Biofilms in endodontic infectionJOSÉ F. SIQUEIRA JR, ISABELA N. RÔÇAS & DOMENICO RICUCCI

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

Received 19 May 2011; accepted 13 November 2011.

Introduction

The infectious etiology of apical periodontitis has beenwell established over the last four decades and a myriadof studies, first using advanced anaerobic culture tech-niques and then sophisticated molecular microbiologymethods, has contributed to define the microbialspecies associated with the disease (1). These studieshave demonstrated that endodontic infections areinvariably caused by a mixed community whose diver-sity may vary according to the type of infection andclinical manifestation of the disease (2). Some infectedroot canals, especially those associated with large apicalperiodontitis lesions, have been shown to harbor bac-terial population densities in the same order of mag-nitude as human populations inhabiting largecountries (3–7). Thus, bacterial diversity and densityin root canals have been a matter of continuous studyas this knowledge may have an impact on the under-standing of the disease pathogenesis and response totreatment. Also in this regard, knowledge concerningthe patterns of microbial colonization in the infectedsite is of paramount importance, not only for a betterunderstanding of the disease process, but also for theestablishment of therapeutic strategies that are effec-

tive in reaching and eliminating as many bacterial cellsas 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 oftypical biofilms (8–12). These structures represent oneof the most successful strategies for microorganismsto 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 inendodontic infections permits apical periodontitis tobe included in the set of biofilm-induced oral diseases,along with caries and marginal periodontitis. In someaspects, mixed (multi-species) biofilm communitiescan act as a multi-cellular organism, with a resultantcollective pathogenic effect on the host. Thiscommunity-as-pathogen concept may well be appliedto the etiology of apical periodontitis (13).

This review paper focuses on the concept of apicalperiodontitis as an infectious disease caused by bacte-rial communities primarily organized in intraradicularbiofilms. Several aspects of the biofilm lifestyle, devel-oping dynamics, collective pathogenicity, prevalence,

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Endodontic Topics 2012, 22, 33–49All rights reserved

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and morphology are discussed as they relate to apicalperiodontitis.

The biofilm lifestyle: an overviewIt is now widely recognized that the vast majorityof microorganisms in nature invariably grow andbehave as members of metabolically integratedcommunities—biofilms (14–15). The study of micro-bial biofilms assumes a great importance in differentsectors of industrial, environmental, and medicalmicrobiology. For instance, microbial biofilms areinvolved in the corrosion of pipes and the degradationof submerged structural components in off-shore oilplatforms and boats, and can threaten the safety ofdrinking water by growing in water distribution pipes(16). The importance in medical microbiology arisesfrom the fact that estimates indicate that biofilm infec-tions are responsible for 65–80% of human infectionsin the developed world (17). In the oral cavity, caries,gingivitis, and marginal periodontitis are typicalexamples of diseases caused by bacterial biofilms in theform of supragingival or subgingival dental plaque(18). Recent evidence tends to include apicalperiodontitis in this set of biofilm-induced oraldiseases (9).

Biofilm can be defined as a sessile multi-cellularmicrobial community characterized by cells that arefirmly 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 separatedby water channels (19–22). In most biofilms, while thebacterial populations account for less than 10% of thedry mass, the EPS matrix can account for over 90%(23). Dental biofilms can exhibit up to 300 or morecell layers of thickness (22).

The EPS matrix confers unique characteristics to thebiofilm 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, veryoften acting as a “biological glue”;

(ii) it provides mechanical stability to the biofilm;

(iii) it allows for extracellular enzymes to accumulateand exert important activities, which includenutrient acquisition and co-operative degrada-tion of complex macromolecules;

(iv) it keeps biofilm cells in close proximity, thusallowing for interactions including quorumsensing, genetic exchanges, and pathogenicsynergism;

(v) in periods of nutrient deprivation, it can serve asa nutrient source, although some componentsof the matrix may be only slowly or partiallydegradable;

(vi) it retains water and maintains a highly hydratedmicroenvironment surrounding the biofilmpopulations;

(vii) it plays a protective role against host defensecells and molecules as well as antimicrobialagents.

The biofilm community represents a complex bio-logical system that is structurally and dynamically or-ganized. Populations of cells are strategically positionedfor optimal metabolic interaction and the resultantarchitecture favors the physiology and ecological role ofthe community. The properties displayed by a multi-species biofilm community are mostly dictated by theinteractions between populations, which create novel

Fig. 1. Endodontic bacterial biofilm. Section taken atthe apical third of the mesial root of a mandibular molarwith necrotic pulp and apical periodontitis lesion. Thisis typically a sessile multi-cellular bacterial communitycharacterized by cells attached to the dentin surface andenmeshed in a self-produced matrix of extracellular poly-meric substances (Taylor’s modified Brown & Brenn,original magnification 1,000 ¥). Reproduced with per-mission from Ricucci D. Patologia e Clinica Endodontica.Bologna, Italy: Edizioni Martina, 2009.

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physiological functions that cannot be observed withindividual components. Consequently, in some aspectsthe biofilm community behaves in a complex waythat is somewhat similar to multi-cellular organisms(24–25).

It is also noteworthy that bacteria living in biofilmspresent a different pattern of gene expression. It hasbeen demonstrated by proteomic techniques or DNAarrays that genes expressed by cells organized in bio-films differed by 20–70% from those expressed by thesame cells growing in planktonic cultures (26–28).Consequently, biofilm cells exhibit a different pheno-type that has been found to be more resistant toantimicrobial agents, stress, and host defenses whencompared to their counterparts living in a planktonicstate. This contributes to biofilms being a greatproblem with respect to treatment susceptibility.

Several aspects of the biofilm, such as encasementand immobilization of the populations in close prox-imity, favor co-operation and communication betweenthe community members. Co-operation is of utmostimportance in making nutrients available and may alsocontribute to provide resistance to environmentalstresses. Communication is especially important tovirulence and survival (29). One of the most studiedcommunication mechanisms is that of quorum sensing(30–32). By this mechanism, bacteria are able to esti-mate the total number of individuals in their localcommunity and act accordingly. For instance, by regu-lating the growth of the overall community, nutrientscan be conserved during periods of starvation (32). Asfor virulence, quorum sensing can delay the produc-tion of virulence factors until the population reaches asufficient level or quorum to cause damage and resistthe host defenses (31).

To summarize, a number of advantages have beenrecognized for bacteria living in biofilm communities.They are as follows (18–20,22,25,33–35):(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 enzymaticco-operation to degrade complex nutrients;

(iii) protection from competing microorganisms,host defenses, antimicrobial agents, and environ-mental stresses;

(iv) facilitated cell–cell communication (quorumsensing), which can influence survival andvirulence;

(v) facilitated genetic exchanges, which may includegenes encoding antibiotic resistance and virulencefactors;

(vi) enhanced pathogenicity, especially due to syner-gism between different bacterial species in amixed community. This results in a net patho-genic effect (36). In fact, even the ability toform biofilms has been regarded as a virulencefactor (25).

The community-as-pathogenconceptSince 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 certainlyvalid for many classic diseases caused by exogenousand true pathogens. However, this concept may not besuccessfully applicable to most human endogenousinfections, for which no single pathogen but a set ofspecies usually organized in mixed biofilm communi-ties has been associated with disease causation. Thesediseases have very often been regarded as having apolymicrobial etiology.

There is a current trend to accept a more holisticapproach regarding the etiology of endogenous infec-tions in that the microbial community as a whole isindeed the unit of pathogenicity (13,37–38). Accord-ing to this concept, the whole can very often begreater than the simple sum of its parts and any com-ponent cannot be thoroughly understood except inrelation 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 behaviorof the community and the outcome of the host–bacterial community interaction will depend upon thespecies comprising the community and how themyriad of associations that can occur within the com-munity affect and modulate the virulence of thespecies. The virulence ability of a given species is alleg-edly different when it is in pure culture, in pairs, or aspart of a large bacterial “society” (community) (13,38–42).

Conceivably, the pathogenesis of apical periodontitisrequires the concerted action of bacteria in a multi-species community. Bacterial virulence factors involvedin the pathogenesis of apical periodontitis consist ofa summation of structural cellular components,

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antigens, and secreted substances that accumulate inthe biofilm (43). The concentration and virulence ofthis bacterial “soup” will depend upon the populationdensity, species composition, and bacterial interactionsin the community. Once the biofilm forms in the apicalcanal, this “soup” of antigens and virulence factorsbecomes 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 communitiesin apical periodontitisIt has been demonstrated that, whereas associations ofany 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 thedifferent clinical presentations of apical periodontitis(13). Community profiles are essentially determinedby species richness and abundance. Many molecularmicrobiology techniques used to profile communitiesin different environments have been applied to thestudy of the microbial communities associated withhuman healthy and diseased sites. Some of these tech-niques have been used in endodontic microbiologyresearch and the results of community profiling analy-ses of the endodontic microbiota can be summarizedbelow.(i) The different types of endodontic infections

were confirmed as being composed of multi-species bacterial communities (44–46). This alsoapplies to persistent/secondary infections associ-ated with treated teeth (3,46–51), which hadbeen traditionally considered as being composedof only one or two species based on culturestudies.

(ii) Some underrepresented as-yet-uncultivated bac-teria can be commonly found in infected rootcanals as part of endodontic communities(52–53).

(iii) There is a great individual-to-individual variabilityin the diversity of endodontic bacterial commu-nities associated with the same clinical disease(45–46,51). In other words, each individualharbors a unique endodontic microbiota in termsof species richness and abundance. The fact thatthe composition of the endodontic microbiotadiffers consistently between individuals sufferingfrom the same disease (44–45,47) indicates a

heterogeneous etiology for apical periodontitis,where multiple species combinations can lead tosimilar disease outcomes.

(iv) Bacterial communities seem to follow a specificpattern according to the clinical condition(asymptomatic apical periodontitis, acute apicalabscesses, and post-treatment apical periodonti-tis) (45,47,54). Therefore, disease severity (inten-sity of signs and symptoms) or response totreatment may be related to the bacterial commu-nity composition. In other words, from the per-spective of the “single-pathogen” concept, apicalperiodontitis can be considered as of no specificmicrobial etiology. However, based on the“community-as-pathogen” concept, it is possibleat this point to infer that some bacterial commu-nities are more related to certain forms of thedisease than others (45,54).

(v) There seems to be a geography-related pattern incommunity profiles. Inter-individual variability islower amongst individuals residing in the samegeographical location when compared to thatobserved for individuals living in distant countries(44–45,47,55).

(vi) The apical portion of the root canal harbors amicrobiota that is significantly different in com-position than that occurring in the more coronalaspects of the root canal (56–57). The apical bac-terial communities are as diverse as those occur-ring at the middle/coronal thirds. A highvariability is observed for both inter-individual(samples from the same root canal region butfrom different patients) and intra-individual(samples from different root canal regions of thesame tooth) comparisons (56).

Biofilm and apical periodontitisThe evidence that apical periodontitis is a biofilm-induced disease comes from in situ investigationsusing optical and/or electron microscopy (8,10–12,58–59). These studies have allowed observations ofbacteria colonizing the root canal system in primaryor persistent/secondary infections as sessile biofilm-like structures usually covering the dentinal root canalwalls (Fig. 2). Apical ramifications, lateral canals, andisthmuses connecting main root canals have all beenshown to harbor bacterial cells frequently organizedin biofilms (60–62) (Fig. 3). Moreover, biofilms

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adhered to the apical root surface (extraradicular bio-films) (Fig. 4) have been reported and regarded as apossible cause of post-treatment apical periodontitis(63–65).

Although the concept of apical periodontitis as abiofilm-induced disease has been built upon theseobservations, 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

untreated teeth (primary infections) and treated teeth(persistent/secondary infections) with apical peri-odontitis and looked for associations betweenendodontic biofilms and clinical/histopathologicalconditions. Some of the most important findings ofthis study can be summarized as follows:

Fig. 2. Apical intraradicular biofilm. Mandibular secondpremolar subjected to endodontic surgery and extractedthree months later due to recurrence of symptoms andsinus tract. (a) Section cut on a bucco-lingual plane. Theoverview shows that the surgical approach was inappro-priate, leaving the lingual canal (on the right) unde-tected and untreated (Taylor’s modified Brown &Brenn, original magnification 16 ¥). (b) Magnificationof 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 magnificationof the rectangular area in (b). Note the abundance ofextracellular matrix. Some polymorphonuclear neutro-phils are attracted to the biofilm surface (original mag-nification 400 ¥).

Fig. 3. Biofilm extending to a lateral canal. (a) Man-dibular second premolar with necrotic pulp. There havebeen several pain episodes with severe swelling andthe tooth was clinically deemed as non-restorable. Theradiograph shows a large radiolucency, located on themesial aspect of the root. (b) Longitudinal sectionpassing through the main canal, encompassing a lateralcanal at the transition between the apical and middlethirds. A bacterial biofilm fills half of the lateral canaland is faced by a severe concentration of inflammatorycells (Taylor’s 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 tothe dentinal walls. The lateral canal content is character-ized by amorphous necrotic debris heavily colonized bybacterial “flocs” (original magnification 400 ¥).

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(i) Intraradicular biofilm arrangements were ingeneral observed in the apical segment of 77%of the root canals of teeth with apical periodon-titis (80% in untreated canals and 74% in treatedcanals). There were cases in which the biofilmeven formed on the inflamed soft tissue near theapical foramen (Fig. 5).

(ii) Morphologically, intraradicular bacterial bio-films were usually thick and composed ofseveral layers of bacterial cells. Different mor-photypes were commonly seen per biofilm. Therelative proportions between bacterial cells/populations and the extracellular matrix werehighly variable. Therefore, endodontic biofilm

Fig. 4. Extraradicular biofilm. (a–b) 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 (Taylor’s modified Brown & Brenn, original magnification 25 ¥). (d–e) Highermagnifications of the left and right calculus in (c) showing different bacterial concentrations. Inset shows a densebiofilm with predominantly filamentous forms at the outer layer of calculus (original magnification 100 ¥; originalmagnification of the inset 400 ¥).

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morphology differed consistently from indi-vidual to individual.

(iii) Dentinal tubules underneath biofilms coveringthe walls of the main apical canal were very ofteninvaded by bacteria from the bottom of thebiofilm structure. In addition, biofilms were alsocommonly seen covering the walls of apical rami-fications, lateral canals, and isthmuses. All ofthese areas usually represent a challenge forproper disinfection because of the difficult (oreven impossible) access to instruments andirrigants.

(iv) Bacterial biofilms were visualized in 62% and 82%of the root canals of teeth with small and largeapical periodontitis lesions, respectively. All ofthe root canals associated with very large lesions(>10 mm in radiographic diameter) were foundto harbor intraradicular biofilms. Because it takestime for apical periodontitis to develop andbecome radiographically visible, it seems reason-able to surmise that large lesions represent along-standing pathological process caused by aneven “older” intraradicular infection. In a long-standing infectious process, the involved bacteriamay have had enough time and appropriate con-

ditions in order to adapt to the environment andcreate a mature and organized biofilm commu-nity. The fact that infected root canals of teethwith large lesions harbor a large number of cellsand species almost always organized in biofilmsmay help to explain the long-standing conceptthat the treatment outcome may be influencedby lesion size (66).

(v) The prevalences of intraradicular biofilms inteeth associated with apical cysts, abscesses, andgranulomas were 95%, 83%, and 69.5%, respec-tively. Biofilms were significantly associatedwith epithelialized lesions. Because apical cystsdevelop as a result of epithelial proliferation insome granulomas (67), it may be anticipatedthat the older the apical periodontitis lesion, thegreater the probability of it becoming a cyst.Similarly to teeth with large lesions, the age ofthe pathologic process may also help to explainthe high prevalence of biofilms in associationwith cysts.

(vi) No correlation was found between biofilms andclinical symptoms or sinus tract presence.

(vii) Extraradicular biofilms were very infrequent,being observed in only 6% of the cases. Except

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 spaceappears completely clogged with a dense bacterial biofilm formed onto the canal walls and also on the inflamed softtissue near the apical foramen (Taylor’s modified Brown & Brenn, original magnification 100 ¥). (c) Detail from theright wall in (b). The biofilm is composed mainly of filamentous forms (original magnification 400 ¥). Modified withpermission from Ricucci D. Patologia e Clinica Endodontica. Bologna, Italy: Edizioni Martina, 2009.

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for one case, they were always associated withintraradicular biofilms. All of the cases showingan extraradicular biofilm exhibited clinical symp-toms. Thus, it seems that extraradicular infec-tions in the form of biofilms or planktonicbacteria are not a common occurrence, areusually dependent on intraradicular infection,and are more frequent in symptomatic teeth.

(viii) Bacteria were also seen in the lumen of themain canal, ramifications, and isthmuses as flocsand planktonic cells, either intermixed with

necrotic pulp tissue or possibly suspended in afluid phase (Fig. 6). Bacterial flocs in clinicalspecimens may originate from the growth ofcell aggregates/co-aggregates in a fluid or theymay have detached from biofilms (68). Flocsmay exhibit many of the same characteristics asbiofilms (25), and are sometimes regarded as“planktonic biofilms.” Along with planktonicbacterial cells, flocs may play a role in thepathogenesis of acute clinical forms of apicalperiodontitis (13).

Fig. 6. Bacteria occurring in the canal lumen as flocs and planktonic cells. Mesio-buccal root apex of an untreatedmaxillary molar. (a) Section encompassing the main apical canal and the entrance of a ramification (Taylor’s modifiedBrown & Brenn, original magnification 16 ¥). (b) Higher magnification of the canal content in the area indicated bythe 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 ofnecrotic tissue (original magnification 630 ¥).

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Apical periodontitis as abiofilm-induced disease

The following criteria have been proposed by Parsek &Singh (69) to establish a causal link between biofilmsand a given infectious disease:(i) The infecting bacteria are adhered to or associ-

ated with a surface (by “associated with” theymeant that bacterial aggregates/co-aggregatesdo not need to be firmly attached to thesurface).

(ii) Direct examination of infected tissue shows bac-teria forming clusters or micro-colonies encasedin an extracellular matrix.

(iii) The infection is generally confined to a particularsite and although dissemination may occur, it is asecondary event.

(iv) The infection is difficult or impossible to eradicatewith antibiotics despite the fact that the respon-sible microorganisms are susceptible to killing inthe 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 thehost tissue associated with host inflammatory cells.Accumulation of polymorphonuclear neutrophilsand macrophages surrounding bacterialaggregates/co-aggregates in situ considerablyincreases the suspicion of biofilm involvementwith disease causation.

Ricucci & Siqueira (9) proposed another criterion:(vi) Elimination or significant disruption of the

biofilm structure and ecology leads to remissionof the disease process.

These criteria have obvious limitations, but it iswidely assumed that they provide general characteris-tics by which to consider the role of biofilms in thepathogenesis of a certain disease (68). The findingsfrom Ricucci & Siqueira’s study (9) showing biofilmstructures in the great majority of cases of primary andpost-treatment apical periodontitis, along with theobserved morphological features of these biofilms,were considered to fulfill four of the six criteria.Bacterial agreggates/co-aggregates were observedadhered to or at least associated with the root canaldentin surface (criterion 1). Bacterial colonies wereseen in the huge majority of the specimens encased inan amorphous extracellular matrix (criterion 2). Endo-

dontic biofilms were often confined to the root canalsystem, in only a few cases extending to the externalroot surface, but dissemination through the lesionnever occurred (criterion 3). In the great majority ofcases, biofilms were directly facing inflammatory cellsaccumulated 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 inthe planktonic cell state are susceptible to currently-used antibiotics (70–72). The lack of efficacy of sys-temic antibiotics against intraradicular infections is dueto the fact that the drug cannot reach the bacterialpathogens because they are located in an avascularnecrotic space. The recognition of biofilms as the mainmode of bacterial establishment in the root canalsystem further strengthens the explanations for thelack of antibiotic effectiveness against endodonticinfections. As for criterion 6, the frequent observationof biofilms in treated canals with post-treatmentdisease may at least suggest that there is a potential forfulfillment of this criterion.

The dynamics of endodonticbiofilm formation—a theoryAt first, one might think of endodontic biofilmsforming in a way similar to many other biofilms innature, i.e. colonization of a solid surface by plank-tonic bacterial cells floating in a fluid phase that bathesthat surface. In the root canal, for biofilm formation tofollow that dynamics, the pulp would have to becomenecrotic and liquefied before bacterial invasion.However, if one takes into account the dynamics ofroot canal invasion by bacteria present in caries lesionsthat exposed the pulp tissue, a different dynamics forbiofilm 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 doesthe biofilm that causes it (Fig. 7). Eventually, when thelast dentin layer is destroyed in advanced caries lesions,the pulp becomes exposed primarily to the cariesbiofilm (and also to planktonic bacteria floating insaliva) (Fig. 8). As a result of exposure, the pulpportion beneath the carious lesion becomes severelyinflamed, necrotic, and eventually the frontline of

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infection advances to involve first the tissue in the pulpchamber and then moves inward in the pulp in anapical direction. The biofilm is what is present at thefrontline of the infection (Fig. 9). These events ofbacterial aggression, inflammation, necrosis, and infec-tion occur by compartments of pulp tissue and gradu-ally move in an apical direction. Ultimately, the apicalcanal will become necrotic and infected. Therefore, itis reasonable to assume that the process of biofilmformation occurs gradually in the canal as the infec-tious process migrates in an apical direction.

In the advanced front of a root canal infection, thebiofilm enters into contact with the host tissue, whichis inflamed (Fig. 9). This inflamed area is expected tocontain large amounts of inflammatory exudate,which is rich in proteins and glycoproteins that serveas an optimal source of nutrients for the advancingbacterial community. In histobacteriological sections,biofilms can be observed not only adhered to thecanal walls, but also covering the surface of theinflamed tissue in the forefront of the infection(Fig. 9) (9).

Fig. 7. Caries biofilm. (a) Maxillary first molar with severe caries destruction in an 11-year-old girl. The pulpresponded 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 subjacentpulp tissue. Note the amount of irritation dentin formed under the carious attack, which is penetrated by bacteria inthe mesial pulp horn (on the right) (Taylor’s modified Brown & Brenn, original magnification 16 ¥). (c) Detail fromthe carious cavity surface with several infected dentin spicules (original magnification 100 ¥). (d) Higher magnificationof the dentin spicule indicated by the arrow in (c) showing a dense biofilm is adhered all over to its circumference. Onone side this appears to be composed mainly of filamentous forms. Dentinal tubules and their branches are completelyfilled by bacteria (original magnification 400 ¥). (e) High power view of the interface between dentin surface andbacterial biofilm (original magnification 1,000 ¥). Modified with permission from Ricucci D. Patologia e ClinicaEndodontica. Bologna, Italy: Edizioni Martina, 2009.

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Fig. 8. Pulp exposed to caries biofilm. (a) Mandibular first molar with advanced periodontal disease and a deep distalradicular caries approaching the pulp. The patient requested extraction because of severe spontaneous pain. (b) Sectionpassing in the area where the carious lesion exposes the pulp tissue (Taylor’s modified Brown & Brenn, originalmagnification 25 ¥). (c) Detail from (b) (original magnification 100 ¥). (d) Detail from the perforation in the dentinwall from (c). A biofilm can be seen on the dentin surfaces (original magnification 400 ¥). (e) Magnification of thebacterial colony in the pulp tissue in (c). This is surrounded by a concentration of neutrophils (original magnification400 ¥). Reproduced modified with permission from Ricucci D. Patologia e Clinica Endodontica. Bologna, Italy:Edizioni Martina, 2009.

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In addition to microscopic observations, microbio-logical studies of the composition of the microbiota indentinal caries and primary endodontic infectionsseem to indirectly support the theory that biofilmsform on the canal walls as the infection moves apically.Several bacterial species/phylotypes found in biofilmsfrom deep dentinal caries lesions are also present in

primary intraradicular infections (74–78). This sug-gests that these species participate in the processes ofpulp inflammation and necrosis and are the pioneercolonizers of the necrotic root canal. As the processadvances apically, it is highly possible that theseadvancing bacteria receive “reinforcements” of new-comers from saliva. After the infection reaches the

Fig. 9. Biofilms at the frontline of infection. (a) Mandibular molar with a severe carious process. The pulp wasclinically necrotic and radiolucencies were present on both mesial and distal roots (Taylor’s modified Brown & Brenn,original magnification 2 ¥). (b) Detail of the distal root canal at the middle third. Transition from necrotic/infectedto vital/non-infected pulp tissue can be observed (original magnification 16 ¥). (c) Magnification of the area indicatedby the rectangle in (b). The tissue is severely inflamed, but no bacterial colonization can be seen (original magnification100 ¥). (d) Detail from the coronal root canal in (b). Dense bacterial biofilms can be seen at this level, surrounded bya severe concentration of polymorphonuclear neutrophils (original magnification 100 ¥). (e–f) High power views ofthe areas indicated respectively by the rectangle and the arrow in (d) (original magnification 400 ¥).

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apical part of the canal, the possibility obviously existsthat latecomers now present in the fluid phase in thenecrotic canal may gain entry into the biofilm com-munity, provided they are competitively competentand bring ecological advantages to the overall commu-nity physiology. With the passage of time, the biofilmstructure becomes more and more organized andreaches a state of community homeostasis andbalance—a climax community. Once this state is estab-lished, the composition of the biofilm community isexpected to remain stable over time, unless it is mark-edly challenged by environmental changes.

Microbial diversity inendodontic biofilmsCulture 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 ofmagnitude higher with the advent of massively paral-lel DNA pyrosequencing techniques (80). Specifically,the diversity of the endodontic microbiota has alsobeen unravelled by numerous culture and molecularstudies. Collectively, more than 400 different micro-bial species/phylotypes have been identified in endo-dontic samples from teeth with different forms ofapical periodontitis (2). These taxa are usually foundin combinations involving many species/phylotypesin primary infections and fewer ones in secondary/persistent infections (81).

At high phylogenetic levels, endodontic bacteria fallinto 15 phyla, with the most common representativespecies/phylotypes belonging to the phyla Firmicutes,Bacteroidetes, Actinobacteria, Fusobacteria, Proteo-bacteria, Spirochaetes, and Synergistetes (4,51,54,82–85). In addition to bacteria, other micro-organisms can be found in endodontic infections.Archaea and fungi have been only occasionally foundin intraradicular infections (86–89), though the lattercan be more prevalent in treated teeth with post-treatment disease (90).

Endodontic infections develop in a previously sterileplace which does not contain a normal microbiota.Therefore, any species found in the root canal has thepotential to be an endodontic pathogen or at least toplay a role in the ecology of the microbial community.Despite the long list of bacterial taxa so far detected inendodontic infections, no more than 20–30 bacterial

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 theroot canal become important, even those communitymembers found in low numbers that can be dispro-portionate to their ecological role (13).

Studies of the endodontic microbiota are plaguedwith technical limitations. For instance, the greatmajority of microbiological studies have examinedsamples taken by paper points from the main rootcanal. 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 toreach areas distant from the main canal, includingramifications and isthmuses, which can also harbormicrobial cells. A couple of studies have recently usedthe entire apical root portion sectioned off fromextracted teeth and then cryogenically ground (56–57). This approach permits a more selective analysis ofthe apical microbiota and incorporates all of thedifficult-to-reach anatomical areas in the final sample.Therefore, members of the apical bacterial biofilms arecertainly included in the sample for identification.

Following the recognition that biofilm communitiesare very common in the apical root canal and areinvolved in disease causation, it seems important toidentify the species composition of these apical bacte-rial communities, excluding the portion of the micro-biota that remains planktonic. It is highly likely thatplanktonic bacterial cells floating in the main canalmay have been dettached from the biofilms, but theymay also be by-standers, latecomers, or individualsthat did not succeed in joining the community due toa lack of competitiveness. Approaches involvingmicroscopy in association with either in situ hybrid-ization or open-ended molecular microbiology tech-niques can be of great value in helping to decipher thespecific composition of endodontic biofilms. This willaid in the understanding of the ecology of these patho-genic communities. These and other sophisticatedmethods for the study of biofilms have yet to beapplied to the study of endodontic biofilms.

Concluding remarksBacterial biofilms are very prevalent in the apical rootcanals of teeth with primary and post-treatment apicalperiodontitis. The pattern of bacterial community

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45

arrangement in the canal fulfills acceptable criteria toinclude apical periodontitis in the set of diseases causedby biofilms.

Future research should address several issues thatarise from these observations. The ultrastructure ofendodontic biofilms should be studied so as to providea better understanding of its physiology, ecology,pathogenicity, and response to treatment. Unravellingthe specific composition of endodontic biofilms willrequire the integration of sophisticated microscopicand molecular microbiology approaches. This knowl-edge can be of utmost importance not only in pro-moting a refined understanding of endodonticbiofilms, but also in helping to develop better strate-gies for treatment. Moreover, community profilingtechniques applied directly to endodontic biofilmsamples may provide information as to the occurrenceof specific communities related to disease. Forinstance, it may help identify communities that aremore related to symptoms or that are more resistant totreatment.

As many studies have been used to answer “who isthere,” the time has now come to determine “what arethey doing there.” The physiology and pathogenicityof biofilms can be inferred by methods that provide agenome-wide analysis of DNA obtained directly fromthe environment (metagenomics), and a profile ofRNA (transcriptomics), proteins (proteomics), andmetabolic end-products (metabolomics) expressed orreleased by the community. These methods also havethe potential to disclose patterns of molecules associ-ated with clinical conditions, which may serve a role asoutcome predictors.

There is also an urgent need to evaluate the effects oftreatment on real endodontic biofilms, using in vivo orex vivo (recently extracted teeth with apical periodon-titis) models. Moreover, it is very important to inves-tigate the resilience, recovering ability, and fate ofbiofilm communities that are only partially affected ordisrupted by treatment. Another potential focus ofstudy is the susceptibility of the biofilm matrix to theeffects of treatment and its fate if left behind, so as toshed 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 innature are developed and become more and moreadvanced, the potential exists for further details of thebiofilm lifestyle to be revealed. Endodontic scientists

should keep abreast of the literature on this topic as itrelates to other areas in order to expand their views onthe concepts and methods applied to the study andtreatment of biofilms.

AcknowledgementsThis study was partially supported by grants from FundaçãoCarlos Chagas Filho de Amparo à Pesquisa do Estado do Riode Janeiro (FAPERJ) and Conselho Nacional de Desenvolvi-mento Científico e Tecnológico (CNPq), Brazilian govern-mental institutions.

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