canals and 74% in treated
canals). There were cases in which the biofilm
even formed on the inflamed soft tissue near the
apical foramen (Fig. 5).
(ii) Morphologically, intraradicular bacterial bio-
films were usually thick and composed of
several layers of bacterial cells. Different mor-
photypes were commonly seen per biofilm. The
relative proportions between bacterial cells/
populations and the extracellular matrix were
highly variable. Therefore, endodontic biofilm

38
 Biofilms and apical periodontitis

Fig. 5. Apical level of infection. (a) Radicular fragment of a maxillary second premolar destroyed by a carious process,
showing an apical radiolucency. (b) Section cut approximately at the center of the main foramen. The canal space
appears completely clogged with a dense bacterial biofilm formed onto the canal walls and also on the inflamed soft
tissue near the apical foramen (Taylors modified Brown & Brenn, original magnification 100 ). (c) Detail from the
right wall in (b). The biofilm is composed mainly of filamentous forms (original magnification 400 ). Modified with
permission from Ricucci D. Patologia e Clinica Endodontica. Bologna, Italy: Edizioni Martina, 2009.

morphology differed consistently from indi-
vidual to individual.
(iii) Dentinal tubules underneath biofilms covering
the walls of the main apical canal were very often
invaded by bacteria from the bottom of the
biofilm structure. In addition, biofilms were also
commonly seen covering the walls of apical rami-
fications, lateral canals, and isthmuses. All of
these areas usually represent a challenge for
proper disinfection because of the difficult (or
even impossible) access to instruments and
irrigants.
(iv) Bacterial biofilms were visualized in 62% and 82%
of the root canals of teeth with small and large
apical periodontitis lesions, respectively. All of
the root canals associated with very large lesions
(>10 mm in radiographic diameter) were found
to harbor intraradicular biofilms. Because it takes
time for apical periodontitis to develop and
become radiographically visible, it seems reason-
able to surmise that large lesions represent a
long-standing pathological process caused by an
even older intraradicular infection. In a long-
standing infectious process, the involved bacteria
may have had enough time and appropriate con-
ditions in order to adapt to the environment and
create a mature and organized biofilm commu-
nity. The fact that infected root canals of teeth
with large lesions harbor a large number of cells
and species almost always organized in biofilms
may help to explain the long-standing concept
that the treatment outcome may be influenced
by lesion size (66).
(v) The prevalences of intraradicular biofilms in
teeth associated with apical cysts, abscesses, and
granulomas were 95%, 83%, and 69.5%, respec-
tively. Biofilms were significantly associated
with epithelialized lesions. Because apical cysts
develop as a result of epithelial proliferation in
some granulomas (67), it may be anticipated
that the older the apical periodontitis lesion, the
greater the probability of it becoming a cyst.
Similarly to teeth with large lesions, the age of
the pathologic process may also help to explain
the high prevalence of biofilms in association
with cysts.
(vi) No correlation was found between biofilms and
clinical symptoms or sinus tract presence.
(vii) Extraradicular biofilms were very infrequent,
being observed in only 6% of the cases. Except

39
Siqueira et al.

Fig. 6. Bacteria occurring in the canal lumen as flocs and planktonic cells. Mesio-buccal root apex of an untreated
maxillary molar. (a) Section encompassing the main apical canal and the entrance of a ramification (Taylors modified
Brown & Brenn, original magnification 16 ). (b) Higher magnification of the canal content in the area indicated by
the arrow in (a). Necrotic tissue colonized by sparse bacterial cells and faced with an accumulation of polymorpho-
nuclear neutrophils (original magnification 100 ). (c) Higher magnification of the bacterial front/neutrophils
(original magnification 400 ). (d) Higher magnification of the bacterial flocs and planktonic cells in the context of
necrotic tissue (original magnification 630 ).

for one case, they were always associated with
intraradicular biofilms. All of the cases showing
an extraradicular biofilm exhibited clinical symp-
toms. Thus, it seems that extraradicular infec-
tions in the form of biofilms or planktonic
bacteria are not a common occurrence, are
usually dependent on intraradicular infection,
and are more frequent in symptomatic teeth.
(viii) Bacteria were also seen in the lumen of the
main canal, ramifications, and isthmuses as flocs
and planktonic cells, either intermixed with
necrotic pulp tissue or possibly suspended in a
fluid phase (Fig. 6). Bacterial flocs in clinical
specimens may originate from the growth of
cell aggregates/co-aggregates in a fluid or they
may have detached from biofilms (68). Flocs
may exhibit many of the same characteristics as
biofilms (25), and are sometimes regarded as
planktonic biofilms. Along with planktonic
bacterial cells, flocs may play a role in the
pathogenesis of acute clinical forms of apical
periodontitis (13).

40

Apical periodontitis as a
biofilm-induced disease
The following criteria have been proposed by Parsek &
Singh (69) to establish a causal link between biofilms
and a given infectious disease:
(i) The infecting bacteria are adhered to or associ-
ated with a surface (by associated with they
meant that bacterial aggregates/co-aggregates
do not need to be firmly attached to the
surface).
(ii) Direct examination of infected tissue shows bac-
teria forming clusters or micro-colonies encased
in an extracellular matrix.
(iii) The infection is generally confined to a particular
site and although dissemination may occur, it is a
secondary event.
(iv) The infection is difficult or impossible to eradicate
with antibiotics despite the fact that the respon-
sible microorganisms are susceptible to killing in
the planktonic cell state.
A fifth criterion was further added by Hall-Stoodley
& Stoodley (68):
(v) Ineffective host clearance, which may be evidenced
by the location of bacterial colonies in areas of the
host tissue associated with host inflammatory cells.
Accumulation of polymorphonuclear neutrophils
and macrophages surrounding bacterial
aggregates/co-aggregates in situ considerably
increases the suspicion of biofilm involvement
with disease causation.
Ricucci & Siqueira (9) proposed another criterion:
(vi) Elimination or significant disruption of the
biofilm structure and ecology leads to remission
of the disease process.
These criteria have obvious limitations, but it is
widely assumed that they provide general characteris-
tics by which to consider the role of biofilms in the
pathogenesis of a certain disease (68). The findings
from Ricucci & Siqueiras study (9) showing biofilm
structures in the great majority of cases of primary and
post-treatment apical periodontitis, along with the
observed morphological features of these biofilms,
were considered to fulfill four of the six criteria.
Bacterial agreggates/co-aggregates were observed
adhered to or at least associated with the root canal
dentin surface (criterion 1). Bacterial colonies were
seen in the huge majority of the specimens encased in
an amorphous extracellular matrix (criterion 2). Endo-
Biofilms and apical periodontitis
dontic biofilms were often confined to the root canal
system, in only a few cases extending to the external
root surface, but dissemination through the lesion
never occurred (criterion 3). In the great majority of
cases, biofilms were directly facing inflammatory cells
accumulated in the most apical canal, in ramifications,
and in isthmuses (criterion 5).
Although criterion 4 was not assessed in that study,
it is widely known that intraradicular endodontic infec-
tions cannot be effectively treated by systemic antibi-
otic therapy, even though most endodontic bacteria in
the planktonic cell state are susceptible to currently-
used antibiotics (7072). The lack of efficacy of sys-
temic antibiotics against intraradicular infections is due
to the fact that the drug cannot reach the bacterial
pathogens because they are located in an avascular
necrotic space. The recognition of biofilms as the main
mode of bacterial establishment in the root canal
system further strengthens the explanations for the
lack of antibiotic effectiveness against endodontic
infections. As for criterion 6, the frequent observation
of biofilms in treated canals with post-treatment
disease may at least suggest that there is a potential for
fulfillment of this criterion.
The dynamics of endodontic
biofilm formationa theory
At first, one might think of endodontic biofilms
forming in a way similar to many other biofilms in
nature, i.e. colonization of a solid surface by plank-
tonic bacterial cells floating in a fluid phase that bathes
that surface. In the root canal, for biofilm formation to
follow that dynamics, the pulp would have to become
necrotic and liquefied before bacterial invasion.
However, if one takes into account the dynamics of
root canal invasion by bacteria present in caries lesions
that exposed the pulp tissue, a different dynamics for
biofilm formation can be envisioned, which is sup-
ported by histobacteriological observations.
Caries is a disease caused by biofilms (Fig. 7) (73).
As the caries lesion advances toward the pulp, so does
the biofilm that causes it (Fig. 7). Eventually, when the
last dentin layer is destroyed in advanced caries lesions,
the pulp becomes exposed primarily to the caries
biofilm (and also to planktonic bacteria floating in
saliva) (Fig. 8). As a result of exposure, the pulp
portion beneath the carious lesion becomes severely
inflamed, necrotic, and eventually the frontline of
41
Siqueira et al.

Fig. 7. Caries biofilm. (a) Maxillary first molar with severe caries destruction in an 11-year-old girl. The pulp
responded normally to sensitivity tests. The patient and her parents did not accept any treatment aimed at conser-
vation of the tooth, and requested extraction. (b) Overview of the heavily infected carious dentin and the subjacent
pulp tissue. Note the amount of irritation dentin formed under the carious attack, which is penetrated by bacteria in
the mesial pulp horn (on the right) (Taylors modified Brown & Brenn, original magnification 16 ). (c) Detail from
the carious cavity surface with several infected dentin spicules (original magnification 100 ). (d) Higher magnification
of the dentin spicule indicated by the arrow in (c) showing a dense biofilm is adhered all over to its circumference. On
one side this appears to be composed mainly of filamentous forms. Dentinal tubules and their branches are completely
filled by bacteria (original magnification 400 ). (e) High power view of the interface between dentin surface and
bacterial biofilm (original magnification 1,000 ). Modified with permission from Ricucci D. Patologia e Clinica
Endodontica. Bologna, Italy: Edizioni Martina, 2009.

infection advances to involve first the tissue in the pulp
chamber and then moves inward in the pulp in an
apical direction. The biofilm is what is present at the
frontline of the infection (Fig. 9). These events of
bacterial aggression, inflammation, necrosis, and infec-
tion occur by compartments of pulp tissue and gradu-
ally move in an apical direction. Ultimately, the apical
canal will become necrotic and infected. Therefore, it
is reasonable to assume that the process of biofilm
formation occurs gradually in the canal as the infec-
tious process migrates in an apical direction.
In the advanced front of a root canal infection, the
biofilm enters into contact with the host tissue, which
is inflamed (Fig. 9). This inflamed area is expected to
contain large amounts of inflammatory exudate,
which is rich in proteins and glycoproteins that serve
as an optimal source of nutrients for the advancing
bacterial community. In histobacteriological sections,
biofilms can be observed not only adhered to the
canal walls, but also covering the surface of the
inflamed tissue in the forefront of the infection
(Fig. 9) (9).

42
 Biofilms and apical periodontitis

Fig. 8. Pulp exposed to caries biofilm. (a) Mandibular first molar with advanced periodontal disease and a deep distal
radicular caries approaching the pulp. The patient requested extraction because of severe spontaneous pain. (b) Section
passing in the area where the carious lesion exposes the pulp tissue (Taylors modified Brown & Brenn, original
magnification 25 ). (c) Detail from (b) (original magnification 100 ). (d) Detail from the perforation in the dentin
wall from (c). A biofilm can be seen on the dentin surfaces (original magnification 400 ). (e) Magnification of the
bacterial colony in the pulp tissue in (c). This is surrounded by a concentration of neutrophils (original magnification
400 ). Reproduced modified with permission from Ricucci D. Patologia e Clinica Endodontica. Bologna, Italy:
Edizioni Martina, 2009.

43
Siqueira et al.

Fig. 9. Biofilms at the frontline of infection. (a) Mandibular molar with a severe carious process. The pulp was
clinically necrotic and radiolucencies were present on both mesial and distal roots (Taylors modified Brown & Brenn,
original magnification 2 ). (b) Detail of the distal root canal at the middle third. Transition from necrotic/infected
to vital/non-infected pulp tissue can be observed (original magnification 16 ). (c) Magnification of the area indicated
by the rectangle in (b). The tissue is severely inflamed, but no bacterial colonization can be seen (original magnification
100 ). (d) Detail from the coronal root canal in (b). Dense bacterial biofilms can be seen at this level, surrounded by
a severe concentration of polymorphonuclear neutrophils (original magnification 100 ). (ef) High power views of
the areas indicated respectively by the rectangle and the arrow in (d) (original magnification 400 ).

In addition to microscopic observations, microbio-
logical studies of the composition of the microbiota in
dentinal caries and primary endodontic infections
seem to indirectly support the theory that biofilms
form on the canal walls as the infection moves apically.
Several bacterial species/phylotypes found in biofilms
from deep dentinal caries lesions are also present in
primary intraradicular infections (7478). This sug-
gests that these species participate in the processes of
pulp inflammation and necrosis and are the pioneer
colonizers of the necrotic root canal. As the process
advances apically, it is highly possible that these
advancing bacteria receive reinforcements of new-
comers from saliva. After the infection reaches the

44

apical part of the canal, the possibility obviously exists
that latecomers now present in the fluid phase in the
necrotic canal may gain entry into the biofilm com-
munity, provided they are competitively competent
and bring ecological advantages to the overall commu-
nity physiology. With the passage of time, the biofilm
structure becomes more and more organized and
reaches a state of community homeostasis and
balancea climax community. Once this state is estab-
lished, the composition of the biofilm community is
expected to remain stable over time, unless it is mark-
edly challenged by environmental changes.
Microbial diversity in
endodontic biofilms
Culture and culture-independent molecular microbi-
ology studies have identified more than 1,000 differ-
ent bacterial species/phylotypes in the oral cavity
(79), but this number may actually be an order of
magnitude higher with the advent of massively paral-
lel DNA pyrosequencing techniques (80). Specifically,
the diversity of the endodontic microbiota has also
been unravelled by numerous culture and molecular
studies. Collectively, more than 400 different micro-
bial species/phylotypes have been identified in endo-
dontic samples from teeth with different forms of
apical periodontitis (2). These taxa are usually found
in combinations involving many species/phylotypes
in primary infections and fewer ones in secondary/
persistent infections (81).
At high phylogenetic levels, endodontic bacteria fall
into 15 phyla, with the most common representative
species/phylotypes belonging to the phyla Firmicutes,
Bacteroidetes, Actinobacteria, Fusobacteria, Proteo-
bacteria, Spirochaetes, and Synergistetes (4,51,54,
8285). In addition to bacteria, other micro-
organisms can be found in endodontic infections.
Archaea and fungi have been only occasionally found
in intraradicular infections (8689), though the latter
can be more prevalent in treated teeth with post-
treatment disease (90).
Endodontic infections develop in a previously sterile
place which does not contain a normal microbiota.
Therefore, any species found in the root canal has the
potential to be an endodontic pathogen or at least to
play a role in the ecology of the microbial community.
Despite the long list of bacterial taxa so far detected in
endodontic infections, no more than 2030 bacterial
Biofilms and apical periodontitis
species are considered to be major candidate endodon-
tic pathogens based on prevalence studies. However,
as one considers the community as the unit of patho-
genicity, virtually all species/phylotypes present in the
root canal become important, even those community
members found in low numbers that can be dispro-
portionate to their ecological role (13).
Studies of the endodontic microbiota are plagued
with technical limitations. For instance, the great
majority of microbiological studies have examined
samples taken by paper points from the main root
canal. This sampling approach does not allow for dis-
tinctions of the root canal portion that is sampled
(apical, middle, or coronal portion), nor is it able to
reach areas distant from the main canal, including
ramifications and isthmuses, which can also harbor
microbial cells. A couple of studies have recently used
the entire apical root portion sectioned off from
extracted teeth and then cryogenically ground (56
57). This approach permits a more selective analysis of
the apical microbiota and incorporates all of the
difficult-to-reach anatomical areas in the final sample.
Therefore, members of the apical bacterial biofilms are
certainly included in the sample for identification.
Following the recognition that biofilm communities
are very common in the apical root canal and are
involved in disease causation, it seems important to
identify the species composition of these apical bacte-
rial communities, excluding the portion of the micro-
biota that remains planktonic. It is highly likely that
planktonic bacterial cells floating in the main canal
may have been dettached from the biofilms, but they
may also be by-standers, latecomers, or individuals
that did not succeed in joining the community due to
a lack of competitiveness. Approaches involving
microscopy in association with either in situ hybrid-
ization or open-ended molecular microbiology tech-
niques can be of great value in helping to decipher the
specific composition of endodontic biofilms. This will
aid in the understanding of the ecology of these patho-
genic communities. These and other sophisticated
methods for the study of biofilms have yet to be
applied to the study of endodontic biofilms.
Concluding remarks
Bacterial biofilms are very prevalent in the apical root
canals of teeth with primary and post-treatment apical
periodontitis. The pattern of bacterial community
45
Siqueira et al.
arrangement in the canal fulfills acceptable criteria to
include apical periodontitis in the set of diseases caused
by biofilms.
Future research should address several issues that
arise from these observations. The ultrastructure of
endodontic biofilms should be studied so as to provide
a better understanding of its physiology, ecology,
pathogenicity, and response to treatment. Unravelling
the specific composition of endodontic biofilms will
require the integration of sophisticated microscopic
and molecular microbiology approaches. This knowl-
edge can be of utmost importance not only in pro-
moting a refined understanding of endodontic
biofilms, but also in helping to develop better strate-
gies for treatment. Moreover, community profiling
techniques applied directly to endodontic biofilm
samples may provide information as to the occurrence
of specific communities related to disease. For
instance, it may help identify communities that are
more related to symptoms or that are more resistant to
treatment.
As many studies have been used to answer who is
there, the time has now come to determine what are
they doing there. The physiology and pathogenicity
of biofilms can be inferred by methods that provide a
genome-wide analysis of DNA obtained directly from
the environment (metagenomics), and a profile of
RNA (transcriptomics), proteins (proteomics), and
metabolic end-products (metabolomics) expressed or
released by the community. These methods also have
the potential to disclose patterns of molecules associ-
ated with clinical conditions, which may serve a role as
outcome predictors.
There is also an urgent need to evaluate the effects of
treatment on real endodontic biofilms, using in vivo or
ex vivo (recently extracted teeth with apical periodon-
titis) models. Moreover, it is very important to inves-
tigate the resilience, recovering ability, and fate of
biofilm communities that are only partially affected or
disrupted by treatment. Another potential focus of
study is the susceptibility of the biofilm matrix to the
effects of treatment and its fate if left behind, so as to
shed light on the issue of the remaining biofilm
carcass in some way negatively influencing per-
iradicular tissue healing (10).
As the technologies for the study of biofilms in
nature are developed and become more and more
advanced, the potential exists for further details of the
biofilm lifestyle to be revealed. Endodontic scientists
46
should keep abreast of the literature on this topic as it
relates to other areas in order to expand their views on
the concepts and methods applied to the study and
treatment of biofilms.
Acknowledgements
This study was partially supported by grants from Fundao
Carlos Chagas Filho de Amparo Pesquisa do Estado do Rio
de Janeiro (FAPERJ) and Conselho Nacional de Desenvolvi-
mento Cientfico e Tecnolgico (CNPq), Brazilian govern-
mental institutions.
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