Review Article

Redefining the Persistent Infection in Root Canals:

Possible Role of Biofilm Communities
Luis Chvez de Paz, DDS, MS, PhD
Abstract
Current concepts suggest that persisting infections subsequent to endodontic therapy are caused by one or
two bacterial species that are too robust to be eliminated by conventional treatment measures. As a consequence, numerous studies are exploring the characteristics of these most resistant organisms to define
an effective treatment strategy to eradicate them from
root canals. By taking an ecological perspective, the
main objective of this review is to present evidence that
the nature of persisting endodontic infections depends
not on the robustness of the organisms in the infected
site, but on their capability of adapting their physiology
to the new environmental conditions set by the treatment. Changes in the environment, such as an increase
in pH by calcium hydroxide or the effect of antimicrobials, are capable of triggering genetic cascades that
modify the physiological characteristics of bacterial
cells. Surface adherence by bacteria to form biofilms is
a good example of bacterial adaptation and one that is
pertinent to endodontic infections. Increasing information is now available on the existence of polymicrobial
biofilm communities on root canal walls, coupled with
new data showing that the adaptive mechanisms of
bacteria in these biofilms are significantly augmented
for increased survival. This ecological view on the persisting infection problem in endodontics suggests that
the action of individual species in persisting endodontic
infections is secondary when compared to the adaptive
changes of a polymicrobial biofilm community undergoing physiological and genetic changes in response to
changes in the root canal environment. (J Endod 2007;
33:652 662)

Key Words
Bacterial adaptation, Enterococcus faecalis, microbial
ecology, pathogens, physiological changes

From the Department of Oral Biology, Faculty of Odontology, Malm University, Malm, Sweden.
Address requests for reprints to Dr. Luis Eduardo Chvez
de Paz Villanueva, Department of Oral Biology, Faculty of
Odontology, Malm University, SE-205 06 Malm, Sweden.
E-mail address: luis.chavez.de.paz@od.mah.se.
0099-2399/$0 - see front matter
Copyright 2007 by the American Association of
Endodontists.
doi:10.1016/j.joen.2006.11.004

652

Chvez de Paz

traditional concept that explains infectious processes occurring in humans suggests that diseases are produced as the result of the aggressive invasion of harmful
microorganisms, which battle with the human hosts defenses, triggering mechanisms
that release antibodies and immune cells. The impact of such an approach generates a
predisposition to search for those most dangerous microorganisms that can cause/
trigger the most severe damage to the host. In line with this view, infectious processes
of the oral cavity were proposed to be caused by a relatively small number of organisms
from the diverse collection of species found in the human mouth (1). In caries, for
example, the frequent isolation of Streptococcus mutans from carious lesions (2 4)
generated a considerable number of studies to explore the ex vivo features of this
bacterium. Research findings showing the significant acid-tolerant capabilities of S.
mutans defined this organism as the agent responsible for initial enamel and dentine
demineralization. Similarly, in periodontal disease, the frequent recovery of proteolytic
microorganisms from deep periodontal pockets, such as Porphyromonas gingivalis,
increased the attention of periodontists to these bacteria because they were considered
key etiological agents of the disease (5, 6). The main disadvantage with this traditional
view of the infectious process, especially in oral infections, is that the determination of
true cause-and-effect relationships is not always possible. Consequently, the predominance of certain microorganisms at a given site may be the result of the disease itself
rather than that of the initiating agent (7). Recently, the ecological plaque hypothesis
(8 13) has improved on these classic infectious concepts to explain the etiology of
caries and periodontal disease. This hypothesis suggests that the organisms associated
with the disease may also be present at sound sites, but at levels too low to represent a
clinical threat. In other words, disease is produced as the result of changes in the local
environmental conditions that will shift the balance of the resident flora.
Root canal infections have a different nature than that of caries or periodontitis
because they become established in originally sterile compartments of the oral cavity. In
many cases, this led to the concept that the etiology of root canal infections involves only
a single pathogen. For example, the predominance of certain proteolytic black-pigmented anaerobic organisms in cultures from infected root canals associated with acute
symptoms suggested that these organisms are foremost etiological agents in such cases
(14, 15). Recently, the frequent recovery of Enterococcus faecalis in root canals
associated with persistent infections brought about an intense research interest in this
bacterium. E. faecalis has become the ideal organism to test different irrigants, medicaments, and antiseptic solutions used in endodontics ex vivo, with findings that revealed its innate resistance capacity (16 18). This extensive interest in E. faecalis,
perhaps driven by its ability to grow under almost any laboratory condition (19),
resulted in the concept that the organism is the sole etiological agent for chronic
endodontic infections. Consequently, the focus on E. faecalis resulted in much less
information on the existence of other organisms in such infections that may possess
similar tolerating characteristics to E. faecalis and that would shed light on the existence of a polymicrobial persisting community. Thus, it is not surprising that ecological
parameters in root canal infections are not often discussed.
From an ecological perspective, the root canal can be considered a highly controlled environment with a limited number of niches. Although niches are composed by
a variety of environmental factors that limit the growth of one species relative to others
(20), the main limiting factors in root canal niches that influence bacterial colonization
are, for instance, oxygen and nutrient availability (21). After root canal treatment, other

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Figure 1. Different niches and limiting factors in the root canal environment.

limiting factors become involved, such as pH and the short-/long-term

effects of the antibacterial medicaments applied. The limiting factors for
three different niches in root canals are depicted in Fig. 1, from which
it can be reasonably assumed that bacterial survival in such controlled
environments, especially after root canal treatment, is based on the
capacity of organisms to adapt to the existing conditions.
Although traditional views suggest that the organisms surviving
root canal treatment are a selected group of the most robust organisms, the application of ecological parameters indicates that bacterial
survival after root canal treatment will depend not on the robustness of
the organisms, but on how good an adaptor the organism is to the new
limiting factors in their corresponding niches. Furthermore, as in every
natural microenvironment, the adaptive capabilities of individual organisms are exponentially augmented when growing in biofilm communities. As proposed in this review, data now exist (Fig. 2) that provide an
argument for the inclusion of the biofilm concept in the etiology of
persisting endodontic infections. The foundation for this ecological approach to endodontic infections suggests that the most dangerous
pathogen is not an individual species, but a polymicrobial entity that
undergoes physiological and genetic changes triggered by changes in
the root canal environment.

Pathogens and Virulence

The accepted definitions of microbial pathogen and microbial virulence were formulated largely from the study of infections caused by a
single etiological agent, although both represent combinations of highly
complex parameters. As a result, classifications of microbial pathogens
place the primary responsibility for causing a determined disease at all
times on the microorganism [for a review see Casadevall and Pirofski
(22)]. Likewise, the virulence of a pathogen is generally defined as the
degree of pathogenicity or ability of the organism to cause disease
measured by an experimental procedure (23).
The theory of the single pathogen as the etiological agent of a
specific disease is derived from Robert Kochs Postulates, which are
based on the work accomplished by Koch and his coworkers on diseases, such as, tuberculosis, diphtheria, anthrax, and cholera, that were
determined to be caused by specific microbial entities at a time when the
most prominent medical and scientific communities denied their importance [see Kaufmann and Schaible (24)]. By current standards,
however, there are very few microorganisms to which the term pathogen can be applied invariably (25). A common example is group A
streptococci, which are etiological agents of acute rheumatic fever,

JOE Volume 33, Number 6, June 2007

rheumatic heart disease, post-streptococcal glomerulonephritis, and

invasive infections causing at least 517,000 deaths each year (26). Not
included in such a traditional pathogenic categorization are the various microorganisms that we encounter on a daily basis coexisting
peacefully with humans and from which a small number of them may be
capable of a pathogenic phase. For example, Escherichia coli is ubiquitous, asymptomatically colonizing the human intestines and is widely
distributed in the environment, yet, after experiencing specific genetic
variations, this organism can cause epidemic dysentery and neonatal
meningitis, among other diseases (27). Another example is Listeria
monocytogenes, which is well adapted as a saprophyte for peaceful
survival in soil and decaying vegetation, but it has also another phase
where it acts as an intracellular invader capable of causing serious
infections in humans (28 30). Current research suggests that the ability of these opportunistic organisms to switch from the harmless to the
pathogenic state appears to occur in response to environmental
changes that are mediated through complex regulatory pathways, which
reversibly modulate the expression of virulence factors. The advent of
bacterial genomic studies has significantly increased our understanding
of the pathogenic state of many microbes with the on/off virulence
switch, in fact, constituting a valuable marker of individual microbial
pathogens (3133).

Oral Pathogens
Historically, much of the earlier research into dental caries and the
various periodontal diseases was focused on correlating a single specific organism with the disease to satisfy Kochs Postulates. We now
know that not all diseases are the result of the action of a single organism and this is particularly true of the oral cavity where all of the microbial diseases associated with tissue destruction involve more than
one type of organism and are, therefore, mixed infections (34). This
polymicrobial nature of oral disease has its basis in the characterization
of the organisms present in dental plaque and their potential roles in
dental caries, gingivitis, and periodontitis (3537). Early data (38)
recognized the association of mutans streptococci, including Streptococcus mutans and S. sobrinus, with the initial phase of human dental
caries because their acidogenic and aciduric properties permitted them
to create a low-pH environment in dental plaque after the ingestion of
sugars. In addition, lactobacilli and certain acid tolerant non-mutans
streptococci can now be considered virulent with respect to dental
caries (39, 40). In periodontal disease, the use of animal models suggested that Actinomyces naeslundii was involved in the destructive
alveolar bone loss characteristic of advanced periodontitis (41),
whereas evidence was also presented in the 1970s that black-pigmented
organisms, such as Porphyromonas gingivalis, were directly involved
in periodontitis (36).
Marsh (7) proposed the ecological plaque hypothesis to explain
changes in the ecology of dental plaque that lead to the development of
caries or periodontal disease. This hypothesis constitutes a dynamic
model in which plaque-mediated diseases are the consequence of imbalances in the resident microflora resulting from an enrichment within
the microbial community of the above mentioned oral pathogens
(9 11, 13). In caries, for example, potentially cariogenic bacteria may
be found naturally in dental plaque, but at neutral pH and with a conventional low-sugar diet, the levels of such potentially cariogenic bacteria are clinically insignificant. If the intake of fermentable carbohydrates increases, the low pH provoked in plaque favors the proliferation
of acidogenic and aciduric bacteria, such as mutans streptococci and
lactobacilli, which promote enamel demineralization.
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Figure 2. Preparation of a tooth apex associated with chronic infection as observed with scanning electron microscope. Overview in (a) at 40 magnification shows
the main root canal obtured with gutta percha (arrow). Magnifications of 150 in (b) and 300 in (c) show a gap between the gutta-percha and the root canal wall,
a section is marked in (c) for higher magnification. Images (d), (e), and (f) show the marked section at magnifications of 2,500, 7,000, and 10,000,
respectively. Accumulations of bacterial cells adhered in the gap between the gutta percha and the root canal wall are observed. The specimen was provided by Dr.
Olle Henningsson, Department of Endodontics, Malm University, and the images were obtained with a Hitachi electron microscope.

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Planktonic cells
pH 7

Biofilms
pH 10.5

E. faecalis

pH 10.5

Viable cells: 81-93%

Viable cells: 90-98%

Viable cells: 98-100%

Viable cells: 90-98%

Viable cells: 70-98%

Viable cells: 98-100%

Viable cells: 0-3%

Viable cells: 84-95%

S. anginosus

L. paracasei

Viable cells: 98-100%

Figure 3. Fluorescence micrographs using Live/Dead fluorescence staining for

bacterial viability. Cells stained fluorescent green represent viable cells,
whereas cells stained fluorescent red are nonviable or damaged. In the first
column, images show planktonic cells of three root canal strains at neutral
media (pH 7). The middle column shows planktonic cells after exposure to pH
10.5 for 4 hours, and the right column shows biofilm cells exposed to alkaline
challenge (pH 10.5) for 4 hours. Bars, 2 m. Images are published with
permission of Blackwell Publishing. International Endodontic Journal, Chvez
de Paz et al. (65).

Endodontic Pathogens
Currently, there is no substantial evidence indicating that certain
microorganisms of the microbial flora in root canal infections are more
virulent than others. With this in mind, Sundqvist and Figdor (42) stated
that a proper definition for endodontic pathogens should include every
organism capable of inducing the tissue destruction in apical periodontitis. In reality, however, the majority of endodontic-microbiology studies refer to the endodontic pathogen as the bacterium isolated from a
symptom-associated root canal that grows in the laboratory in a specific
media. By this approach, the most frequently recovered species will
assume the role of major endodontic pathogen. In persistent root canal
infections, for example, the frequent occurrence of monocultures of E.
faecalis has raised suspicion that this bacterium may be the sole organism persisting in the root canals. Considering that mono-infections
rarely if ever occur in nature, it is possible that the apparent pure
cultures of E. faecalis could be the result of sampling and culturing
techniques that favor it over other organisms at the site that were either
in low numbers or were physiologically inactive or dormant (see later).
For instance, in a commonly cited study (43), from the total 100 rootfilled teeth with apical periodontitis sampled E. faecalis was reported as
the most frequently recovered organism (32%), although in 32% of the
cases with persistent lesion no microbe could be isolated. In yet nine
root-filled teeth without periapical lesion that showed bacterial growth,
the organism was found in one case. In a similar study, 25 root-filled
teeth requiring retreatment were sampled and E. faecalis was found in
14 of those 20 teeth with bacterial growth (44). However, it would seem
that this study was focused primarily in proving the occurrence of E.
faecalis in root-filled teeth rather than in exploring the microbial flora
in persisting infections. Similarly, in a recent study using a sophisticated
nested PCR technique, the target bacterium E. faecalis was found in 41
of 50 (82%) untreated root canals and in 38 of 50 (76%) treatmentfailureassociated root canals (45). As in other related works (46
49), PCR methodology seems to be exclusively directed to find only E.
JOE Volume 33, Number 6, June 2007

faecalis, ignoring the rest of the flora present that may be as important
as E. faecalis in provoking the treatment failures.
On the other hand, recent investigations have confirmed the
polymicrobial nature of root canal infections (50, 51). In a study with
monkeys (50), different combinations of bacteria were experimentally
inoculated in root canals and periapical lesions were induced. The teeth
were treated endodontically and followed-up radiographically and histologically for 2 to 2.5 years. In the root canals with bacteria present
when the root filling was removed, 30 of the 31 canals had persisting
periapical lesions. Importantly, more of these nonhealed lesions were
associated with various combinations of bacterial strains, that is, mixed
infections, than single strains. Previously, the same research group (52)
also found that when an eight-strain collection of species, derived
from one infected root canal, was re-inoculated in equal proportions
into other monkey teeth, species such as Bacteroides oralis (now Prevotella oralis) dominated in mixed infections and showed a more potent capacity for tissue destruction. Furthermore, B. oralis could not be
re-isolated from inoculated root canals after the experimental period
when inoculated as a pure culture. In another study using the tissue cage
model implanted subcutaneously in the backs of rabbits, the same collection of eight bacterial strains from monkey root canals were inoculated in different combinations and individual species. The combination
of B. oralis, Fusobacterium necrophorum, Peptostreptococcus
anaerobius, and Streptococcus milleri was the most predominant and
induced higher titers of circulating antibodies than that obtained with
individual inoculations, such as E. faecalis (53).
Even if we accept the polymicrobial nature of root canal infections,
one of the major problems in understanding endodontic infections is
that we still extrapolate between individual organisms growing in liquid
(planktonic) cultures and the in vivo situation. A significant literature
now exists demonstrating that the physiology of a bacterium in planktonic culture is profoundly different from that of the same organism
growing on a surface in a biofilm [see review by Costerton et al. (54)].
For instance, planktonic bacteria are more sensitive to antimicrobial
agents because of their ease of diffusion within the bulk fluid, whereas
biofilm bacteria are notably resistant to these agents (5559). In this
context, the study of biofilms in root canal infections has included
biofilms formed by mixed cultures of anaerobic bacteria in extracted
teeth (60, 61) or by pure cultures of E. faecalis (62, 63). Biofilms of
five root canal isolates have also been used to test the antimicrobial
efficacy of endodontic irrigants, such as sodium hypochlorite (NaOCl)
(2.25%), 0.2% chlorhexidine, 10% povidone iodine, and 5 ppm colloidal silver, with NaOCl shown to be the most effective agent of this
group (64). In addition, our research group tested the alkaline tolerance of species isolated from chronically infected root canals and found
that E. faecalis and other Gram-positive organisms, such as Lactobacillus paracasei, Olsenella uli, or Streptococcus gordonii, shared
similarly high alkaline-tolerant capabilities when growing in planktonic
conditions. S. anginosus, S. oralis, and F. nucleatum, on the other
hand, were greatly affected by the alkaline stress (see Fig. 3) (65). Of
importance, however, was the observation that this difference in alkaline tolerance was not apparent when the strains were tested in biofilms
because all seven strains showed a similar high tolerance to alkaline pH
(Fig. 3). These findings not only show the capacity of root canal bacteria
other than E. faecalis to adapt to alkaline stress, but also provide further
evidence that bacteria in surface-adhered biofilm consortia are more
resistant to environmental stress than when grown in liquid culture.

Physiological Status of Bacteria

The previous discussion relative to the adaptation and survival of
oral bacteria under environmental stress indicates the importance

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of the physiological state of bacteria with respect to the potential level of
activity in disease processes. However, the exact description of the status
of a microorganism can be complex given the numerous terms used
to describe different physiological states, such as, dead, moribund,
starved, dormant, resting, quiescent, viable-but-not-culturable, injured,
sublethally damaged, inhibited, resuscitable, living, active, and vital
(66). Many of these terms are used conceptually and do not reflect the
actual knowledge of the state of the organism in question (67). That this
gap in information exists is apparent from recent bacterial genomic
sequencing data that indicate how little we still know about bacterial
physiology. This can be seen in studies with Haemophilus influenzae,
which has 736 genes of unknown function in its genome out of a total of
1,743 genes (68).
Viability of bacteria is conventionally defined as the capacity of
cells to perform all cell functions necessary for survival under given
conditions (66). The simplest method we have used to assess bacterial
viability is the plate count method, where the number of viable cells
approximates the number of colony-forming units. Bacteria, therefore,
have been classified into two physiological groups: those that can and
those that cannot readily be grown to detectable levels in vitro (67). In
root canal infections, we have cultivated microbial strains from root
canal samples in the laboratory, using growth media that contained a
specific substrate, and then identified the cultivated bacteria at the physiological, biochemical, and, more recently, at the molecular level. The
metabolic properties of these bacterial isolates were then used to infer
the potential roles of these and related microorganisms in the environment. Under some circumstances, however, such methods may underrepresent the number of viable bacteria for a variety of reasons, such as
cases where slightly damaged organisms are present (69), the laboratory growth media used are deficient for one or more essential nutrients
required for the growth of some bacteria in the sample (70), or viable
cells are present that have lost their ability to form colonies (71). Furthermore, if the bacteria exist in a biofilm they may assume a status of
low-metabolic activity similar to stationary-phase planktonic growth for
the majority of time (72, 73). The bacteria in such low active states may
be undetectable by regular culture techniques.
Currently, a variety of methods have been developed to assess the
physiological status of bacterial cells, including metabolic activity, the
integrity of the cell, or the presence of nucleic acids (Fig. 4). Perhaps
the most useful are fluorescent probes that target different cellular
functions, such as membrane potential, enzyme activity, nucleic acids,
and membrane integrity [see review by Joux and Lebaron (74)]. Based
on the detection of the amount of rRNA in bacterial cells, fluorescence
in situ hybridization (FISH) has been applied to identify and, to an
extent, determine the physiological state of different species in their
natural environments (75). One weakness of FISH is the generation of
false negative results with bacteria possessing low physiological activity
because they exhibit low amounts of rRNA, resulting in low signal intensity (76). In endodontics, FISH has been used to visualize and identify bacteria from periapical lesions of asymptomatic root-filled teeth
(77). Alternative fluorescent probes to test bacterial viability are the
Live/Dead BacLight kit and the tetrazolium salts 2-(4-iodophenyl)-3(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT) and 5-cyano-2,3ditolyl tetrazolium chloride (CTC). The Live/Dead kit tests the integrity
of the cell membrane by applying two nucleic acid stains, SYTO-9 and
propidium iodide (PI), which can simultaneously detect dead/injured
(fluorescent red by stain with PI) and intact cells (fluorescent green by
staining with SYTO-9) (78, 79). This fluorescent probe has been used to
assess the viability of root canal strains ex vivo (65) and to determine the
autoaggregation and coaggregation of bacteria isolated from teeth with
acute endodontic infections (80). Furthermore, the outcome of such a
procedure can be seen in Fig. 5 with a histological preparation of a root
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Figure 4. Different approaches used in the assessment of bacterial viability.

apex specimen obtained after surgical removal of a tooth associated

with persistent radiographic signs and symptoms. The filamentous organisms observed in the histological images (Fig. 5a and b), are also
observed under the fluorescent microscope, where their viability
can be assessed (cells stained fluorescent green in Fig. 5c and d).
The tetrazolium salts INT and CTC are often used as markers of bacterial respiratory activity, as well as viability [for a review see Creach et al.
(81)]. With these relatively simple methods, a good correlation between the number of INT/CTC-positive cells and the CFU count can be
obtained.
In conclusion, the physiological state of bacteria in root canals,
whether they are viable, dormant, injured, or in another state of their life
cycle, is a crucial marker that measures their involvement in periapical
inflammation rather than only the identification of species from DNA
fragments.

Mechanisms of Adaptation
There are a number of different mechanisms used by bacteria that
permit them to adapt to the environment. Biofilm formation (55), physiological modification (82), stress response (83), and the creation of
subpopulations of cells (84) are among some of the adaptive mechanisms used by bacteria along with various mechanisms involving the
exchange of genetic material between bacteria (85). The exploration of
these mechanisms can aid us in understanding the bacterial survival
under the limiting environments, such as that found in the root canal.
One of the most relevant features of adaptation for oral bacteria is the
adhesion to surfaces that leads to the formation of plaque biofilms,
which serves not only to aid in their retention in the oral cavity, but also
results in increased survival (86). Interestingly, this ability to form
complex biofilm communities is not lost when oral organisms colonize
other sites in the human body. For example, species from viridians oral
streptococci, such as Streptococcus oralis and S. gordonii, have been
found to form plaque biofilms on the endocardium and valve leaflets
and are thus considered major etiological agents in endocarditis (87,
88). Similarly, oral microorganisms are able to colonize root canals by
adhering to the dentine walls as shown in microphotographs of a tooth
apex associated with a chronic infection taken with a scanning electron
microscope (Fig. 6). Aggregations of microorganisms can be seen adhering to the inner walls of an accessory canal under high magnification, thus demonstrating the retention of these biofilm communities
even after root canal treatment.

Biofilms
Given that surface-associated microbial communities are the main
form of colonization and retention by oral bacteria in the mouth, it is not
unreasonable to assume that biofilms also form in root canals having the
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a

x 1000

Figure 5. Histological preparation of a section of a tooth associated with chronic infection. Overview in (a) shows the site of the root canal with a condensation of
bacteria, stained with a Taylor-modified Brown and Brenn staining. The high magnification (1,000) in (b) shows a number of filamentous organisms surrounded
by neutrophils. Images (c), (d), and (e) show sections stained with Live/Dead BacLight, where the viability of filamentous bacteria could be noticed. Fluorescent green
cells represent viable cells, whereas fluorescent red cells are injured or dead cells. The histological specimen was provided by Dr. Domenico Ricucci and the images
produced are material of the manuscript in preparation (Chvez de Paz LE, Ricucci D, Svenster G. Viability of bacteria in histological specimens of infected root
apexes).

same properties as the parent communities colonizing the enamel and

cementum surfaces (see Figs. 2 and 6). Biofilms form when planktonic
bacteria in a natural liquid phase are deposited on a surface containing
an organic conditioning polymeric matrix or conditioning film (see
Fig. 7). In this dynamic process many other organisms co-adhere to the
surface and grow with some cells detaching from the biofilm over time
(89 92). Biofilm formation in root canals, as hypothesized by Svenster and Bergenholtz (93), is probably initiated at some time after the
first invasion of the pulp chamber by planktonic oral organisms after
some tissue breakdown. At this point, the inflammatory lesion frontage
that moves successively toward the apex will provide the fluid vehicle for
the invading planktonic organisms so these can multiply and continue
attaching to the root canal walls. Interestingly, bacteria have been observed to detach from inner root canal surfaces and occasionally mass
in the inflammatory lesion per se (94). This observation could explain
how the inflammatory lesion front serves as a fluid source for bacterial
biofilm detachment and colonization of other inaccessible sites in the
root canal. Thus, when biofilms are formed on surfaces located beyond
the reach of mechanical removal and the effects of antimicrobials, hostderived proteins from remaining necrotic tissues and bacterially produced adhesive substances will provide the proper prerequisites for the
survival of microbes.
Biofilms in root canals have been confirmed by examinations of
extracted teeth with periapical lesions. For example, when sections

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were viewed by transmission electron microscopy, dense aggregates of

cocci and rods embedded in an extracellular matrix were observed
along the walls (95), whereas studies using scanning electron microscopy showed microcolonies of cocci, rods, and filaments on root canal
walls (93, 96, 97). Such root canal biofilms can be seen in Figs. 2 and 6.
Introducing this biofilm concept to endodontic microbiology is a
major step forward in our understanding of root canal infections, especially those of the persistent kind, because microorganisms growing
in biofilms are better protected from adverse environmental changes
and other antimicrobial agents (56, 98). Apart from the physical protection provided by the extracellular matrix [see review by Branda et al.
(99)], additional protection is afforded by physiological changes initiated by the bacteria after their adhesion to the surface (55, 100, 101).
These phenotypic changes by the biofilm bacteria usually result in increased resistance to antimicrobial agents, in some cases up to 1,000fold greater than that of the same microorganisms living planktonically
(56, 57). Evidence already exists showing that biofilms of oral bacteria
are more resistant to chlorhexidine, amine fluoride, amoxycillin, doxycycline, and metronidazole than planktonic cells (58, 59). As mentioned previously, our group found that the viability of susceptible root
canal strains to alkaline stress in planktonic cultures was considerably
increased when these strains were exposed to the same alkaline stress in
biofilms (65). The increased resistance of the strains in biofilms compared to planktonic cultures of the same organism thus raises questions

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Figure 6. Preparation of a tooth apex associated with chronic infection as observed with scanning electron microscope (also depicted in Figure 2). Overview in (a)
at 80 magnification shows an accessory root canal (arrow). Magnification of 400 in (b) shows a wall of the accessory canal with evidence of a microbial biofilm,
a section is marked for higher magnification. Images (c) and (d) show the marked section at a magnification of 3,000. Accumulations of bacterial cells adhered
to the root canal wall are observed. The specimen was provided by Dr. Olle Henningsson, Department of Endodontics, Malm University, and the images were obtained
with a Hitachi electron microscope.

I) Protein
adsorption.
Formation of a
conditioning film
on the surface

seconds

II) Adhesion of
planktonic bacteria.
Primary colonizers

minutes

III) Co-adhesion.
Secondary
colonizers

hours

IV) Growth and

detachment of
adherent bacteria.

days - weeks

Surface

Figure 7. Stages of biofilm formation [modified from Svenster and Bergenholtz (93)].

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Figure 8. One-day-old biofilms of Lactobacillus paracasei isolated from an infected root canal associated with persistent symptoms. (a) Shows the biofilm cells
growing in optimal conditions, 37C in the rich media peptone yeast glucose (PYG). Biofilm cells were stained with Live/Dead BacLight. (b) Shows the biofilm
cells of L. paracasei after exposure for 1 day with a commercial sodium fluoride solution (0.2%) and after 1 day of reactivation in optimal conditions, 37C
in PYG. As observed, the cellular morphology of the biofilms cells of L. paracasei after stress was reduced, from an original area of 3 m2 to an area of 1.5
m2. The images are material from the manuscript in preparation (Chvez de Paz LE, Hamilton IR, Svenster G. Dormancy of oral bacteria in biofilms).

as to the validation of studies using exclusively liquid-grown cultures in

laboratory tests.
During the various stages of biofilm development, as well as
throughout the various sections of the biofilm, cells are in different
physiological states. Cells at the base of the film, for example, may be
dead or lysing, whereas those near the surface may be actively growing;
however, even with such physiological diversity, it can be argued that
the majority of time cells in biofilms are in a status equivalent to cells
in the stationary phase of growth (72, 73, 102). From the perspective of
the persisting root canal flora, however, one might imagine that such
stationary-phase cells might maintain a low but sufficient metabolic
activity to provoke periapical inflammation. Thus, from the metabolic
perspective, they will not be dead but would, theoretically, be able to
contribute to the persistence of inflammation.

Morphological Changes
Adaptation of cells to environmental shift in some cases may trigger phenotypic changes in cell morphology. Figure 8 illustrates the
morphologic changes undertaken by a root canal strain of Lactobacillus paracasei grown in biofilms exposed to a commercial rinsing solution of sodium fluoride at a concentration of 0.2%. Column A shows
phase contrast and Live/Dead images of the 1-day-old biofilms cells of L.
paracasei growing in optimal conditions in peptone yeast glucose media (PYG) at 37C. In Column B, images of biofilms of L. paracasei,
exposed to a 0.2% sodium fluoride solution for 1 day and then reactivated in PYG at 37C for 1 day, show changes in the cellular morphology
of L. paracasei. Similar changes were observed with the root canal
strain of Streptococcus anginosus. The explanation for this phenomenon is scant; however, such changes are consistent with stress responses triggering changes while the cells are dividing. For example,
colonies of an antibiotic-resistant strain of Pseudomonas aeruginosa
were found to have different morphologies when exposed to kanamycin
with the agent resulting in the formation of smaller and rougher colonies than wild-type cells. However, when this P. aeruginosa strain was
grown on antibiotic-free media for 5 days, colonies appeared smooth
and large just as their wild-type. The authors suggested that these phenotypic variations observed in the resistant variants were transient (82).
With the advent of tools such as confocal laser scanning microscopy,
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numerous structural proteins have been shown to be involved in bacterial morphological alterations. Some of these proteins have a good
molecular similarity to tubulin and are thus capable of forming scaffolding-like filamentous assemblies in bacteria (103, 104). For instance, the structural proteins, crescentin and MreB, were recently implicated as scaffolds responsible for maintaining shape within
Caulobacter crescentus (105, 106). In any case, it is clear that environmental stress results in phenotypic changes to bacterial cells and
such modifications constitute important markers of bacterial adaptation.

Proteomics
A variety of environmental changes can trigger bacterial adaptation
resulting in the coordinated induction of stress proteins or heatshock proteins. Such proteins have been well characterized for diverse
oral species, such as S. mutans and S. oralis among others, as generated when exposed to both heat and acid stress (83, 107109). Intracellular expression of different proteins was also reported for E.
facealis upon alkaline stress (110, 111). These specific bacterial adaptive pathways also include the expression of cytosolic proteins that are
released outside the cell as shown by the release of glyceraldehyde-3phosphate dehydrogenase (GAPDH) from S. gordonii after a change in
pH from 6.5 to 7.5 (112). Furthermore, in a recent study, we observed
that selected cytosolic proteins were as well induced and released to the
external media in cultures of root canal isolates exposed to sublethal
alkaline stress (65). The proteins Dnak, Hpr, and fructose-1,6-bisphosphate aldolase (FBA) were released extracellularly at high levels by root
canal strains of S. anginosus, S. oralis, and S. gordonii. Interestingly,
these copious protein excretions were more intense with planktonic
cultures than with biofilms, and thus such sublethal effects of a stress
may be altered when an organism assumes growth in a biofilm.
Formation of Subpopulations
Bacterial adaptation also includes the creation of subpopulations
of cells at the population level. This generation of diversity within a
population is achieved either by genetic pathways involving constitutive
or transient mutators and contingency loci, or by modifications of cellular phenotypes (113, 114). Groups of cells have been found to persist
Persistent Infection in Root Canals

659

Review Article
after exposure to lethal doses of antibiotics and new growing populations appear in the culture (115, 116). These persister cells (1) may
represent cells in some protected part of their cell cycle, (2) are capable of rapid adaptation, (3) are in a dormant state, or (4) are unable to
initiate programmed cell death in response to the stimulus (84). Thus,
such persisters cells represent a recalcitrant subpopulation that will not
die and are capable of initiating a new population with normal susceptibility once the antibacterial effect has been dissipated (117119). To
date, these cells have been reported to occur only after the exposure of
a bacterial population to high doses of a single antimicrobial agent,
which triggered the appearance of persister cells exhibiting multiple
drug resistance (120, 121). The frequency of persister occurrence and
the mechanism(s) involved in their appearance is unclear, although
one hypothesis with E. coli suggests that persister cells are regulated by
the expression of chromosomal toxinantitoxin genes (122). In this
case, the operon HipA seems to be responsible for tolerance to ciprofloxacin and mitomycin C in stationary-phase planktonic cells and E.
coli biofilms (122). It was also previously proposed that the expression
of toxins drives bacteria reversibly into the slow growing, multiple drugtolerant phenotypes by shutting down antibiotic targets (84). In the
context of root canal bacteria, the formation of such persisting populations that are capable of surviving imposed endodontic treatment measures, as the increase of alkaline levels resulting from application of
calcium hydroxide (65), would explain how organisms are able to
survive and remain in the environment until the effects of noxious stimuli have dissipated.

Concluding Remarks and Directions for

Future Research
The survival of bacteria in root canals after treatment is based on
the capability of the individual organisms to adapt to the environment
within the consortium. Therefore, the study of the adaptive mechanisms
used by organisms to survive in such a highly controlled environment,
with limiting nutrients plus the effects of the antibacterial medicaments,
is important to our understanding of persisting root canal infections.
The ability of the organisms in such infections to form biofilms can be
seen as the most important adaptive mechanism used by bacteria to
survive the environmental changes resulting from the treatment protocol. Thus, from the realization that all oral microorganisms are capable
of forming a biofilm and that such surface-associated communities exist
in root canals, it is possible to apply the biofilm concept to clinical
treatment; that is, efforts should not be directed to specific individual
organisms, but to a group of well-adapted organisms undoubtedly possessing increased resistance to a variety of antimicrobial agents.
We should obtain much better understanding of the characteristics
and properties of bacterial biofilms in root canals and the degree to
which such microbial communities enhance survival from environmental changes. A glance at how a biofilm consortium of root canal strains
cope with alkaline stress is shown in Chvez de Paz (123). Prospectively, the application of molecular biology will be invaluable to identifying, for instance, biofilm-expressed genes by root canal colonizers,
and permit the identification of genes involved in adaptation signaling.
From this knowledge we should come to identify key adaptive events
occurring in the root canal microflora while organisms are exposed to
root canal treatment measures. This path of research should aid in
finding mechanisms that can block such processes.

Acknowledgments
The work was supported by a research grant from the Knowledge Foundation (KK-stiftelsen, BiofilmsResearch Center for
Biointerfaces). The author expresses gratitude to Dr. Ian R. Hamil660

Chvez de Paz

ton, Professor Emeritus, Department of Oral Biology, University of

Manitoba, Canada, who contributed exceptional guidance and help
with editing throughout this review.

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