Essential endodontology: prevention and treatment of apical periodontitis. 3rd ed

Chapter 4. Microbiology of Apical Periodontitis

José F. Siqueira Jr and Isabela N. Rôças

4.1 Introduction

Infection of the dental root canal system is the major cause of apical periodontitis (Figure 4.1). Although chemical and physical factors can induce inflammation in the periradicular tissues, a large body of scientific evidence indicates that microorganisms are essential to the progression and perpetuation of apical periodontitis [99, 137, 291]. Fungi, archaea, and viruses have been found in association with apical periodontitis, but bacteria are the major microorganisms implicated in the etiology of this disease.

Root canal infection only develops in teeth with partial or total pulp necrosis or in canals that had the pulps removed during previous treatment. Pulp necrosis is a sequel to caries, trauma, periodontal disease, or iatrogenic operative procedures. These conditions create pathways by which oral bacteria can gain access to the root canal system. A necrotic pulp lacks active circulation and can there- fore no longer mobilize inflammation and protect itself against invasion and colonization by oral bacteria. After an endodontic infection is established, bacteria contact the periradicular tissues via apical and lateral foramina. As a consequence, inflammatory changes take place at the periradicular tissues and give rise to the diverse forms of apical periodontitis.

Because apical periodontitis is regarded as an infectious disorder caused by bacterial infection of the root canal system, successful treatment of this disease is contingent upon complete elimination or effective control of the infecting microbiota. A thorough understanding of the disease's etiology is essential for successful treatment. In this context, knowledge of the microbiological aspects of apical periodontitis is of utmost importance for endodontic practice of high quality and founded on solid scientific basis.

This chapter discusses several aspects related to the microbiology of endodontic infections and pathogenesis of apical periodontitis. For a better understanding of several terms used, Table 4.1 depicts definitions for many of them.

4.2 Microbial Causation of Apical Periodontitis

The first recorded observation of bacteria in the root canal dates back to the 17th century, when the Dutch amateur microscope builder Antony van Leeuwenhoek [1632-1723] wrote: “The crown of this tooth was nearly all decayed, while its roots consisted of two branches, so that the very roots were uncommonly hollow and the holes in them were stuffed with a soft matter. I took this stuff out of the hollows in the roots and mixed it with clean rain water, and set it before the magnifying glass so as to see if there were as many living creatures in it as I had aforetime discovered; and I must confess that the whole stuff seemed to me to be alive” [38]. However, at that time, the role of Leeuwenhoek's animalcules as infectious agents was unknown. It took over 200 years before his observations were confirmed and a cause-and-effect relationship between bacteria and apical periodontitis was suggested.

Figure 4.1 Bacteria colonizing the root canal system are the major causative agents of apical periodontitis lesions.

In 1894, Willoughby Dayton Miller, an American dentist who developed his seminal experiments in oral microbiology inspired by Robert Koch, in Berlin, Germany, published a milestone study reporting on the association of bacteria with apical periodontitis, after the analysis of material collected from root canals [131]. By means of bacterioscopy of the canal samples, Miller found the three basic bacterial morphotypes, i.e., cocci, bacilli and spirilla (Figure 4.2). He wrote: “We assume, in a general way, that bacteria must in some manner be connected with these processes (pulp diseases). There are, then, as I have already pointed out, different species of bacteria in the diseased pulp that have not yet been cultivated on artificial media, and of whose pathogenesis we know nothing definite. Their great numbers in some pulps, and especially the repeated occurrence of spirochaetes, justify the sup- position that, under certain circumstances, they may play an important role in suppurative processes”

Miller raised the hypothesis that bacteria were the causative factors of apical periodontitis and that the species composition of the microbiota in the coronal, middle and apical parts of the root canal was clearly different. Some bacteria from root canal samples that were seen under light microscopy could not be cultivated using the methods available at that time. Most of those bacteria were conceivably anaerobic bacteria. Nonetheless, in spite of the considerable technologic advances in the last century regarding bacterial cultivation, it is widely recognized that a large number of microbial species still remain uncultivated [3, 179].

Miller's findings, although pioneering, were only suggestive of a causal relationship between bacteria and apical periodontitis. Two events occurring simultaneously do not necessarily imply a cause-and-effect relation- ship. It was not until approximately 70 years after Miller's classic findings that the causal relationship between bacteria and apical periodontitis was unequivocally demonstrated by an elegant study in germ-free rats from Kakehashi et al. [99]. These authors exposed the dental pulps of conventional and germ- free rats to the oral cavity and observed the pulp and periradicular responses. Histologic evaluation performed at intervals ranging from 1 to 42 days postoperatively revealed that in conventional animals, without exception, all older specimens showed complete pulp necrosis with periapical inflammatory lesions. On the other hand, the dental pulps of germ-free animals repaired themselves by dentinal bridge formation, which was already evident at 14 days; by 21 and 28 days, the newly formed hard tissue was completely sealing the previously exposed area, regardless of the angle or severity of exposure. In every instance, the pulp tissue remained vital beneath the dentinal bridge.

Table 4.1 Definitions of terms used in this chapter.

Term	Definition
16S ribosomal RNA (rRNA)	A component of the smaller (30S) prokaryotic ribosomal subunit. The gene that encodes this structure possesses hypervariable regions containing species- specific signature sequences, which are useful for bacterial and archaeal identification at the species level. It also contains conserved universal regions that permit amplification of part or the entire 16S rRNA gene of virtually all bacteria or archaea
Amensalism	A relationship between two species of organisms in which one is inhibited and the other is unaffected
Archaea	A domain containing prokaryotic microorganisms different from bacteria, which compose another domain (Bacteria). Eukarya (containing organisms with nucleated cells) is the third domain of life.
Asaccharolytic	A microorganism that cannot metabolize carbohydrates and consequently needs other carbon sources, such as peptides, for obtaining energy
Biofilm	A sessile multicellular microbial community characterized by cells firmly attached to a surface and emmeshed in a matrix of extracellular polymeric substances produced by themselves
Community Community profile Community structure Density Diversity Ecology Endogenous infection Exogenous infection	A group of interacting populations Species richness and abundance in a community Species richness and abundance in a community Number of cells (individuals) Measure of the richness and relative abundance of species in a community Study of the interrelationships between organisms and their environments Infection caused by members of the normal human microbiota Infection caused by microorganisms that do not belong to the normal microbiota and that were introduced in the host
Food chain	A linear succession of organisms in a community that are linked to each other through the transfer of nutrients and energy
Food web	A network of interconnected food chains. It shows how different species are connected by different metabolic paths
Horizontal (or lateral) gene transfer	The exchange of genetic material between contemporaneous organisms as opposed to vertical transfer, which represents inheritance of genetic material from an ancestor. Horizontal transfer permits that one species transfers its genetic material to another species.
Infection	Invasion and proliferation of microorganisms in a place where they are not expected to be present. Infection does not necessarily result in disease
Infectious disease	Development of signs and symptoms after microbial infection and damage to host tissues
Inflammophilic bacteria	Bacterial species that flourish in contact with inflamed tissues, deriving nutrients from inflammatory exudates and tissue breakdown products
Load	Number of cells (individuals)
Metaproteomics	The study of all proteins recovered directly from environmental or clinical samples
Microbiota	A collective term for microorganisms forming a complex and diverse community in a given anatomical site

(Continued)

Table 4.1 (Continued)

Term	Definition
Modulin	Bacterial structural components or released products with the ability to modulate the host immune response, especially by stimulating synthesis and release of cytokines
Niche	Functional role (metabolic functions) of an individual in the community
Opportunistic pathogen	A microorganism that causes disease only when host defenses are impaired
Pathogen	A microorganism that causes disease
Pathogenicity	Ability to cause disease
Phylotype	As-yet-uncultivated species that are known only by a 16S rRNA gene sequence rather than by phenotypic characteristics
Polymicrobial infection	An infection caused by several different species
Population	A group of growing cells (microcolony)
Primary or true pathogen	A microorganism that often causes disease within a given host
Putative or candidate pathogen	A microorganism that has been found in association with disease in cross- sectional studies, but evidence from longitudinal observations is lacking
Relative abundance	Proportion of each species within a community
Richness	Number of different species in a community (composition)
Synergism	Interaction between two or more species or factors that results in an effect greater than the sum of their individual effects
Virulence	Degree of pathogenicity
Virulence factor	Microbial products, structural components or strategies that contribute to pathogenicity

Figure 4.2 Drawings from Miller's classic article [131] showing different bacterial forms in a root canal sample observed by microscopy.

In 1976, Sundqvist used anaerobic cultivation methods to evaluate the bacteriology of pulps of human intact teeth that had become devitalized after trauma [291]. His findings revealed that whereas the necrotic pulps of teeth without apical periodontitis lesions were bacteria-free, those showing apical periodontitis lesions were virtually always infected. Anaerobic bacteria predominated, comprising more than 90% of the isolates. It could also be inferred from his findings that the necrotic pulp tissue itself and stagnated tissue fluid in the root canal are not able to induce and maintain apical periodontitis.

Strong evidence about the causal relation- ship between bacteria and apical periodontitis was also provided by Moller et al. [137]. In a study in monkey's teeth, these authors demonstrated that only devitalized pulps that were infected caused apical periodontitis, whereas devitalized but uninfected pulps showed an absence of pathological changes in the periradicular tissues. In addition to corroborating the importance of bacteria for the development of apical periodontitis, this study also showed that the necrotic pulp tissue, in the absence of infection, is not able to induce and perpetuate periradicular inflammation.

Bacteria colonizing the root canal system in association with apical periodontitis are primarily organized in biofilm structures. Several morphologic studies have reported on bacterial organizations resembling biofilms in root canal infections [138, 148, 233, 276], but it was not until the study of Ricucci and Siqueira [183] that the strong association of bacterial biofilms present in the apical portion of the canal and primary and post-treatment apical periodontitis was demonstrated. Given the cross-sectional nature of most endodontic microbiology studies, it is still not clear whether bacterial organization in biofilms is essential for apical periodontitis to develop. However, from a clinical standpoint, it is important to recognize that bacterial biofilms are highly prevalent in the apical canal of untreated and treated teeth with apical periodontitis and may represent a challenge for proper antimicrobial treatment.

4.3 Endodontic Biofilms and the Community-as-Pathogen Concept

Biofilm can be defined as a sessile multicellular microbial community characterized by cells firmly attached to a surface and emmeshed in a matrix of extracellular polymeric substances (EPS) [28, 39] (Figure 4.3). The vast majority of microbial species in nature live in metabolically integrated biofilm communities, and the human body is no exception [28, 126].

While most acute medical infections that require rapid diagnosis and intervention to avoid dramatic damage or death are caused by planktonic bacteria, chronic infections associated with sustained inflammation and gradual tissue damage are usually related to biofilms [168, 273, 330]. Biofilm infections are suggested to be responsible for 65-80% of the human infectious diseases, including endocarditis, middle ear infections, osteomyelitis, prostatitis, and orthopedic device- related infections [27, 30, 168]. In the oral cavity, caries, gingivitis, and marginal periodontitis are typical examples of diseases caused by bacterial biofilms [125]. Apical periodontitis has also been recently introduced in the set of biofilm-induced oral diseases [183].

Figure 4.3 Bacterial biofilm in a tooth with primary apical periodontitis. Notice the bacterial cells adhering to dentin (blank arrow) and embedded in a thick extracellular matrix (black arrow) (Brown and Brenn staining, original magnification 400x).

4.3.1 Structure

Cells in bacterial biofilms are commonly aggregated in microcolonies (populations) embedded and non-randomly distributed throughout the EPS matrix [29, 39, 283, 286]. Distribution of the populations along the biofilm structure is dictated by diverse factors, including arrival time, positive metabolic interactions among the community members, and proximity to convenient nutrient sources. Populations are separated by water channels that travel throughout the biofilm structure, carrying water and nutrients and draining waste products. The largest accumulations of cells commonly occur at the bottom of the biofilm structure, close to the surface to which the biofilm is adhered.

The EPS matrix is produced by the com- munity members themselves and account for the largest portion of the biofilm dry mass (>90%); cells constitute <10% of the biofilm mass [58]. The EPS matrix is mostly composed by polysaccharides, but proteins, nucleic acids, and lipids can also be present [28]. It serves numerous essential functions to the biofilm community, as it mediates adhesion to surfaces, provides mechanical stability, allows for accumulation of extracellular enzymes involved with nutrient acquisition and defense against antimicrobials, favors positive intercellular interactions by keeping cells in close proximity, serves as a reserve of nutrients, retains water, and protects the community against antimicrobial agents and host defenses [58, 239]. Bacteria in biofilms exhibit increased resistance against phagocytosis by neutrophils [94] and macrophages [297].

Biofilm cells exhibit a different phenotype in comparison with their counterparts occurring in the planktonic state. This is largely due to differential gene expression between the two states [16, 154, 228]. The changed gene expression and low growth rate of the biofilm phenotype also generally confers increased resistance to antimicrobial agents, environmental stress, and host defenses [120].

4.3.2 Bacterial Interactions and Intercommunication in Biofilms

In multispecies biofilms, cells are in close contact with one another, which favors inter- actions and communication between them. Interactions affect the overall function and physiology of the biofilm community, as well as its resistance to external threats and virulence [20]. Interactions between biofilm bacteria may enable them to behave collectively as a group.

Communication between bacteria is often referred to as quorum sensing and may occur with both Gram-positive and Gram-negative species [44, 159, 329]. Quorum sensing involves the production, release and subsequent detection of chemical signaling molecules called autoinducers. As a bacterial population producing and releasing autoinducers multiplies, the extracellular concen- tration of autoinducers also increases. When autoinducer concentration reaches a crucial threshold level, the group responds by changing gene expression [159]. This gives bacteria a means to perform specific behaviors and functions only when living in groups. Communication signals are known to be much more relevant among members of a given population than between distinct pop- ulations, but the possibility exists that the signals released by cells present in separate populations may also affect each other [20]. Quorum-sensing systems are important for regulation of virulence, resistance to starvation and host defenses, production of secondary metabolites, and biofilm formation. For instance, some opportunistic pathogens express virulence factors in response to sensing their own cell density. Several candidate endodontic pathogens have been demonstrated to produce quorum-sensing signal molecules [61, 167, 324, 334].

Communication among bacteria in multispecies biofilms can lead to reciprocal transcriptomic and proteomic responses with consequent regulation of nutrient acquisition, metabolic processes and expression of virulence factors [81, 144]. Based on the numerous possible interactions between community members, it is possible to infer that the higher the community diversity, the higher its complexity.

The high cellular density and the stable conditions for cell-cell contact in biofilms also favor horizontal gene transfer between community members [20]. Therefore, genes responsible for antimicrobial resistance or related to virulence properties may be trans- ferred to other members of the community.

Occurrence of syntrophy between community members, in which one species depends on the products released by another, helps shape the organization of populations throughout the biofilm structure. In this sense, cooperative interactions for break- down of complex substrates also assumes rel- evance (see section “Microbial ecology and the root canal ecosystem”).

4.3.3 Community-as-Pathogen Concept

Biofilms associated with most endogenous infections are usually composed of several different species. Consequently, a polymicrobial etiology for these diseases has been proposed. With the biofilm concept, the current trend is to interpret the bacterial community as a whole as the unit of pathogenicity for these diseases [93, 109, 273]. Thus, rather than the specific factors released by one or a few of the colonizing microorganisms, it is the total species composition and relative abundance of each species in the biofilm community as well as the myriad of bacterial interactions that modulate virulence.

It seems that the pathogenesis of apical periodontitis requires the concerted action of bacteria in a multispecies community [273]. The types and levels of the community members as well as the interactions between them result in the accumulation of a multi- tude of virulence factors in the biofilm matrix [269]. As the biofilm reaches the apical region of the root canal system, diffusion of the virulence factors from its biomass evokes and maintains inflammation in the periradicular tissues [258].

Biofilm infections generally represent a persistent source of aggression to the tissues. In root canal infections, persistence is aggravated by the inaccessibility of host defenses to the anatomic site of infection. The juxtaposition of bacterial biofilms to the periradicular tissues triggers destructive inflammatory responses.

Biofilm collective pathogenicity is influenced by synergistic interactions among community members, and the resulting outcome can be more severe than expected for the individual components. As noted earlier, synergistic interactions enhance protection against host defenses and antimicrobial agents and increases virulence of the multispecies community. Furthermore, survival and persistence of the biofilm in an inflamed environment may actually be favored in some circumstances: In fact, it has been suggested that oral bacterial communities associated with long-standing inflammatory processes can be regarded as “inflammophilic” [80]. This means that the community members not only have the ability to survive the attack of the inflammatory response, but also to take advantage of this condition.

Inflammation is an essential process to combat infection, but in biofilm infections it usually does not succeed in eradicating the infection, resulting in tissue damage without elimination of its source. Inflammation can represent an important source of nutrients to some of the bacteria occurring in biofilms in the apical root canal. Nutrients are in the form of glycoproteins from the exudate and tissue breakdown products, such as degraded collagen and heme-containing compounds (haptoglobin, hemopexin and hemoglobin) [80, 258]. It has been shown that the bacterial load in periodontal biofilms is higher with increasing inflammation [1], and the periodontitis-associated community exhibits increased expression of genes related to iron acquisition and proteolytic activity [45].

After the development of apical periodontitis, inflammation-derived products become the main source of nutrients to intracanal bacteria. As a consequence, asaccharolytic and proteolytic bacteria that obtain iron and use these products (peptides and amino acids) as their main source of energy dominate the microbiota in the apical root canal system [258].

Therefore, inflammation acts as a double-edged sword. On the one hand, persistent biofilm intraradicular infection simultane- ously activates both innate and adaptive immune responses that play an important role in preventing the root canal infection from reaching bone and spreading to other body areas. On the other hand, neither of these responses can eliminate the intraradic- ular biofilm and the result is tissue damage (see Mechanisms of bacterial pathogenicity below). In addition, persistent inflammation introduces a new and sustainable source of substrate for growth and survival of inflam- mophilic bacteria in the apical canal.

Based on the community-as-pathogen concept, the pathogenic endodontic bacterial community becomes enriched in virulence factors, which accumulate in the biofilm and are gradually released to stimulate and sustain periradicular inflammation. The community also is adapted to survive in the presence of inflammation, from which it can derive an important source of nutrients for its maintenance.

4.3.4 Biofilms in Teeth with Apical Periodontitis

Several morphological studies have described bacterial organizations resembling sessile biofilm structures in the root canals of teeth with apical periodontitis [22, 138, 148, 186, 229, 276]. While many bacterial cells are sus- pended in fluid or enmeshed in the pulp necrotic tissue in the main root canal lumen (planktonic state) (Figure 4.4), dense bacterial biofilm communities have been observed adhering to the root canal walls to a varying degree [148, 276] (Figure 4.5). Planktonic bacterial cells in the main canal may be newcom- ers or they may be cells detached from the biofilm structures adhering to the canal walls. Biofilms are found not only in the main canal, but they can also propagate to apical ramifications, lateral canals, isthmi, and recesses of the system [22, 147, 182, 184]. Occasionally, they can also reach the apical foramen/ina and extend to the outer root surface, forming an extraradicular biofilm [54, 185, 298].

Figure 4.4 Planktonic bacteria occurring in the canal lumen. Cells are usually suspended in fluid and enmeshed with the necrotic tissue (Brown and Brenn staining, original magnification 400x).

The high prevalence of biofilms in the apical root canal and their strong association with apical periodontitis were demonstrated in a histobacteriological study by Ricucci and Siqueira [183]. Intraradicular biofilms were found in the apical canal of 80% of teeth with primary apical periodontitis and 74% of teeth with post-treatment apical periodontitis. Endodontic biofilms were usually thick and multilayered, and the relative matrix-cell proportions were highly variable (Figure 4.5). Whereas most of the walls were heavily colo- nized, there were areas in which colonization was slight or even absent.

Figure 4.5 (a) Thick bacterial biofilm in a tooth with extensive coronal destruction and apical periodontitis, which was open to the oral cavity and had a history of several exacerbations before extraction (Taylor- modified Brown and Brenn staining, original magnification 50x). (b) Higher magnification showing dense population of bacterial cells faced by accumulation of inflammatory cells, especially polymorphonuclear neutrophils (arrow) (400x). Courtesy of Dr Domenico Ricucci.

Biofilms have been more frequently found in teeth with large lesions and those histo- pathologically diagnosed as cysts [183]. This is possibly related to the fact the large lesions and cysts represent long-standing pathological processes. Consequently, the intraradicular infection, which is the cause of the lesion, is even “older” in these cases. Bacteria involved in these processes are expected to have had sufficient time and conditions to organize themselves in a mature biofilm community. The observation that the treatment outcome is influenced by the size of apical periodontitis [287] can be justified by the difficulties to control an infection that is complex and composed by large numbers of cells and species [196, 291], usually organ- ized in biofilms [183].

A particular finding from the Ricucci and Siqueira's study [183] was the observation of bacterial flocs in some clinical specimens. Flocs are large bacterial colonies surrounded by EPS matrix and regarded as “planktonic biofilms”. These structures may originate from growth of aggregates/coaggregates of planktonic cells or they may have detached from biofilms adhered to a surface [82].

Bacterial invasion of dentinal tubules is commonly seen underneath biofilm structures [183]. The diameter of dentinal tubules is large enough to permit penetration of most oral bacteria. Dentinal tubule infection can occur in about 50-80% of teeth with apical periodontitis [79, 130]. A shallow penetration is more common, but bacterial cells can sometimes be observed as deep as 300 m in dentin [276]. A study in monkeys showed that infection may even reach the entire length of the dentinal tubules and cause changes in the periodontal ligament, especially when cementum is lost by resorption [302]. While some tubules can be heavily infected, adjacent tubules may be free of infection (Figure 4.6). Motility does not appear to be a necessary bacterial attribute for dentinal invasion, since most bacteria so far identified in tubules are non-motile species [130, 165]. Dividing cells are frequently observed within tubules in in situ investigations [276] (Figure 4.7), indicating that bacteria can derive nutrients within tubules, probably from degrading odontoblastic processes, denatured collagen, bacterial cells that die during the course of infection, and intracanal fluids that enter the tubules by capillarity. Actually, several candidate endodontic pathogens have been shown to be capable of penetrating dentinal tubules in in vitro [116, 163, 260] and in vivo studies [130, 165].

Figure 4.6 (a) Bacteria invading dentinal tubules in a tooth with apical periodontitis (Taylor-modified Brown and Brenn staining, original magnification 100x). (b) Higher magnification (400x). Courtesy of Dr Domenico Ricucci.

The occurrence of biofilms attached to the outer root surfaces around apical or lateral foramina is rather uncommon. However, when present, extraradicular biofilms are usually associated with symptoms or sinus tract (see further discussion on “Extraradicular infection”).

4.3.5 Dynamics of Endodontic Biofilm Formation - a Theory

The mechanisms of biofilm formation on different surfaces in nature are similar to dental plaque. Basically, the process starts by colo- nization of a solid surface by planktonic bacterial cells floating in a fluid that bathes that surface. Initially, a conditioning film forms on the tooth surface as a result of adsorption of macromolecules (proteins and glycopro- teins) from saliva to the surface. Bacteria occurring in the planktonic state approach and adhere to the surface by non-specific and specific means. EPS matrix is produced and permits stronger adhesion. Finally, these pioneer species grow and co-aggregate to others, including newcomers, to form populations that organize themselves in a multispecies community - biofilm.

These mechanisms may contribute to biofilm formation in cases that a pulpless canal is open to the oral cavity and saliva seeps into it. However, in most circumstances, the process of biofilm formation in root canals is expected to follow a difference course of events. Pulp inflammation, necrosis and infection is usually a sequel to caries - a disease also caused by biofilms [127]. The biofilm associated with caries gradually advances towards to the pulp as the process destroys dentin. When the pulp tissue is exposed to the caries biofilm, severity of inflammation intensifies and may result in localized areas of tissue necrosis (Figure 4.8). Then, the biofilm advances to occupy these areas, moving in apical direction as the bacterial cells adhere and grow along the dentinal canal walls. Cells detaching from the biofilms and late-coming planktonic cells arriving from the oral cavity will also occur in the canal lumen with necrotic tissue. The events of bacterial aggression, inflammation, necrosis and infection occur by compartments of pulp tissue and gradually move towards to the apical portion of the canal. Consequently, the process of biofilm formation in necrotic root canals occurs gradually as the biofilm in the frontline of the advancing infection moves apically.

Figure 4.7 Bacterial infection of the root canal in a tooth with apical periodontitis. Scanning electron micrographs showing (a) dense bacterial aggregates colonizing the root canal walls and some cells invading the dentinal tubules (original magnification 1800x), and (b) dividing bacterial cells within tubules (5500x).

Figure 4.8 (a) Caries biofilm reaching the pulp and causing extensive inflammation (Taylor-modified Brown and Brenn staining, original magnification 16x). (b) Higher magnification showing bacterial invasion of the tertiary dentin (blank arrow). Bacteria are also seen in the pulp (black arrow), which is severely inflamed (100x). Courtesy of Dr Domenico Ricucci.

4.3.6 Apical Periodontitis is a Disease Caused by Biofilms

Apical periodontitis fulfils the criteria commonly used to establish a causal link between an infectious disease and biofilms [82, 160, 183]. They are as follows.

1) The infecting bacteria are adhered to or associated with a surface.

Findings for apical periodontitis: As dis- cussed above, bacteria infecting the root canal are often organized in communities attached to dentinal walls of the main canal and other areas of the system [22, 138, 148, 183, 186, 229, 276].

2) Direct examination of infected tissue shows bacteria forming populations encased in an extracellular matrix. Findings for apical periodontitis: Histo- bacteriological studies have shown that bacterial communities adhering to the canal walls are enmeshed in an amorphous extracellular matrix with varying thickness [183, 186, 239], a structure similar to biofilms reported in other human sites, including dental plaque.

3) The infection is generally confined to a particular site and although dissemination may occur, it is a secondary event. Findings for apical periodontitis: In endodontic infections, biofilms are frequently restricted to the root canal system (intraradicular biofilms) [183]. In rare conditions, the biofilm is found extending to the external root surface, but dissemination beyond this area or the apical periodontitis lesion is rarely, if ever, reported in chronic cases.

4) The infection is difficult or impossible to eradicate with antibiotics despite the fact that the responsible microorganisms are suscep- tible to killing in the planktonic cell state. Findings for apical periodontitis: Endodontic infections cannot be successfully treated by systemic antibiotics. Antibiotics are prescribed usually in conditions where there are signs of dissemination of the infecting bacteria. The reason for this inefficacy is that the bacteria are located in an avascular necrotic space, in which antibiotics cannot penetrate in adequate concentration to be effective. In addition, bacterial cells living in biofilms are reported to be 100 to 1000 times more resistant to antibiotics than their plank- tonic counterparts [153].

5) Ineffective host clearance, which may be evidenced by the location of bacterial col- onies in areas of the host tissue associated with inflammatory cells.

Findings for apical periodontitis: accumulations of inflammatory cells, mostly pol- ymorphonuclear neutrophils, are often seen facing endodontic biofilms [183].

6) Elimination or significant disruption of the biofilm structure and ecology leads to remission of the disease process.

Findings for apical periodontitis: if the endodontic treatment succeeds in ren- dering the root canal free of detectable bacteria at the time of filling, the success rate is significantly increased [50]. Moreover, the frequent observation of biofilms in root canal-treated teeth with apical periodontitis, and a lack thereof in teeth with healthy periapical areas [183, 187], indicates that there is a potential for fulfilment of this criterion.

4.3.7 Requirements for an Endodontic Pathogenic Community

The biofilm community should fulfil the fol- lowing requisites to initiate and maintain disease:

1) The community density must be high enough to reach a pathogenic load.

2) The community must exhibit an array of antigens and virulence factors, which should be expressed during root canal infection, accumulated in the community EPS matrix, and released to the environment.

3) The community must be spatially located in the root canal system in such way that cells and/or virulence factors and antigens can gain access to the periradicular tissues.

4) The community must contain pathogenic species that are well integrated and organ- ized in a synergic relationship with the other biofilm species.

5) The host must mount a defense strategy at the periradicular tissues, which inhibits the spread of the infection to the bone and beyond, but which also results in tissue damage.

4.4 Mechanisms of Bacterial Pathogenicity

The ability of microorganisms to cause disease is regarded as pathogenicity. Virulence denotes the degree of pathogenicity of a microorganism, and virulence factors are the microbial products, structural components or strategies that contribute to pathogenicity.

The collective pathogenicity of the bacterial community will depend on the overall population density, species composition, and the synergistic interactions between them. Bacterial organization in biofilms permits the accumulation of virulence factors and their gradual release from the EPS matrix to affect the adjacent host tissues. Different virulence factors usually act in combination at various stages of infection, and a single factor may have several functions in different stages. Virulence factors may be involved in attachment to host surfaces, tissue and host cell invasion, spread through the host tissues, direct and indirect tissue damage, and survival strategies, including evasion of host defense responses.

Bacteria exert their pathogenicity by destroying host tissues through direct and/or indirect mechanisms. Direct harmful effects caused by bacteria usually involve secreted products, including enzymes (proteases, peptidases, hyaluronidase, etc), exotoxins, and metabolites (butyrate, propionate, ammonium, polyamines, indole, volatile sulfured compounds, etc) [245]. In apical periodontitis, the indirect mechanisms dominate in the development of tissue damage. Thus, it is the host immune response to the bacterial challenge that ultimately causes the tissue destruction typical of apical periodontitis. Stimulation and activation of the host immune responses is caused by bacterial antigens and structural components (modu- lins), including lipopolysaccharide (LPS or endotoxin), peptidoglycan, lipoteichoic acids, fimbriae, flagella, outer membrane proteins, and exopolysaccharides [86, 303].

Bone resorption illustrates this. Inflammatory and non-inflammatory host cells are stimulated by bacterial components to release chemical mediators such as cytokines and prostaglandins, which are involved in the induction of bone resorption characteristically observed in chronic apical periodontitis lesions [226]. Bacterial DNA also activates macrophages and dendritic cells and triggers the release of pro-inflammatory cytokines [108]. Pro-inflammatory cytokines stimulate osteoclastic bone resorption either by enhancing the proliferation and differentiation of osteoclast precursors or by promoting activation of mature osteoclasts, or both [213].

Another example of indirect damage caused by bacteria is pus formation in acute apical abscesses. Although some bacterial products may cause tissue damage and play a direct role in the generation of pus, the main cause of connective tissue destruction and liquefaction is products released by polymorphonuclear neutrophils (PMNs) in the response to the infection. Hyperactive, supernumerary, or dysregulated PMNs cause tissue damage through the release of toxic substances such as oxygen-derived free radicals or tissue-degrading lysosomal enzymes.

The sheltered location of the root canal microbiota implies that for bacteria to exert their pathogenicity, they must either invade the periradicular tissues or their products and/or structural components must penetrate the tissues and evoke a response by the host tissues. Bacteria invade tissues either by means of their motility or by growth. Motile bacteria, such as treponemes, can escape phagocytes by a rapid movement. However, most endodontic bacteria are non-motile and tissue invasion by growth requires that the rate of reproduction overcomes the host defense mechanisms. Frank invasion of the periradicular tissues is rather uncommon and, when it occurs, bacteria are usually rapidly eliminated. However, in some instances, massive invasion of the periradicular tissues by bacteria results in severe inflammation and abscess formation. The presence of virulent species or strains, and a highly virulent multispecies community predisposes to abscess formation (see “Symptomatic infections”).

It is assumed that the oral microbiota contains only a few truly pathogenic species, and most of them exhibit low virulence. This is consistent with the chronic progressive nature of the most common forms of apical periodontitis. Therefore, because bacterial infection of the periradicular tissues rarely occurs (except for abscesses), direct or indirect damage to the tissues is caused by bacterial secreted products or structural components that diffuse out from the intraradicular biofilm or are released by bacterial cells that reach the periradicular tissues, but which are quickly destroyed therein.

Nearly all virulence factors are tightly reg- ulated, and their expression is linked to environmental signals or cues. Biochemical and physical parameters that affect virulence factor regulation include starvation, populational density, pH, temperature, iron availability, oxygen tension, and redox potential. Therefore, upon receiving the appropriate environmental signals, different sets of virulence genes can be turned on or turned off. This affords bacteria the ability to adapt themselves to different and varying environmental conditions.

Stressful conditions, such as starvation, may stimulate the virulence apparatus in certain pathogens [110, 129]. Periods of starvation are commonly experienced by living bacteria in their natural environments. Once starvation genes are expressed, bacteria shift their behavior in order to survive in conditions of nutrient depletion. The major induced mechanisms include control of the energy generation during starvation and enhancement of the scavenging ability of the scarce nutrient. These mechanisms may allow bacteria to survive in root canal-treated teeth and induce post-treatment apical periodontitis lesions.

4.4.1 Bacterial Virulence Factors Released in the Canal Milieu

The pathogenic ability of multispecies biofilm communities is related to the accumulation and gradual release of virulence factors and antigens from the different component species in the EPS matrix. Evaluation of the bacterial substances released during the course of a multispecies infection thus provides valuable information about the physiologic and pathogenic behavior of the community.

LPS is one of the dominating constituents of the outer membrane of most Gram-negative bacteria and is composed by a hydrophilic polysaccharide and a hydrophobic glycolipid component (lipid A) [193]. Most of its biological effects are related to the lipid A portion, which is exposed after cell death or during multiplication. Interaction of LPS with host cells cause numerous biological effects, including: activation of macrophages/monocytes with consequent synthesis and release of pro-inflammatory cytokines, prostaglandins, nitric oxide, and oxygen-derived free radicals [86, 303, 328]; activation of the complement system [96, 332]; stimulation of osteoclast differentiation and bone resorption [345]; and activation of pattern recognition receptors expressed on trigeminal afferent neurons, triggering intracellular signaling cascades that lead to peripheral release of neuropeptides and central nociceptive neurotransmission [316]. The content of LPS in infected root canals is higher in teeth with primary infections, symptomatic apical periodontitis, large periradicular bone destruction, and in cases with persistent exudation [31, 69, 91, 92, 230, 231]. LPS from endodontic pathogens has been suggested to play an important role in tissue damage associated with abscesses [142] and bone resorption [32].

Although LPS has been widely studied and regarded as an important virulence factor from Gram-negative bacteria with regard to the pathogenesis of apical periodontitis, it seems too simplistic to attribute to this sub- stance all the biological effects of endodontic infections related to causation of apical periodontitis. In a multispecies community like endodontic biofilms, many other virulence factors are expected to be produced and par- ticipate in disease pathogenesis. Indeed, many other potential virulence factors are released in the root canal milieu during the course of infection.

Lipoteichoic acid (LTA) is an anionic polymer that is a major component of the Gram-positive cell wall. Given its biological effects, LTA may be considered as the counterpart of LPS for Gram-positive bacteria. LTA stimulates macrophages/monocytes to release of pro-inflammatory cytokines [303, 321] and activate the complement system [66]. A study quantified LTA in teeth with post-treatment apical periodontitis and found this molecule in all cases examined [9].

Several end-products of the bacterial metabolism are released to the extracellular environment and may be toxic to host cells, cause degradation of constituents of the extracellular matrix of the connective tissue, and interfere with host defense processes [46, 73, 304]. Among them, short-chain fatty acids (SCFAs) are well known metabolic end products of anaerobic bacteria that have been regarded as potential virulence factors [150, 175]. Provenzano et al. [172] evaluated the occurrence of SCFAs in primarily infected root canals before and after treatment and revealed that butyric and propionic acids were the most frequently found. Both SCFAs were also found after chemomechanical procedures. Lactic acid was not present in detectable levels before treatment, but was very frequent after calcium hydroxide medication.

Polyamines are produced by bacteria fol- lowing enzymatic decarboxylation of amino acids. Examples of polyamines include putrescine, spermidine, spermine, and cadaverine. Polyamines can dysregulate apoptosis of PMNs and lead to premature cell death [122]. A study found greater amounts of polyamines in root canals of teeth with spontaneous pain and swelling when compared with asymptomatic teeth [121].

Community profiling studies have revealed a high inter-individual variability in the bacterial diversity of endodontic infections [225, 238], with innumerable bacterial combinations leading to the same disease outh. In these cases, despite the differences in species composition between individuals, the bacterial communities are likely to exhibit a similar physiologic and pathogenic behavior. Disease-associated biofilm communities have been shown to exhibit conserved metabolic gene expression profiles, despite the high subject-to-subject variability in species composition [97]. Thus, even though com- munities associated with a given disease vary in their species composition, they may behave similarly towards host tissues. This suggests that there is some redundancy in terms of bacterial physiology and function in disease-associated communities, in which the overall diversity of bacterial products released is lower than the species diversity. In this context of functional redundancy, a given product that is essential to the physiology or pathogenicity of a community may be provided by different species in different individuals. This helps explain the high variability in community species composition from different individuals with the same disease (e.g., asymptomatic apical periodontitis, acute apical abscess.

Metaproteomics technologies have been used for large-scale characterization of the entire protein complement of microbial communities at a given point in time [327]. Therefore, the products of gene expression (proteins) are identified directly in samples. Some studies have used metaproteomics to evaluate endodontic infections. Nandakumar et al. [149] identified bacterial proteins in cases of primary or persistent infections and found proteases, virulence factors, autolysins, and proteins involved with adhesion, conjugation, and antibiotic resistance. Provenzano et al. [171] evaluated the metaproteome of primary infections associated with acute apical abscesses and asymptomatic apical periodontitis and reported an overall greater number of proteins in the former. The large majority of microbial proteins were related to metabolic and housekeeping processes, including protein synthesis, energy metabolism and DNA processes, indicating the occurrence of viable and active cells. Several proteins related to pathogenicity and resistance/survival were identified, including proteins involved with adhesion, biofilm formation and antibiotic resistance, stress proteins, exotoxins, invasins, proteases and endopeptidases (mostly in abscesses), and an archaeal protein linked to methane production.

In another study, Provenzano et al. [173] evaluated the bacterial metaproteome in root apices and their associated inflammatory lesions from teeth with post-treatment apical periodontitis. Proteins from viable and metabolically active bacterial cells were detected both in the apices and in the associated lesions. Several bacterial proteins related to pathogenicity and resistance/survival were identified in both apices and lesions, including proteins involved with antibiotic resist- ance, proteolytic function, stress-response, adhesion, and virulence.

4.5 Microbial Ecology and the Root Canal Ecosystem

A root canal containing necrotic pulp tissue provides a space for colonization and affords bacteria a moist, warm, nutritious and anaer- obic environment, which is by and large protected from the host defenses because of lack of active blood circulation in the necrotic tissue. Also, the root canal walls are non- shedding surfaces, being conducive to persis- tent colonization and formation of complex sessile biofilm communities.

Intuitively, the root canal system can be considered a rather lush environment for bacterial growth. Consequently, one might assume that colonization should be easy for virtually all oral bacterial species. Over 700 different bacterial species have been reported to occur in the oral cavity and about 100 to 300 species can make up one individual's oral microbiota [37, 106]. Theoretically, these species share similar opportunities to invade and colonize root canals. Nonetheless, the relative dominance of 10 to 30 species found in primarily infected root canals argues otherwise. Even without the significant presence of host defense factors, the necrotic root canal provides a rather selective environment for bacteria to adapt and colonize. Environmental pressures must occur in the root canal that favor the establishment of some species and inhibit others.

4.5.1 Ecological Determinants

The ecological factors that influence the establishment of a colonizing microbiota in most environments include: oxygen tension and redox potential (Eh); available nutrients; microbial interactions; host defense factors; temperature; pH; and receptors for bacterial adhesions.

4.5.1.1 Oxygen Tension and Redox Potential

The root canal infection is a dynamic process and different bacterial species dominate the community at different phases of the infec- tious process. In the earliest stages of root canal infection, the number of bacterial species and cells colonizing the root canal system are low. Pulp infection is frequently a sequel to caries. The bacterial species in the front-line of the caries biofilm are the first ones to reach the pulp. Except for lactobacilli, most species frequently detected in advanced caries lesions associated with pulp exposure and irreversible pulpitis are also found in endodontic infections [209, 210] (Figure 4.9). Early colonizers or pioneer species set the stage for further colonization by other species.

Shifts in the composition of the microbiota are observed over time. They are largely due to changes in environmental conditions, including oxygen tension and redox potential. A study in monkeys revealed that facultative bacteria predominate in the very initial phases of the pulp infectious process [49]. After a few days or weeks, oxygen is depleted within the root canal as a result of pulp necrosis and consumption by facultative bacteria. Further oxygen supply is interrupted because of loss of blood circulation in the necrotic pulp tissue. An anaerobic milieu with consequent low redox potential develops, which is highly conducive to the survival and growth of obligate anaerobic bacteria. With the passage of time, anaerobic conditions become even more pronounced, particularly in the apical segment of the root canal. Several anaerobic bacterial species have been identified in the apical segment of the root canals of teeth with primary apical periodontitis [12, 207].

Figure 4.9 Average relative abundance of bacterial genera in deep dentinal caries lesions in teeth with symptomatic irreversible pulpitis. Data according to Rôças et al. [210].

4.5.1.2 Available Nutrients

The selective physical environment of the root canal system is characterized by an apparent excellent access to nutrients, which however may be limited depending on the spatial location of a given bacterial population and the stage of infection. The large majority of nutrients available for endodontic bacteria are derived from the host. However, certain essential nutrients to some species are not provided by the host and must be delivered by other species in the infected site (discussed below in “Microbial Interactions”). Because the root canal system may not be rich in nutrients, there will be competition for the amounts available. Oral bacterial species have different nutritional demands and competence in acquiring or scavenging for nutrients. As a consequence, bacterial species that can best utilize and compete for nutrients in the root canal system will best succeed in colonization.

In the root canal system, bacteria can utilize the following nutrient sources: 1) necrotic pulp tissue, containing remnants of dead pulp cells and other degenerated constituents of the pulp connective tissue; 2) components (usually proteins and glycoproteins) of tissue fluids and exudate that seep into the root canal system via apical and lateral foramina; 3) saliva that may coronally penetrate into the canal; and 4) products of the metabolism of other bacterial species (discussed below). Because the largest amount of nutrients is available in the main canal lumen, which is the most voluminous part of the root canal system, most of the infecting microbiota, particularly fastidious anaerobic species, is expected to be located in this region.

The dynamics of nutrient utilization by microbial species can also induce shifts in the infecting microbiota of the root canal system, with saccharolytic species dominating the very early stages of the infectious process but being soon outnumbered by asaccharolytic species, which will dominate later stages. For better understanding, an analogy can be made based on studies of nutrient uti- lization in serum [295]. In the initial phases of the infectious process, the low amount of carbohydrates available in serum is rapidly consumed by saccharolytic bacteria. In an intermediary phase, carbohydrates are split off from glycoproteins and their content is completely consumed. Proteins are hydrolyzed and some amino acid fermentation takes place. This indicates a shift from a saccharolytic community to a proteolytic one. Species from the genera Prevotella, Porphyromonas, and Fusobacterium predominate in this phase. In a final phase, progressive protein degradation and extensive amino acid fermentation is observed, with Parvimonas, Prevotella, Porphyromonas, and Fusobacterium nucleatum prevailing over other species [295].

The necrotic pulp tissue represents a finite source of substrate for bacterial growth, given the small volume of tissue occupying the root canal system. However, development of periradicular inflammation ensures a sustainable source of nutrients in the form of proteins, glycoproteins, degraded collagen and iron-containing compounds carried into the canal by seepage of the inflammatory exudate via apical and lateral foramina. At this stage, bacteria that have a proteolytic capacity or establish a cooperative interaction with those that can utilize this substrate in the metabolism start dominating the com- munity. As the infectious process reaches the stage of induction of periradicular inflammation, proteins become the principal source of nitrogen and carbon, particularly in the apical portion of the canal, favoring the establishment of anaerobic species that utilize peptides and/or amino acids in their metabolism. Thus, the root canal environment in untreated necrotic teeth affords bacteria a shifting pattern of nutrient availability and type over time, which will impact on the composition of the microbiota.

4.5.1.3 Bacterial Interactions

The structure of the endodontic microbiota can also be shaped by ecological relation-ships between the species that invade the root canal system. Because endodontic infections are characterized by multispecies biofilm communities, different bacterial species are in close proximity with one another and a multitude of interactions become inevitable. These interactions can be positive or negative.

Positive interactions enhance the survival capacity ofthe interacting species. Sometimes different species coexist in habitats where neither could exist alone. Positive bacterial interactions in multispecies communities include interbacterial nutritional interactions (food chains/webs, and concerted action to break down complex substrates); local environmental modification; collective protection against external threats; cell-cell signaling (quorum-sensing systems); and horizontal gene transfer.

Interbacterial nutritional interactions are mainly represented by food chains/webs that include utilization of metabolic end-prod- ucts from one species by another in a relationship of mutualism or commensalism (Figure 4.10). Mutualism between two species occurs when both benefit from the relationship, as for instance the bi-directional use of metabolites. Commensalism is a uni- directional relationship between bacteria, in which one species benefits and the other is unaffected.

Figure 4.10 Nutritional relationships that may occur between bacterial species in an endodontic multispecies community.

Some species can modify the environment and thereby provide growth conditions favorable to other species. For instance, by reducing the oxygen tension in the environment, pioneer facultative bacteria can favor the establishment of anaerobes. Also, by releasing proteases or antibiotic-inactivating enzymes in the environment, some species can provide protection to the entire community from host defenses (e.g., antibodies and complement) and antibiotics, respectively.

Negative interactions act as feedback mechanisms that limit population densities. Examples that may occur in the endodontic milieu include competition and amensalism. Competition occurs when two species are striving for the same resource and focuses on available nutrients and space for colonization. Amensalism (antagonism) occurs when one species produces a substance (bacteri- ocin or metabolic end-product) that inhibits another species. Pioneer species colonizing a habitat may inhibit the establishment of competitive latecomers. The simplest way to avoid inhibition factors released by one bacterial species is to find sites that are not colonized by antagonistic species. Another method is to counterattack the antagonistic species by producing inhibitory or killing factors against it.

4.5.1.4 Other Ecological Determinants

Host defense factors. In the initial process of pulp infection, invading bacteria face the attack from the host immune defenses in the inflamed pulp. Only those that endure will successfully colonize the canal. When the whole pulp has become necrotic, the root canal microbiota is relatively protected from the host defenses because of the lack of an active circulation. However, in the apical part of the canal, the microbiota may still be influenced by specific host defense components entering the canal with the inflammatory exudate. Only the species capable to overcome these defenses can survive. The ability to form biofilms is one important survival strategy for bacteria in this regard.

Temperature. The temperature levels within the root canal likely range from 35-38°C, which is conducive to colonization by virtually all oral and many environmental bacteria.

pH. The pH in the necrotic pulp ranges from 6.4 to 7.0 [299], but it can rise slightly as a result of the metabolism of proteins by some bacterial species. Some putative endodontic pathogens grow better at slightly alkaline pH values and present marked alterations in the enzyme profile under such conditions. For instance, trypsin-like activity in Porphyromonas gingivalis is maximal at pH 8.0 [128]. Bacteria vary in regard to their pH tolerance, with most species best growing within a range of 6 to 9 pH [7]. Fungi generally exhibit a slightly wider pH range, growing within a range of 5 to 9 pH [7].

Receptors for bacterial adhesins. Adhesins are bacterial molecules involved with specific host tissue recognition and adhesion as well as cell-cell specific binding between bacte- ria. They are usually chemical components of fimbriae, pili, cell walls, and capsules and play an important role in the strong adhesion of bacteria to the surfaces for initial biofilm formation. Cell-cell binding also favors the attachment of latecomers with the consequent establishment of complex multispecies bacterial communities. Because adhesins bind to specific complementary receptors on host surfaces, the types of host molecules expressed influence the species that will adhere and colonize the surface. While this factor is an important determinant of the bacterial species that colonize the tooth surfaces for dental plaque formation, there is only scarce information on the types of receptors for bacterial adhesins involved with intracanal biofilm formation. Plasminogen, a major plasma protein that can coat the dentinal walls following tissue fluid seepage in the canal, and collagen type I from predentin and dentin may serve as receptors for bacterial adhesion in necrotic canals [107, 117, 139]. In cases where saliva leaks into the root canal, a conditioning film composed of salivary proteins can form on the canal walls and favor bacterial adhesion and biofilm formation [65].

4.5.2 Ecology of the Endodontic Biofilm Community

As noted earlier, a dynamic bacterial biofilm community establishes itself in the necrotic root canal system and is shaped over time by gradual species succession [49, 294]. The pioneer species influence the pattern of bacterial succession within the root canal. With the passage of time, the number of species gradually changes and increases and the community becomes more complex. Community members may be joined or replaced by other species.

The spatial organization of the bacterial populations within the endodontic biofilm is unlikely to be at random. Species that are metabolic interdependent will likely be located in close proximity to each other.

In the late stages of the pulp infectious process, certain groups of anaerobic bacteria start dominating the microbiota. Eventually, a stable situation and a high level of community organization may be reached, with pop- ulations co-existing in harmony and balance with their environment. This means that the community reach a climax in its maturity and organization [124, 284]. A multitude of niches (metabolic functions) may take place in the biofilm consortium. Consequently, physiologically different species can coexist indefinitely provided they are functionally compatible.

Environmental conditions within the root canal system are not uniform and differences will influence the composition of the infecting community and the establishment of different ecological niches. Organization of populations in the endodontic community are conceivably dictated by the ecological determinants occurring in different parts of the root canal system. Environmental conditions may vary along the entire extent of the root canal system, with differences being more pronounced at the two ends (coronal and apical). Oxygen and nutrient gradients arguably set up in root canals exposed to the oral environment [285]. In the coronal region of the canal, given the proximity with the oral cavity, the oxygen tension is higher than in other areas of the canal. As a consequence, facultatives and aerotolerant anaerobes are expected to prevail in this area, whereas the anaerobic conditions in the apical segment are highly conducive to the establishment of a microbiota dominated almost exclusively by obligately anaerobic bacteria [49]. Similarly, bacteria located in the most coronal aspects of the canal can utilize carbohydrates from the host diet and saliva, which can seep into the canal via coronal exposure. Bacteria in the most apical area of the root canal system utilize protein- and glycoprotein-rich tissue fluids and exudate which penetrate in the canals via apical and lateral foramina. Therefore, the dominance of different bacterial groups along the biofilm structure occurring in different parts of the root canal system can be predicted based mostly on oxygen tolerance and nutrient requirements.

4.6 Types of Endodontic Infections

Endodontic infections can be classified according to their anatomical location (intraradicular or extraradicular infection) and the time participating bacteria gained entry into the root canal (primary, secondary, or persis- tent infection) [245]. The composition of the microbiota may vary depending on the different types of infection and different forms of apical periodontitis.

4.6.1 Intraradicular Infection

As the name implies, this is caused by bacteria colonizing the root canal system. it can be subdivided into three categories.

4.6.1.1 Primary Intraradicular Infection

Primary intraradicular infection is caused by bacteria that invaded and colonized the necrotic pulp tissue. It has also been referred to as initial infection and is the cause of pri- mary apical periodontitis. Primary infections are characterized by a mixed consortium composed of 10 to 30 bacterial species per canal [140, 180, 196, 267, 272, 277]. The number of bacterial cells in an individual infected canal may vary from 10³ to 10⁸ [18, 222, 250, 291, 311]. Teeth with sinus tracts and/or large apical periodontitis lesions have been shown to harbor a microbiota that is more complex in terms of number of species (richness) and cells (density) [196, 251, 291]. The microbiota of primary infections is conspicuously dominated by anaerobic bacteria, particularly Gram-negative species belonging to the genera Fusobacterium, Treponema, Tannerella, Dialister, Porphyromonas, Prevotella, and Campylobacter. Gram-positive anaerobes from the genera Parvimonas, Filifactor, Actinomyces, Olsenella, and Pseudoramibacter, as well as facultative or microaerophilic streptococci are also commonly found in primary intraradicular infections.

4.6.1.2 Secondary Intraradicular Infection

Secondary intraradicular infections are caused by microorganisms that were not present in the primary infection, but were introduced in the root canal system at some time during or after professional intervention. In any circumstance, if penetrating microorganisms succeed in surviving and colonizing the root canal, a secondary infection is established.

The main sources of secondary infections during treatment include: remnants of dental plaque, calculus or caries on the tooth crown; leaking rubber dam; contamination of endodontic instruments; and contamination of irrigant solutions.

Microorganisms can also enter the root canal system between appointments. This may occur following leakage along the temporary restorative material; breakdown, fracture or loss of the temporary restoration; fracture of tooth structure; and exposure to the oral environment in teeth left open for drainage.

Finally, microorganisms can penetrate the root canal system after placement of the root canal filling in the following situations: leak- age through the temporary or permanent restorative material; breakdown, fracture, or loss of the temporary/permanent restoration; fracture of the tooth structure; recurrent decay exposing the root canal filling material; or loss of seal provided by the temporary material as a consequence of delay in placement of the permanent restoration.

The species causing secondary infections will depend on the source of contamination. Nonoral species may be found, including Pseudomonas aeruginosa, Staphylococcus species, enteric rods, Candida species, and Enterococcus faecalis [77, 176, 177, 262, 279, 318]. If the cause of secondary infection is coronal leakage or any other condition that exposes the root canal to saliva, then the species involved will come from the subject's own oral cavity.

4.6.1.3 Persistent Intraradicular Infection

Persistent intraradicular infections are caused by bacteria that in some way resisted intracanal antimicrobial procedures and endured periods of nutrient deprivation in an infected canal during treatment. The cultivable micro- biota associated with persistent infections is usually composed of fewer species than pri- mary infections, and Gram-positive facultative bacteria are the most prevalent [135, 169, 266, 271, 272, 292]. Fungi can be found in teeth with post-treatment disease in frequencies significantly higher when compared with primary infections [278].

Both persistent and secondary infections are for the most part clinically indistinguishable and can be responsible for several clinical problems, including persistent exudation, persistent symptoms, inter-appointment exacerbations, and post-treatment apical periodontitis (Figure 4.11).

4.6.2 Extraradicular Infection

Extraradicular infection is characterized by bacterial invasion of the inflamed periradicular tissues. While it is almost invariably a sequel to the intraradicular infection, an established extraradicular infection may be dependent on or independent of the intraradicular infection. The most common form of extraradicular infection, which is dependent on the intradicular infection, is the acute apical abscess. It has been suggested that apical actinomycosis, caused by Actinomyces species or Propionibacterium propionicum, may be a form of extraradicular infection independent of the intraradicular infection [145]. This condition accounts for about 2-4% of the apical periodontitis lesions, but its independent nature remains unproven [257]. The question as to whether the extraradicular infection is dependent on or independent of the intraradicular infection assumes special relevance from a therapeutic standpoint, since the former can be successfully managed by root canal therapy while the latter may have to be handled by periradicular surgery.

Figure 4.11 Tooth with post-treatment apical periodontitis. Persistent or secondary intraradicular infections are the main causative agents of this form of the disease.

4.7 Identification of Endodontic Bacteria

Although the final pathogenicity is strongly determined by the biofilm as a community, identification of the species associated with a given infectious disease is still necessary for the understanding of the etiology and pathogenesis of the disease as well as for the development of better strategies to treat and prevent the disease. Microbiological studies for identification of the species participating in endodontic infections can be chronologically divided into five generations on the basis of the different strategic approaches used and their contribution to knowledge [272, 275]:

• First generation: This is represented by studies of the endodontic microbiota using open-ended culture methods to detect virtually all the cultivable species present in the root canal. The great contribution of this generation relates to the disclosure of many cultivable species strongly associated with apical periodontitis, including Fusobacterium nucleatum, Prevotella species, Porphyromonas species, Parvimonas micra, and streptococci.

• Second generation: This encompasses studies that employ closed-ended (speciesor group-specific) DNA-based molecular microbiology methods, such as polymerase chain reaction (PCR) and the conventional checkerboard hybridization approach, to target cultivable bacterial species. Results from these studies generally showed higher prevalences for many cultivable bacterial species and contributed to strengthen their association with apical periodontitis. In addition, some difficult-to-grow species from the genera Tannerella, Dialister, Filifactor, and Treponema were included in the set of candidate endodontic pathogens.

• Third generation: This involves open-ended DNA-based molecular studies, using broad-range PCR followed by cloning and Sanger sequencing, terminal-restriction fragment length polymorphism (T-RFLP) or denaturing gradient gel electrophoresis (DGGE), for detecting cultivable species and as-yet-uncultivated phylotypes in endodontic infections. These molecular methods are generally laborious, time-con- suming, and expensive, resulting in the analysis of only a few samples per study. However, they have permitted cataloging the cultivable and as-yet-uncultivated/ uncharacterized portions of the endodontic microbiota, refining the knowledge of bacterial diversity associated with apical periodontitis.

• Fourth generation: These studies use closed-ended molecular methods, such as speciesor group-specific PCR and the reverse-capture checkerboard assay, for large-scale investigations of the prevalence and levels of cultivable and as-yet-uncultivated bacteria in endodontic infections. Findings from this generation confirmed the association of several cultivable bacteria with apical periodontitis and included some as-yet-uncultivated phylotypes in the set of candidate endodontic pathogens.

• Fifth generation: High-throughput (formerly “next-generation”) sequencing (HTS) approaches have been introduced which have permitted DNA sequencing from samples to a far deeper coverage and higher throughput in comparison with the traditional Sanger sequencing approach [88, 315]. Consequently, bacterial diversity in samples have been explored to reveal even low-abundance components of the community. These methods, especially the pyrosequencing and Illumina approaches, have been used for open-ended analysis of endodontic samples, and substantially expanded the knowledge of the bacterial diversity associated with apical periodontitis.

4.7.1 Taxonomy of Endodontic Infections

Current evidence reveals that over 500 different bacterial species have been identified in endodontic infections; these species fall into 9 of the 13 phyla that have oral repre- sentatives, namely Bacteroidetes, Firmicutes, Spirochaetes, Fusobacteria, Actinobacteria, Proteobacteria, Synergistetes, “Candidatus Saccharibacteria” (formerly TM7), and SR1 [272] (Figure 4.12). However, there may be representatives of at least 10 other phyla in endodontic infections as revealed by HTS methods [90, 114, 156, 225, 255, 259, 301, 308, 339]. Named species that have been regarded as candidate endodontic pathogens include Gram-negative bacteria from the genera Fusobacterium, Porphyromonas, Prevotella, Dialister, Treponema, Tannerella, Pyramidobacter and Campylobacter, and Gram-positive bacteria from the genera Parvimonas, Pseudoramibacter, Streptococcus, Enterococcus, Olsenella, Filifactor, Actinomyces, and Propionibacterium [12, 64, 68, 104, 140, 180, 196, 221, 224, 268, 272, 291] (Table 4.2).

Figure 4.12 Bacterial phyla and their main representatives in endodontic infections.

4.7.2 As-yet-Uncultivated Phylotypes

Phylotype is a term used for those as-yet-uncultivated species that are known only by a 16S rRNA gene sequence identified by molec- ular microbiology methods from the third, fourth and fifth generations. Third-generation studies of the endodontic bacterial diversity have shown that 40% to 60% of the species-level taxa (richness) still remain to be cultivated and validly named [140, 180, 221, 222, 313]. As-yet-uncultivated phylotypes may represent about 30-40% of the endodontic bacterial community in terms of relative abundance [221]. Uncultivated phylotypes detected in endodontic infections have been classified into several known genera, including Dialister, Treponema, Prevotella, Solobacterium, Olsenella, Fusobacterium, Megasphaera, Veillonella, and Selenomonas [140, 195, 212, 219, 221, 224, 248, 268]. One of the most prevalent as-yet-uncultivated phylotypes encountered in endodontic infections in fourth-generation studies is Bacteroidaceae sp. HOT-272 (synonym, Bacteroidetes oral clone X083) [196, 197]. Several members of the Synergistetes phylum have been identified in infected root canals; most of them still remain uncultivated [195, 208, 268, 270]. Some, however, have been recently cultivated using special strategies, which permitted them to be characterized, and formally named as Pyramidobacter piscolens [41] and Fretibacterium fastidiosum [307].

Table 4.2 Bacterial genera and respective common representative species occurring in endodontic infections.

Genus	Common representatives	Primary infections (asymptomatic apical periodontitis)	Primary infections (acute apical abscess)	Persistent/ secondary infections (post-treatment apical periodontitis)
Gram-negative Anaerobic rods
Dialister	D. invisus, D. pneumosintes, uncultivated phylotypesa	+ + +	+ + +	+
Porphyromonas	P. endodontalis, P. gingivalis	+ + +	+ + +	+
Tannerella	T. forsythia	+ + +	+ + +	+
Prevotella	P. intermedia, P. nigrescens, P multissacharivorax, P. baroniae, P. denticola, uncultivated phylotypesa	+ + +	+ + +	+
Alloprevotella	A. tannerae	+ +	+ +	-
Fusobacterium	F. nucleatum, uncultivated phylotypesa	+ + +	+ + +	+
Campylobacter	C. rectus, C. gracilis, C. showae	+ +	+	+
Fretibacterium	F. fastidiosum, uncultivated phylotypesa	+ +	+ +	-
Pyramidobacter	P. piscolens	+ +	+ +	+
Selenomonas	S. sputigena, S. noxia, uncultivated phylotypesa	+ +	+	-
Anaerobic cocci
Veillonella	V. parvula, uncultivated phylotypesa	+ +	+	-
Megasphaera	uncultivated phylotypesa	+	+	-
Anaerobic spirilla
Treponema	T. denticola, T. socranskii, T. parvum, T. maltophilum, T. lecithinolyticum	+ + +	+ + +
Facultative rods
Capnocytophaga	C. gingivalis, C. ochracea	+	-	-
Eikenella	E. corrodens	+ +	+ +	-
Gram-positive Anaerobic rods
Actinomyces	A. israelii, A. gerencseriae, A. meyeri, A. odontolyticus	+ +	+	+ +
Pseudoramibacter	P. alactolyticus	+ + +	+	+ +
Filifactor	F. alocis	+ +	+	+
Peptostreptococcaceae (Eubacterium)	P. infirmum, P. saphenum, P. nodatum, P. brachy, P. sulci	+	+	+

Table 4.2 (Continued)

Genus	Common representatives	Primary infections (asymptomatic apical periodontitis)	Primary infections (acute apical abscess)	Persistent/ secondary infections (post-treatment apical periodontitis)
Mogibacterium	M. timidum, M. pumilum, M. neglectum, M. vescum	+	+	+
Propionibacterium	P. acnes, P. propionicum	++	+	++
Eggerthella	E. lenta	+	+	-
Olsenella	O. uli, O. profusa	++	+	+
Atopobium	A. parvulum, A. minutum, A. rimae	+	-	+
Solobacterium	S. moorei, uncultivated phylotypesc	+	-	+
Anaerobic cocci
Parvimonas	P. micra	+++	+++	++
Peptostreptococcus	P. stomatis, P. anaerobius, uncultivated phylotypesa	+	+	-
Anaerococcus	A. prevotii	+	-	-
Streptococcus	S. anginosus, S. constellatus S. intermedius	+++	+++	+++
Gemella	G. morbillorum	+	+	+
Facultative rods
Actinomyces	A. naeslundii	+	-	-
Facultative cocci
Streptococcus	S. mitis, S. sanguinis, S. gordonii, S. oralis	++	+	+++
Enterococcus	E. faecalis	+	-	+++
Granulicatella	G. adiacens	+	-	-

^a for uncultivated phylotypes, Gram-staining patterns, cell morphology, and relationship to oxygen are estimates based on the general features of the genus.

+++, very frequently found in many studies ++, frequently found in many studies +, found in many studies -, rarely found, if ever

4.8 Endodontic Biofilm Community Profiles

Bacterial community profiles are determined by species richness and abundance. DNA- based molecular methods have been used to profile the biofilm communities associated with different types of endodontic infections and manifestations of apical periodontitis.

These studies have demonstrated that the endodontic bacterial communities associated with primary [26, 119, 277] and post-treatment apical periodontitis [18, 26, 114, 194, 205, 223, 266] are mixed, composed of several different species, including as-yet-uncultivated bacteria [118, 248]. Different profiles have been reported for endodontic bacterial communities associated with different clinical conditions, including asymptomatic apical periodontitis, acute apical abscesses, and post-treatment apical periodontitis (see below) [205, 221, 277, 301].

No two endodontic infecting communities are the same in terms of richness and abundance, resulting in a high inter-individual variability [26, 114, 151, 225, 277]. Even two infected teeth in the same individual show different profiles [2]. This indicates that apical periodontitis has a heterogeneous etiology, and multiple species combinations can lead to similar disease outcomes. In spite of the inter-individual variability, samples from individuals living in the same geographical location are more similar among them when compared to individuals living in distant locations [64, 119, 205, 252, 277]. This geography-related pattern in community profiles raises questions about the effectiveness of specific antimicrobial treatment (antibiotics) protocols for worldwide use.

4.9 Microbiota in the Apical Root Canal

The apical segment of the root canal system is a critical territory for the host, the infecting microbiota, and the clinician. Because the apical foramina are the main portals of exit of bacteria and their virulence factors, the host defenses must be mounted and concentrated near those areas in order to prevent infection from gaining access to the bone and disseminating to other body areas. In the apical canal, bacteria are located in a strategic position to obtain nutrients from and cause damage to the periradicular tissues. The clinician in turn must control the infection especially in this area and create conditions to prevent reinfection in order to succeed with root canal treatment.

The apical canal offers different ecological conditions in terms of oxygen tension and type of nutrients available, favoring the establishment of a microbiota that signifi-cantly differs from the one occurring in the most coronal parts of the canal system [2, 12, 49, 131, 296]. Bacterial counts in the apical canal may range from 10⁴ to 10⁶ cells [12].

The bacterial species identified in this region are highly likely to be the most important ones involved in the etiology of apical periodontitis, because they are in close con- tact with the affected tissues. While most studies have evaluated the microbiota of the entire root canal system, a few studies have looked at the bacterial species occurring exclusively in the apical root canal. Such studies can only be performed in extracted teeth or in teeth subjected to root-end resection. Several candidate endodontic pathogens have been identified in the apical canal segment, including Prevotella species, Porphyromonas species, Pseudoramibacter alactolyticus, Streptococcus species, Olsenella uli, F. nucleatum, P micra, Tannerella forsythia, and Treponema species [12, 40, 207, 247, 253].

A molecular study found a mean number of 28 species in both apical and middle/coronal samples; however, the mean shared species was only 54%, ranging from values as low as 2% to 79% [2]. Therefore, while harboring similar number of species, the composition of the bacterial community in the apical canal was different from the coronal parts. There is also a high variability between subjects in terms of apical microbiota composition [2, 259].

Some species are more frequently found in the apical portion of the canal than in its matched coronal regions, including Prevotella baroniae, T. forsythia, and F. nucleatum, while others, such as Streptococcus species, are more prevalent in middle/coronal samples [207]. The complexity of the apical microbiota has been confirmed by studies using HTS methods [156, 259].

Bacteria in the apical canal of treated teeth with persistent disease are highly likely to be the cause of treatment failure. Molecular analysis of the apical root canal system of adequately treated teeth with persistent apical periodontitis have disclosed highly complex bacterial communities [255] (Figure 4.13). The community composition varied signifi- cantly from individual to individual. The mean bacterial load in cryopulverized apical canal samples of adequately treated teeth with apical periodontitis was about 10⁴ cells [5].

Figure 4.13 Average relative abundance of bacterial genera in root apex samples from teeth with post-treatment apical periodontitis. Data according to Siqueira et al. [255].

4.10 Symptomatic Infections

4.10.1 Factors Influencing the Development of Symptoms

In some cases, bacterial infection of the root canal can give rise to acute forms of apical periodontitis, including symptomatic apical periodontitis and acute apical abscess (Figure 4.14). Endodontic microbiology studies have long looked for association between specific bacterial species and symptomatic disease. Although it has been suggested that some Gram-negative anaerobic bacteria can be associated with symptoms [71, 74, 204, 274, 291, 305, 335], several studies have found similar prevalences of the same species in asymptomatic cases [15, 60, 78, 98, 196, 242, 243].

Figure 4.14 Acute apical abscess with extensive swelling. In cases like this, an extraradicular infection is established.

This suggests that factors other than the mere presence of a given pathogenic species can influence the development of symptoms. Actually, symptomatic manifestations of apical periodontitis are a result of interplay of diverse factors as proposed elsewhere [245, 275]. They are as follows.

4.10.1.1 Difference in Virulence Ability among Clonal Types of the Same Species

Clonal types of a given pathogenic bacterial species can significantly diverge in their virulence ability [8, 56, 75, 143, 155]. A disease ascribed to a pathogen is in fact caused by specific virulent clonal types of that species [143]. Therefore, the presence of virulent clonal types of candidate endodontic pathogens in the root canal may be a predisposing factor for pain. This helps explain why the same species are detected in both symptomatic and asymptomatic cases.

4.10.1.2 Cell Numbers or Infectious Load

Other important factors contributing to the development of symptomatic infections are the overall and specific bacterial loads. Overall bacterial load refers to the total cell numbers in the community, whereas the specific load relates to the counts and relative abundance of certain pathogenic species. The total number of bacterial cells in the community results in a heavy infectious bioburden for the host to cope with, and is characterized by a massive accumulation of virulence factors. Total bacterial counts in acute apical abscesses can vary from 10⁴ to 10⁹ cells per sample [104, 112, 325]. The levels of some specific virulent species are also of importance for the community pathogenicity. The possibility exists that the number of cells of a given species reported to occur in both symptomatic and asymptomatic infections is larger in the former. A molecular study detected T. forsythia in significantly higher density in symptomatic than in asymptomatic endodontic infections [227]. Another molecular study revealed that while the mere presence of the target species/phylotypes was not associated with symptoms (abscessed teeth), the counts of some taxa (Porphyromonas endodontalis, Prevotella baroniae, Treponema denticola, and Streptococcus species) were significantly higher in abscesses than in asymptomatic cases [201] Thus, presence of a potentially virulent pathogen in high counts may increase the virulence of the whole community and give rise to symptoms.

4.10.1.3 Bacterial Interactions and Collective Pathogenicity

Most endodontic pathogens are only capable to cause disease when in association with other species [13, 55, 101, 241, 293, 333]. This is because of synergism occurring among different species in a community. Interactions may influence virulence and play a role in symptom causation. Studies comparing symptomatic and asymptomatic endodontic infections showed different dominant species in the communities, with a significantly higher species richness in symptomatic teeth [220, 221, 225, 277, 337]. The increased diversity in teeth with symptomatic infections is expected to influence the collective pathogenicity of the biofilm, resulting in incalculable synergistic interactions among the community members. Every component of a polymicrobial infection, even species regarded as avirulent and/ or in low numbers in the community, have been suggested to affect virulence of the whole biofilm community [42, 43, 164, 236]. Communication between members of a bacterial community can change the expression of virulence factors by certain pathogenic species and affect the collective pathogenicity [42]. Some pairs of species have been found in association with endodontic symptoms [70, 174]. Actually, some endodontic pathogens form different part- nerships and associations in symptomatic infections in comparison with asymptomatic ones [202], leading to communities that can be more virulent and capable of causing symptoms.

4.10.1.4 Environmental Cues for Expression of Virulence Factors

A pathogenic species does not always express its virulence factors throughout its lifetime. The environment plays an important role in inducing the turning on or the turning off of virulence genes [10, 57, 105, 329]. Bacteria can sense environmental changes and respond accordingly by genetic expression of products that favor adaptation and survival. Several endodontic pathogens, such as trep- onemes, F. nucleatum, P gingivalis, and Prevotella intermedia, have been shown to exhibit gene expression and virulence influenced by environmental cues [61, 102, 103, 336, 343]. The possibility exists that symptoms may also result from root canal environmental conditions that are conducive to the expression of bacterial virulence genes.

4.10.1.5 Host Resistance and Disease Modifiers

Individuals significantly differ in their ability to cope with infections, and differences may even become evident during each individual’s lifetime [132]. Those individuals with reduced resistance to infections may be more prone to develop clinical symptoms. In addition, disease modifiers, such as genetic poly- morphisms and diabetes, may influence the severity of the disease and predispose to symptoms [4, 36, 59].

4.10.1.6 Concomitant Herpesvirus Infection

Herpesvirus infection may be a disease modifier and is usually associated with diminished host resistance. Human cyto- megalovirus (HCMV) and/or Epstein-Barr virus (EBV) have been detected in specimens of apical periodontitis lesions in association with symptoms [215, 217]. Other herpesvi- ruses, such as human herpesvirus (HHV)-8, varicella zoster virus (VZV) and HHV-6 have also been identified in acute apical abscess samples [52, 53]. However, it still remains to be determined whether herpesvirus partici-pate in the pathogenesis of acute infections or they are only bystanders attracted to the area as a consequence or the bacterially-induced inflammation (see further discussion on “Other microorganisms in endodontic infections”).

4.10.2 Shift in the Microbiota Before Symptom Appearance

Symptomatic endodontic infections, including the acute apical abscess, may develop in teeth with or without periradicular radiolucency. In the latter, the initial infection of the canal is assumed to reach significant virulence to cause a rapidly evolving acute periradicular inflammation, before bone resorption develops. This is because bone resorption is usually a slow chronic process associated with long-standing root canal infection. However, when an abscess develops in a tooth with radiographically visible periradicular lesion, this indicates that it resulted from exacerbation of a previously existing chronic process. In these cases, a change in the host-pathogen relationship predisposed to exacerbation. It may be a temporary or definitive decrease in the host immune resistance, as for instance caused by stress or viral infection (e.g., flu, cold, herpesvirus); however, it may also be precipitated by a shift in the structure of the endodontic microbiota.

Cross-sectional studies have shown that the structure (richness and relative abundance) of the endodontic bacterial communities in symptomatic teeth is significantly different from that of asymptomatic teeth [225, 277]. Differences are evident in the type of dominant species, the total number of species (richness) and the bacterial load. This may indicate that ecological rearrangements in the bacterial community structure precede the appearance of symptoms. The ecological succession from asymptomatic to symptomatic condition may be related to the emergence of newly dominant community members, caused either by environmental changes that predispose to the growth of some specific members or even by arrival of novel species. Differences in the type and load of dominant species and the resulting bacterial interactions may be responsible for differences in the pathogenicity degree of the whole biofilm community and lead to symptoms.

4.11 Persistent/Secondary Endodontic Infections

Given the essential role played by bacteria in the etiology of apical periodontitis, endodontic treatment should focus on both elimination of bacterial cells colonizing the root canal system and prevention of reinfection. Sterilization is the ideal goal of the treatment, but the realistic goal with techniques and substances currently available is disinfection. Thus, bacteria must be eliminated to levels that are compatible with periradicular tissue healing [271]. If bacteria are allowed to remain in the root canal at the time of filling, there is an increased risk of adverse treatment outcome [50, 281, 317].

Secondary infections due to canal contam- ination during treatment or coronal leakage after treatment can be the cause of post-treatment apical periodontitis, but bacterial persistence (persistent infections) is the most common cause of treatment failure [271]. It is salient to point out that even when antimicrobial endodontic treatment does not completely eradicate the intraradicular infection, substantial levels of bacteria are eliminated and the root canal environment is markedly disturbed. For bacteria to survive they need to resist or escape intracanal disinfection procedures and rapidly adapt to the drastically altered environment.

Most intracanal bacteria are sensitive to standard treatment procedures. Never- theless, some bacteria may endure treatment procedures, especially if they are located in areas not reached by instruments and the antimicrobial substances used. When treatment is performed below acceptable standards, the risks of bacterial persistence are obviously high. However, areas such as isthmi, dentinal tubules, recesses, lateral canals and apical ramifications are difficult to reach with instruments and irrigants, and bacteria estab- lished therein can remain unaffected by treatment [6, 22, 147, 181, 186, 309, 314].

Bacteria persisting in the root canals after treatment procedures do not always maintain an infectious process and cause post-treatment apical periodontitis [50, 281]. The possible reasons for this are shown in Table 4.3. Actually, for residual bacteria to cause persistent infections and influence the treatment outcome they need to fulfil certain requirements depicted in Table 4.4.

Virtually all root canal-treated teeth with post-treatment apical periodontitis have been demonstrated to harbor an intraradicular infection [115, 186, 200, 266]. This indicates that residual bacteria can in some way acquire nutrients within filled root canals. Because no obturation technique or filling material promote a predictable antibacterial and fluid-tight coronal, lateral, and apical seal of the root canal system [76], persisting bacteria can derive nutrients from saliva (coronally seeping into the canal) or from the periradicular inflammatory exudate (apically or laterally seeping into the canal) [256]. Even though most necrotic pulp tissue is removed during chemomechanical procedures, residual bacteria can utilize some necrotic tissue remnants as nutrient source. These tissue remnants may be localized in isthmi, recesses, dentinal tubules, and lateral canals, which very often remain unaffected by instruments and irrigants [35, 240, 320, 346]. In addition, even in the main root canal lumen, some dentinal walls may remain untouched after instrumentation [240, 331], with different instrumentation techniques leaving up to 50% of the root canal surface area untouched [123, 158, 166, 254].

Table 4.3 Situations in which residual bacteria may not influence treatment outcome.

Residual bacteria do not cause post-treatment disease when:

1) they die after placement of the root canal obturation

2) they remain in quantities and virulence that are subcritical to cause or sustain periradicular inflammation

3) they are located in areas with access denied to the periradicular tissues

Table 4.4 Requisites for residual bacteria to influence treatment outcome.

For residual bacteria to maintain or cause disease they need:

1) to withstand periods of nutrient deprivation, scavenging for low traces of nutrients and/or assuming a state of low metabolic activity

2) to find a steady source of nutrients in order to survive and flourish

3) to resist treatment-induced disturbances in the ecology of bacterial community, including disruption of quorum-sensing systems, food webs/chains and genetic exchanges

4) to reach numbers sufficient to cause damage to the host

5) to have unrestrained access to the periradicular tissues through apical/lateral foramina or lateral perforations to cause damage

6) express virulence factors that reach critical concentrations in the modified root canal environment

If residual bacteria are located in the very apical part of the root canal or in ramifications, they have unrestricted access to nutrients in the form of protein- and glycoprotein-rich tissue fluids and exudate. Actually, histobac- teriological analyses of teeth with post-treatment apical periodontitis have frequently shown persistent infections in areas of apical ramifications [6, 146, 182, 186] and lateral canals [181, 186].

Unlike primary infections, a more restricted group of species has been found in root canal-treated teeth associated with post-treatment apical periodontitis.

E. faecalis has been one of the most frequently identified species in samples taken from root canal-treated teeth with apical periodontitis [48, 83, 87, 135, 141, 161, 169, 170, 203, 206, 232, 266, 292, 326, 344] (Figure 4.15). Fourth generation studies using quantitative real-time PCR analysis reported that this species constitute a median of some 1% (range 0.1% to 100%) of the overall bacterial load in treated canals [200, 232].

Figure 4.15 Prevalence of Enterococcus faecalis in association with post-treatment apical periodontitis according to different studies using either culture or molecular methods for identification.

Figure 4.16 Prevalence of Enterococcus faecalis in endodontic infections associated with different forms of apical periodontitis. CAP, chronic apical periodontitis; AAP, acute apical periodontitis; AAA, acute apical abscess; PTAP, post-treatment apical periodontitis. Data according to Rôças et al. [203].

E. faecalis is not commonly encountered in primary infections [17, 48, 203, 244, 342], and root canal-treated teeth are about nine times more likely to harbor E. faecalis than untreated teeth [203] (Figure 4.16).

This species is rarely, if ever, found as a persister in studies evaluating the antimicrobial effects of treatment and the microbiological conditions of the canal at the time of filling [21, 211, 222, 261, 264, 265, 281, 312, 338]. Enterococci have been reported to occur frequently in cases treated in multiple visits and/or in teeth left open for drainage [279]. They have also been found in high prevalence in root canal-treated teeth with leaking coronal restorations [170]. Because enterococci can be food-born colonizers and have been found in a variety of cheeses [63], their occurrence in the oral cavity has been suggested to be related to food ingestion [178, 340].

Based on many cross-sectional studies, as shown in Figure 4.15, E. faecalis was regarded as possibly the main causative agent of post-treatment apical periodontitis. Recently, however, its status has been questioned by the following findings:

1) E. faecalis is not detected in all studies evaluating the microbiota of root canal- treated teeth with post-treatment disease [25, 212].

2) When present, E. faecalis is rarely the most dominant species in the community [87, 194, 205, 223, 255].

3) E. faecalis has been found in similar prevalence values in root canal-treated teeth with or without apical periodontitis lesions [100, 344].

Streptococcus species have also been commonly found in the canals of teeth with post-treatment apical periodontitis [5, 23, 169, 206, 266]. Their prevalence and relative abundance in treated canals can be even higher than E. faecalis, as demonstrated by fourth and fifth generation studies [5, 200, 338, 339]. Other bacteria found in teeth with post-treatment disease include fastidious anaerobic bacterial species, such as P alactolyticus, Propionibacterium species, Filifactor alocis, Dialister species, Fusobacterium species, P micra, P piscolens, and Prevotella species [72, 135, 194, 200, 205, 223, 266, 268, 339].

As-yet-uncultivated bacteria have also been identified in teeth with post-treatment apical periodontitis. In terms of both rich- ness and relative abundance, uncultivated phylotypes may correspond to about one half of the bacterial community [223]. Some uncultivated phylotypes, such as Bacteroidaceae sp. HOT-272 (Bacteroidetes oral clone X083), are one of the most prevalent uncultivated taxa found in treated canals [223]. The fact that as-yet-uncultivated bacteria can be dominant community members helps explain why some culture studies failed to detect bacteria in treated canals.

Like primary infections, the bacterial com- munity profiles in treated teeth vary from individual to individual, indicating that distinct bacterial combinations play a role in post-treatment disease [205, 223, 255]. Infections associated with treatment failure are characterized by a mixed community, which is however less diverse than primary infections.

Secondary infections with Pseudomonas aeruginosa, enteric rods, and staphylococci leading to prolonged endodontic therapy have been reported [77, 177, 262]. These bacteria are most likely secondary invaders that may gain entry into the root canal due to a breach in the aseptic chain during intracanal intervention.

4.12 Extraradicular Infections

Apical periodontitis is an inflammatory disease that develops in response to intraradicular bacterial infection and usually rep- resents an effective barrier against spread of the infection to the alveolar bone and other body sites. However, bacteria may occasionally succeed in overcoming this defense barrier and cause extraradicular infection. The acute apical abscess is the main example of this condition. There are other forms of extraradicular infection which, unlike apical abscesses, may be characterized by mild or even absence of symptoms. Such infections have been suggested as possible causes of post-treatment apical periodontitis, either by forming a biofilm adhering to the outer apical root surface [152, 189, 190, 298] or by mounting cohesive actinomycotic colonies within the body of the inflammatory lesion [85].

The extraradicular infection may have the following possible origins:

1) It may be an extension of the intraradicular infectious process, caused by bacteria that directly invaded the periradicular tissues and overcame the local host defenses. Most oral bacteria found in endodontic infections are opportunistic pathogens and as such lack a virulence apparatus that allows them to invade the periradicular tissues, subvert the host defenses, and survive within a hostile inflamed environment. However, some candidate oral pathogens, such as Treponema species, Porphyromonas endodontalis, P. gingivalis, T. forsythia, Prevotella species, and F. nucleatum, have been shown to possess such virulence traits [19, 51, 89, 95, 306, 341].

Figure 4.17 Large bacterial colony being attacked by phagocytes within the lumen of a pocket (bay) cyst (original magnification 3300x). Reproduced from Siqueira [256].

2) Bacteria may reach the periradicular tissues by penetrating into the lumen of “bay” cysts, which is in direct communication with the apical foramen/ina (Figure 4.17).

3) Bacteria may persist in the extraradicular space following remission of an acute apical abscess. These persisters would then maintain an extraradicular infection associated with chronic inflammation and an actively draining sinus tract - a chronic apical abscess.

4) Apical extrusion of infected debris during chemomechanical preparation is another way by which bacteria may reach the per- iradicular tissues. Once therein, bacteria embedded in dentinal chips can be physically protected from the host defense mech- anisms and persist in the inflamed tissues.

5) Bacterial colonies or biofilms located in the very apical part of the root canal system may assume an extraradicular location following resorption of the apical root segment.

6) Bacteria occurring in intraradicular biofilms may reach the external apical root surface through dentinal tubules in an area where root surface cementum was resorbed, and then form an extraradicular biofilm.

Presumably, the extraradicular infection can be dependent on or independent of the intraradicular infection [257]. The dependent infection is fostered by the intracanal bacterial community. Thus, if the clinician succeeds in controlling the latter, the host can manage to control the extraradicular infectious component. Independent extraradicular infections in turn are those that are not sustained by an intraradicular infection and consequently do not respond to non-surgical endodontic treatment. Apical actinomycosis, caused by Actinomyces species and Propionibacterium propionicum, has been suggested as another form of independent extraradicular infection (Figure 4.18) [85, 280]. However, its occurrence as a self-sustained pathologic entity involved as exclusive cause of treatment failure remains to be proven by studies using modern sensitive methods to simultaneously evaluate the bac- teriological conditions of the apical root canal system.

Extraradicular biofilms have been found to occur in approximately 6% of the teeth with apical periodontitis [183] (Figure 4.19). Sometimes, these biofilms may exhibit foci of calcification, resembling dental calculus [185, 190]. In the large majority of cases, the extradicular biofilm is associated with an intraradicular biofilm, suggesting a dependent infection [183, 288]. However, the extraradicular biofilm may be occasionally independent of the intraradicular infection and cause treatment failure [189]. Extraradicular biofilms are virtually always associated with symptoms in untreated teeth [183] and may be the cause of persistent symptoms [189] or exudation [190] in teeth undergoing root canal treatment.

A histobacteriologic study evaluating the distribution of infection in teeth with chronic apical abscesses and sinus tracts revealed the extraradicular occurrence of bacteria in 83% of the cases. In 71% of the examined teeth, bacteria were forming a biofilm adhering to the outer root surface. Most of these structures showed some mineralization indicative of calculus formation [191].

Figure 4.18 Apical actinomycosis. (a) Bacterial aggregate in an epithelialized apical periodontitis lesion, suggestive of actinomycosis (Masson trichrome, original magnification 25x). (b) Higher magnification of the actinomycotic aggregate, which is surrounded by inflammatory cells (400x). Courtesy of Dr Domenico Ricucci.

Except for cases with sinus tracts, it is still controversial whether asymptomatic, chronic apical periodontitis lesions can harbor bacteria for very long beyond initial tissue invasion or without being maintained by concomitant root canal infection [11, 182]. Studies have reported the extraradicular occurrence of a mixed infection by anaerobic bacteria in post-treatment lesions [62, 84, 214, 237, 288-290, 300, 323, 337]. As with apical actinomycosis and apart from the discussion as to whether contamination can be effectively prevented during surgical sampling of apical periodontitis, there is no clear evidence showing that these cases of extraradicular infections were independent of an intracanal infection.

Figure 4.19 (a) Extraradicular biofilm (Taylor- modified Brown and Brenn staining, original magnification 25x). (b) Higher magnification showing dense population of bacterial cells adhering to cementum (100x). Courtesy of Dr Domenico Ricucci.

The prevalence of extraradicular infections in untreated teeth with asymptomatic apical periodontitis is low [148, 183, 263], which is coherent with the high success rate of non-surgical endodontic treatment [33, 188]. In teeth with post-treatment disease, for which extraradicular bacteria have been suggested as a possible cause of failure, a histobacteriologic study showed no case suggestive of independent extraradicular infection [186]. Actually, there are only a few cases of post-treatment apical periodontitis reported that may have been caused by an extraradicular infection not associated with infection in the apical canal system [189, 192]. In addition, the high healing rate of apical periodontitis following retreatment [34, 188] indicates that the major cause of post-treatment disease is located intraradicularly and accessible to non-surgical therapeutic procedures. This is confirmed by microbiological studies showing that virtually all teeth with post-treatment apical periodontitis are associated with intraradicular bacterial infection [115, 186, 200, 205, 266].

4.13 Other Microorganisms in Endodontic Infections

Bacteria are the main microorganisms found in endodontic infections. However, other microorganisms have also been sporadically found. They include fungi, archaea, and viruses.

4.13.1 Fungi

Fungi are eukaryotic microorganisms that occur in two basic forms: molds (multicellular filamentous fungi consisting of branching cylindric tubules) and yeasts (unicellular fungi with spherical or oval-shaped cells). Fungi have only occasionally been found in primary root canal infections [272]. However, some studies have detected Candida species in about 20% of the samples from primary root canal infections [14, 133]. Reasons for discrepancies between studies are not evident, but may include differences in the identification methods used and in the general health conditions of the patients.

Fungi have been more frequently detected in studies evaluating the microbiological conditions of root canal-treated teeth.

Figure 4.20 Colonization of the dentinal root canal walls by Candida albicans (scanning electron microscopy, original magnification 600x).

Detection frequencies for Candida species in persistent/secondary infections range from 3% to 18% [25, 47, 135, 136, 162, 169, 194, 266, 292]. Fungi can gain access to root canals via contamination during endodontic therapy or can overgrow after inefficient intracanal antimicrobial procedures that caused an imbalance in the microbiota [278]. Candida albicans is by far the fungal species most commonly detected in teeth with post-treatment disease. This species has been consid- ered as a dentinophilic microorganism due to its ability to colonize and invade dentin [234, 235, 246] (Figure 4.20). In addition to the invading ability, Candida albicans has also been demonstrated to be resistant to some intracanal medicaments, such as cal- cium hydroxide [319]. Whether or not fungi participate in the pathogenesis of apical periodontitis still remains to be clarified.

4.13.2 Archaea

Archaea represents one of the three primary evolutionary domains of life, along with Bacteria and Eukarya. This domain com- prises a highly diverse group of prokaryotes, distinct from bacteria. To date, no member of the Archaea domain has been described as a human pathogen. Methanogenic archaea have been detected in samples from subgin-gival plaque associated with periodontal disease [111]. Although many studies have failed to detect archaea in primary endodontic infections [198, 199, 249], others have detected methanogenic archaea in low prevalence [156, 157, 313], and one notable exception is a study that detected archaea in 25% of the canals with primary infections [310]. In that study, archaeal diversity was limited to a Methanobrevibacter oralis-like phylotype and the relative abundance of archaeal population accounted for up to 2.5% of the total prokaryotic community (i.e., bacteria plus archaea) [310]. Given its overall low prevalence as reported by most studies, it is unlikely that archaea plays a significant pathogenic role in apical periodontitis.

4.13.3 Viruses

Viruses are not cells but particles structurally composed of a nucleic acid molecule (DNA or RNA) and a protein coat. They are inert in the extracellular environment; as obligate intracellular parasites, viruses are totally dependent on living cells to perform life functions. When they infect living cells, the viral nucleic acid molecule directs the repli- cation of the complete virus and assumes control of the metabolic activities of the host cell. Because viruses require viable host cells to infect and use the cellular machinery to replicate the viral genome, they cannot thrive in the root canal containing necrotic pulp tissue. Viruses have been reported to occur in root canals only in teeth with vital pulps. For instance, the human immunodeficiency virus (HIV) has been detected in vital pulps of HIV-seropositive patients [67] and some herpesviruses have been identified in both non-inflamed and inflamed vital pulps [113]. On the other hand, different herpesviruses have been detected in apical periodontitis lesions [52, 53, 215-217], where living cells abound.

It has been suggested that herpesviruses, especially human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV), may be implicated in the pathogenesis of apical periodontitis [282]. This would be either a direct result of virus infection and replication, or a result of virally induced impairment of local host defenses, which might give rise to over-growth of pathogenic bacteria in the apical canal segment. A hypothesis for herpesviruses participation in the pathogenesis of apical periodontitis has been proposed [282]. Bacterial infection of the root canal would cause an influx of herpesvirus infected cells into the periradicular tissues. Reactivation of these herpesviruses by tissue injury caused by bacteria might lead to local impairment of host immune defenses in the periradicular microenvironment, affecting the response against infection. In addition, herpesvirus-infected inflammatory cells are stimulated to release pro-inflammatory cytokines and contribute to increased inflammation [134, 282, 322].

Herpesviruses have been identified in samples from symptomatic apical periodontitis lesions [215, 217], acute apical abscesses [24, 52], large lesions [216, 217], and lesions from HIV-positive patients [218]. However, the mere occurrence of herpesviruses in samples of apical periodontitis does not necessarily imply a role in disease causation. Herpesviruses infecting inflammatory cells may occur chronically throughout the human body. As these virus-infected inflammatory cells are attracted to and accumulate in the inflamed periradicular tissues, these viruses will also be present and detected. Herpesvirus participation in disease etiology could be inferred if high viral titers and/or viral RNA transcripts or proteins were detected in the lesion samples, or the clinical condition improves and the lesion heals after antiviral treatment. Pending such documentation, the role of herpesviruses in the pathogenesis of apical periodontitis remains unknown.

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