Apical periodontitis is essentially an inflammatory disease of microbial etiology primarily caused by infection of the root canal system. Although chemical and physical factors can induce periradicular inflammation, a large body of scientific evidence indicates that endodontic infection is essential to the progression and perpetuation of the different forms of apical periodontitis. Endodontic infection develops in root canals devoid of host defenses, as a consequence of either pulp necrosis (as a sequel to caries, trauma, periodontal disease, or invasive operative procedures) or pulp removal for treatment.
Although fungi and most recently archaea and viruses have been found in association with endodontic infections, bacteria are the major microorganisms implicated in the pathogenesis of apical periodontitis. In advanced stages of the endodontic infectious process, bacterial organizations resembling biofilms can be observed adhered to the canal walls. Consequently, apical periodontitis has been included in the roll of biofilm-related oral diseases. Bacteria colonizing the root canal system enter in contact with the periradicular tissues via apical/lateral foramina or root perforations. As a consequence of the encounter between bacteria and host defenses, inflammatory changes take place in the periradicular tissues and give rise to the development of apical periodontitis. Depending on several bacterial and host-related factors, endodontic infections can lead to acute (symptomatic) or chronic (asymptomatic) apical periodontitis.
The ultimate goal of endodontic treatment is either to prevent the development of apical periodontitis or, in cases where the disease is already present, to create adequate conditions for periradicular tissue healing. The intent is to repair and preserve the tooth and associated periradicular bone. Because apical periodontitis is an infectious disease, the rationale for endodontic treatment is to eradicate the occurring infection or prevent microorganisms from infecting or reinfecting the root canal or the periradicular tissues. The cardinal principle of any health care profession is the thorough understanding of disease etiology and pathogenesis, which provides a framework for effective treatment. In this context, understanding the microbiologic aspects of apical periodontitis is the basis for endodontic practice and should be managed with an evidence-based approach. This chapter focuses on diverse aspects of endodontic microbiology, including pathogenetic, taxonomic, morphologic, and ecologic issues.
Apical Periodontitis as an Infectious Disease
The first recorded observation of bacteria in the root canal dates back to the 17th century and the Dutch amateur microscope builder Antony van Leeuwenhoek (1632-1723). He reported that the root canals of a decayed tooth “were stuffed with a soft matter” and that “the whole stuff” seemed to be alive. At that time, the role of Leeuwenhoek’s “animalcules” in disease causation was unsuspected. It took almost 200 years until his observation was confirmed and a cause-and-effect relationship between bacteria and apical periodontitis was suggested. This occurred specifically in 1894, when Willoughby Dayton Miller, an American dentist working at the laboratory of Robert Koch in Berlin, Germany, published a milestone study reporting the association between bacteria and apical periodontitis after an analysis of samples collected from root canals. By means of bacterioscopy of the canal samples, he found bacterial cells in the three basic morphologies known at the time: cocci, bacilli, and spirilla (or spirochetes) ( Fig. 14-1 ). Morphologically, the endodontic microbiota was clearly different in the coronal, middle, and apical parts of the root canal. Spirochetes were found in high frequencies in abscessed cases, and a pathogenic role was suspected for these bacteria. Most of the bacteria Miller observed under light microscopy could not be cultivated using the technology available at that time. Those bacteria were conceivably anaerobic bacteria, which were only successfully cultivated about 50 to 100 years later with the advent of anaerobic culture techniques. However, it is now widely recognized that a large number of bacterial species living in diverse environments still remain to be cultivated by current technology, and the root canal is no exception (discussed later in this chapter). Based on his findings, Miller raised the hypothesis that bacteria were the causative agents of apical periodontitis.
Approximately 70 years after Miller’s classic study, his assumptions were confirmed by an elegant study from Kakehashi and colleagues. These authors investigated the response of exposed dental pulps to the oral cavity in conventional and germ-free rats. Histologic evaluation was performed and revealed that pulp necrosis and apical periodontitis lesions developed in all conventional rats; however, the exposed pulps of germ-free rats not only remained vital but also repaired themselves with hard-tissue formation. Dentin-like tissue sealed the exposure area and isolated the pulps again from the oral cavity.
The important role of bacteria in the etiology of apical periodontitis was further confirmed by Sundqvist’s classic study. This author applied advanced anaerobic culturing techniques to the evaluation of bacteria occurring in the root canals of teeth whose pulps became necrotic after trauma. Bacteria were found only in the root canals of teeth exhibiting radiographic evidence of apical periodontitis, confirming the infectious cause of this disease. Anaerobic bacteria accounted for more than 90% of the isolates. Findings from Sundqvist’s study also demonstrated that in the absence of infection, the necrotic pulp tissue itself and stagnant tissue fluid in the root canal cannot induce or perpetuate apical periodontitis lesions.
Möller and colleagues also provided strong evidence about the microbial causation of apical periodontitis. Their study using monkeys’ teeth demonstrated that only devitalized pulps that were infected induced apical periodontitis lesions, whereas devitalized and noninfected pulps showed an absence of significant pathologic changes in the periradicular tissues. In addition to corroborating the importance of microorganisms for the development of apical periodontitis, this study also confirmed that necrotic pulp tissue per se is unable to induce and maintain an apical periodontitis lesion.
Microorganisms causing apical periodontitis are primarily organized in biofilms colonizing the root canal system. Nair was possibly the first to observe intracanal bacterial organizations adhered to the root canal walls, resembling biofilm structures. Several other morphologic studies found similar structures, but it was not until the study of Ricucci and Siqueira that the high prevalence of bacterial biofilms were consistently revealed in association with primary and posttreatment apical periodontitis (see the section Spatial Distribution of the Microbiota: Anatomy of Infection for a further discussion of biofilms in endodontic infections).
Routes of Root Canal Infection
Under normal conditions, the pulpodentin complex is sterile and isolated from oral microbiota by overlying enamel, dentin, and cementum. In the event that the integrity of these natural layers is breached (e.g., as a result of caries, trauma-induced fractures and cracks, restorative procedures, scaling and root planning, attrition, abrasion) or naturally absent (e.g., because of gaps in the cemental coating at the cervical root surface), the pulpodentin complex is exposed to the oral environment. The pulpodentin complex is then challenged by microorganisms present in caries lesions, saliva bathing the exposed area, or dental plaque formed on the exposed area. Microorganisms from subgingival biofilms associated with periodontal disease may also have access to the pulp via dentinal tubules at the cervical region of the tooth and lateral or apical foramina (see also Chapter 25 ). Microorganisms may also have access to the root canal any time during or after endodontic intervention, emphasizing the need for effective (fluid tight) use of the rubber dam.
Whenever dentin is exposed, the pulp is put at risk of infection as a consequence of the permeability of normal dentin dictated by its tubular structure ( Fig. 14-2 ). Dentinal tubules traverse the entire width of the dentin and have a conical conformation, with the largest diameter located near the pulp (mean, 2.5 µm) and the smallest diameter in the periphery, near the enamel or cementum (mean, 0.9 µm). The smallest tubule diameter is entirely compatible with the cell diameter of most oral bacterial species, which usually ranges from 0.2 to 0.7 µm. One might assume that once exposed, dentin offers an unimpeded pathway for bacteria to reach the pulp via these tubules. However, it has been demonstrated that bacterial invasion of dentinal tubules occurs more rapidly with a nonvital pulp than with a vital pulp. With a vital pulp, outward movement of dentinal fluid and the tubular contents (including odontoblast processes, collagen fibrils, and the sheathlike lamina limitans that lines the tubules) influence dentinal permeability and can conceivably delay intratubular invasion by bacteria. Because of the presence of tubular contents, the functional or physiologic diameter of the tubules is only 5% to 10% of the anatomic diameter seen by microscopy. Other factors such as dentinal sclerosis beneath a carious lesion, tertiary dentin, smear layer, and intratubular deposition of fibrinogen also reduce dentin permeability and thereby limit or even impede bacterial progression to the pulp via dentinal tubules. Host defense molecules, such as antibodies and components of the complement system, may also be present in the dentinal fluid of vital teeth and can assist in the protection against deep bacterial invasion of dentin. As long as the pulp is vital, dentinal exposure does not represent a significant route of pulpal infection, except when dentin thickness is considerably reduced or when the dentin permeability is significantly increased.
Most of the bacteria in the carious process are nonmotile; they invade dentin by repeated cell division, which pushes cells into tubules. Bacterial cells may also be forced into tubules by hydrostatic pressures developed on dentin during mastication. Bacteria inside tubules under a deep carious lesion can reach the pulp even before frank pulpal exposure. As mentioned, it has been assumed that the pulp will not be infected if it is still vital. The few bacteria that reach the pulp may not be significant, because the vital pulp can eliminate such a transient infection and rapidly clear or remove bacterial products. This efficient clearance mechanism tends to prevent injurious agents from reaching a high enough concentration to induce significant inflammatory reactions. On the other hand, if the vitality of the pulp is compromised and the defense mechanisms are impaired, even a small amount of bacteria may initiate infection.
Direct exposure of the dental pulp to the oral cavity is the most obvious route of endodontic infection. Caries is the most common cause of pulp exposure, but bacteria may also reach the pulp via direct pulp exposure as a result of iatrogenic restorative procedures or trauma. The exposed pulp tissue comes in direct contact with oral bacteria from carious lesions, saliva, or plaque accumulated onto the exposed surface. Almost invariably, exposed pulps will undergo inflammation and necrosis and become infected. The time elapsed between pulp exposure and infection of the entire canal is unpredictable, but it is usually a slow process.
The egress of microorganisms and their products from infected root canals through apical, lateral, or furcation foramina, dentinal tubules, and iatrogenic root perforations can directly affect the surrounding periodontal tissues and give rise to pathologic changes in these tissues. However, there is no consensus as to whether the opposite is true—that is, whether subgingival biofilms associated with periodontal disease can directly cause pulpal disease. Conceptually, microorganisms in subgingival plaque biofilms associated with periodontal disease could reach the pulp by the same pathways intracanal microorganisms reach the periodontium and could thereby exert harmful effects on the pulp. However, it has been demonstrated that although degenerative and inflammatory changes of different degrees may occur in the pulp of teeth with associated marginal periodontitis, pulpal necrosis as a consequence of periodontal disease only develops if the periodontal pocket reaches the apical foramen, leading to irreversible damage to the main blood vessels that penetrate through this foramen ( Fig. 14-3 ). After the pulp becomes necrotic, periodontal bacteria can reach the root canal system via exposed dentinal tubules at the cervical area of the root or via lateral and apical foramina to establish an endodontic infectious process.
It has been claimed that microorganisms can reach the pulp by anachoresis . Theoretically, microorganisms can be transported in the blood or lymph to an area of tissue damage, where they leave the vessel, enter the damaged tissue, and establish an infection. However, there is no clear evidence showing that this process can represent a route for root canal infection. It has been revealed that bacteria could not be recovered from unfilled root canals when the bloodstream was experimentally infected, unless the root canals were overinstrumented during the period of bacteremia, with resulting injury to periodontal blood vessels and blood seepage into the canal. Another argument against anachoresis as a route for pulpal infection comes from the study by Möller and colleagues, who induced pulpal necrosis in monkeys’ teeth and reported that all cases of aseptic necrosis remained bacteria-free after 6 to 7 months of observation.
Bacteria have been isolated from traumatized teeth with necrotic pulps with apparently intact crowns. Although anachoresis has been suggested to be the mechanism through which these traumatized teeth become infected, current evidence indicates that the main pathway of pulpal infection in these cases is dentinal exposure due to enamel cracks. Macro- and microcracks in enamel can be present in most teeth (not only traumatized teeth) and do not necessarily end at the enamel-dentin junction; they can extend deep into the dentin. A large number of dentinal tubules can be exposed to the oral environment by a single crack. These cracks can be clogged with dental plaque and provide portals of entry for bacteria. If the pulp remains vital after trauma, bacterial penetration into tubules is counteracted by the dentinal fluid and tubular contents, as discussed earlier, and pulpal health is not usually jeopardized. But if the pulp becomes necrotic as a consequence of trauma, it loses the ability to protect itself against bacterial invasion, and regardless of dentin thickness, dentinal tubules then will become true avenues through which bacteria can reach and colonize the necrotic pulp.
Whatever the route of bacterial access to the root canal, necrosis of pulp tissue is a prerequisite for the establishment of primary endodontic infections. To reiterate: if the pulp is vital, it can protect itself against bacterial invasion and colonization. If the pulp becomes necrotic due to caries, trauma, operative procedures, or periodontal disease, then it can be easily infected. This is because host defenses do not function in the necrotic pulp tissue, and those in the periradicular tissues do not reach deep into the root canal space.
Another situation in which the root canal system is devoid of host defenses relates to cases in which the pulp was removed for treatment. Microbial penetration in the canal can occur during treatment, between appointments, or even after root canal obturation. The main causes of microbial introduction into the canal during treatment include remnants of dental biofilm, calculus, or caries on the tooth crown; leaking rubber dam; contamination of endodontic instruments (e.g., after touching with the fingers); and contamination of irrigant solutions or other solutions of intracanal use (e.g., saline solution, distilled water, citric acid). Microorganisms can also enter the root canal system between appointments by leakage through the temporary restorative material; breakdown, fracture, or loss of the temporary restoration; fracture of the tooth structure; and in teeth left open for drainage. Microorganisms can penetrate the root canal system even after completion of the root canal obturation by leakage through the temporary or permanent restorative material; breakdown, fracture, or loss of the temporary/permanent restoration; fracture of the tooth structure; recurrent decay contaminating the root canal obturation; or delay in the placement of permanent restorations.
Mechanisms of Microbial Pathogenicity and Virulence Factors
The ability of a microorganism to cause disease is regarded as its pathogenicity . Virulence denotes the degree of pathogenicity of a microorganism, and virulence factors are the microbial products, structural cellular components, or strategies that contribute to pathogenicity. One example of bacterial strategy that contributes to pathogenicity includes the ability to coaggregate and form biofilms, which confers protection against microbial competitors, host defenses, and antimicrobial agents. Some microorganisms routinely cause disease in a given host and are called primary pathogens . Other microorganisms cause disease only when host defenses are impaired and are called opportunistic pathogens . Bacteria that make up the normal microbiota are usually present as harmless commensals and live in balance with the host. One of the greatest beneficial effects of human microbiota is probably the tendency to protect the host from exogenous infections by excluding other microorganisms. Nevertheless, in certain situations, the balance may be disturbed by a decrease in the normal level of resistance, and then the commensal bacteria are usually the first to take advantage. Most bacteria involved with endodontic infections are normal inhabitants of the oral microbiota that exploit changes in the balance of the host–bacteria relationship, becoming opportunistic pathogens.
Bacteria involved with the pathogenesis of primary apical periodontitis may have participated in the early stages of pulp inflammation and necrosis, or they may have gained entry into the root canal space any time after pulpal necrosis. In the former situation, involved bacteria are usually those present in the advanced front of caries lesions and from saliva bathing the affected area. Bacteria in caries lesions form authentic biofilms adhered to dentin ( Fig. 14-4 ). Diffusion of bacterial products through dentinal tubules induces pulpal inflammation long before the tissue is exposed. After pulp exposure, the surface of the tissue can also be colonized and covered by bacteria present in the caries biofilm. The exposed pulp tissue is in direct contact with bacteria and their products and responds with severe inflammation. Some tissue invasion by bacteria may also occur. Bacteria in the battlefront have to survive the attack from the host defenses and at the same time acquire nutrients to keep themselves alive. In this bacteria–pulp clash, the latter invariably is “defeated” and becomes necrotic, so bacteria move forward and “occupy the territory”—that is, they colonize the necrotic tissue. These events advance through tissue compartments, coalesce, and move toward the apical part of the canal until virtually the entire root canal is necrotic and infected ( Fig. 14-5 ). At this stage, involved bacteria can be regarded as the early root canal colonizers or pioneer species.
Early colonizers play an important role in the initiation of the apical periodontitis disease process. Moreover, they may significantly modify the environment, making it conducive to the establishment of other bacterial groups. These new species may have access to the canal via coronal exposure or exposed dentinal tubules, establish themselves, and contribute to a shift in the microbiota. Rearrangement in the proportions of the pioneer species and latecomers occurs, and as the environment changes, some early colonizers are expected to no longer participate in the consortium of advanced disease. With the passage of time, the endodontic microbiota becomes more and more structurally and spatially organized.
Some virulence attributes required for pathogens to thrive in other sites may be of no value for bacteria that reach the root canal after necrosis—for instance, the ability to evade host defenses. This is because latecomers face no significant opposition from host defenses, which are no longer active in the canal after necrosis. Although colonization may appear an easy task for late colonizers, other environmental factors (e.g., interaction with pioneer species, oxygen tension, nutrient availability) will determine whether new species entering the canal will succeed in establishing themselves and join the early colonizers to make up a dynamic mixed community in the root canal. Ultimately, the root canals of teeth with radiographically detectable apical periodontitis lesions harbor both early colonizers that managed to stay in the canals and late colonizers that managed to adapt to the new but propitious environmental conditions.
Bacteria colonizing the necrotic root canal induce damage to the periradicular tissues and give rise to inflammatory changes. In fact, periradicular inflammation can be observed even before the frontline of infection reaches the apical foramen. Bacteria exert their pathogenicity by wreaking havoc on the host tissues through direct or indirect mechanisms. Bacterial virulence factors that cause direct tissue harm include those that damage host cells or the intercellular matrix of the connective tissue. These factors usually involve secreted products, including enzymes, exotoxins, heat-shock proteins, and metabolic end products. Furthermore, bacterial structural components, including lipopolysaccharide (LPS), peptidoglycan, lipoteichoic acid, fimbriae, flagella, outer membrane proteins and vesicles, lipoproteins, DNA, and exopolysaccharides, can act as modulins by stimulating the development of host immune reactions capable not only of defending the host against infection but also of causing severe tissue destruction ( Fig. 14-6 ). For instance, inflammatory and noninflammatory host cells can be 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 asymptomatic (chronic) apical periodontitis lesions. Another example of indirect damage caused by bacteria is the formation of purulent exudate in acute apical abscesses. Host defense mechanisms against bacteria emanating from the root canal appear to be the most important factor involved in the formation of purulent exudate associated with abscesses. Formation of oxygen-derived free radicals, such as superoxide and hydrogen peroxide, alongside the release of lysosomal enzymes by polymorphonuclear leukocytes, gives rise to destruction of the connective extracellular matrix, leading to pus formation. Although direct damage caused by bacterial products may certainly be involved in the pathogenesis of apical periodontitis, bacterial indirect destructive effects seem to be more significant in this regard.
Apical periodontitis is a multifactorial disease that is resultant of the interplay of many host and bacterial factors. Few if any of the putative endodontic pathogens are individually capable of inducing all of the events involved in the pathogenesis of the different forms of apical periodontitis. Probably, the process requires an integrated and orchestrated interaction of the selected members of the mixed endodontic microbiota and their respective virulence attributes. Although LPS is undoubtedly the most studied and quoted virulence factor, it sounds simplistic to ascribe to this molecule all responsibility for apical periodontitis causation. This statement is further reinforced by the fact that some cases of primary infections and many cases of secondary/persistent infections harbor exclusively gram-positive bacteria. Therefore, the involvement of other factors must not be overlooked. In fact, the pathogenesis of different forms of apical periodontitis and even the same form in different individuals is unlikely to follow a stereotyped course with regard to the bacterial mediators involved.
Spatial Distribution of the Microbiota: Anatomy of Infection
Mounting evidence indicates that apical periodontitis, like caries and periodontal diseases, is also a biofilm-related disease. Morphologic studies have shown that the root canal microbiota in primary infections is dominated by bacterial morphotypes that include cocci, rods, filaments, and spirilla (spirochetes) ( Fig. 14-7 ). Fungal cells are sporadically found ( Fig. 14-8 ). Although planktonic bacterial cells suspended in a fluid phase and enmeshed in necrotic pulp tissue can be observed in the main root canal, most bacteria colonizing the root canal system usually grow in sessile multispecies biofilms adhered to the dentinal walls ( Fig. 14-9 ). Lateral canals, apical ramifications, and isthmuses connecting main canals may also be clogged with bacterial biofilms ( Figs. 14-10 and 14-11 ).
Bacterial cells from endodontic biofilms are often seen penetrating the dentinal tubules ( Fig. 14-12 ). Dentinal tubule infection can occur in about 70% to 80% of the teeth evincing apical periodontitis lesions. A shallow penetration is more common, but bacterial cells can be observed reaching approximately 300 µm in some teeth ( Fig. 14-13 ). Dividing cells are frequently observed within tubules during in situ investigations (see Fig. 14-13 ), 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.
Several putative endodontic pathogens have been shown to be capable of penetrating dentinal tubules in vitro, including Porphyromonas endodontalis, Porphyromonas gingivalis, Fusobacterium nucleatum, Actinomyces israelii, Propionibacterium acnes, Enterococcus faecalis, Candida albicans, and streptococci. In their clinical study, Peters and colleagues isolated and identified bacteria present in root dentin at different depths, and the most common isolates belonged to the genera Prevotella, Porphyromonas, Fusobacterium, Veillonella, Peptostreptococcus, Eubacterium, Actinomyces, lactobacilli, and streptococci. Using immunohistologic analysis, Matsuo and associates observed the occurrence of F. nucleatum, Pseudoramibacter alactolyticus, Eubacterium nodatum, Lactobacillus casei, and Parvimonas micra inside dentinal tubules from the canal walls of extracted infected teeth with apical periodontitis.
Whereas bacteria present as planktonic cells in the main root canal may be easily accessed and eliminated by instruments and substances used during endodontic treatment, those organized in biofilms attached to the canal walls or located into isthmuses, lateral canals, and dentinal tubules are definitely more difficult to reach and may require special therapeutic strategies to be eradicated.
Biofilm and Community-Based Microbial Pathogenesis
Individual microorganisms proliferating in a habitat give rise to populations . Such populations often occur as microcolonies in the environment. Populations interact with one another to form a community. Thus, community refers to a unified assemblage of populations that coexist and interact at a given habitat. The community and habitat are part of a larger system called an ecosystem, which can be defined as a functional self-supporting system that includes the microbial community and its environment. In summary, the following hierarchy becomes apparent: ecosystem, community, population, and the single individual (cell).
Populations perform functions that contribute to the overall community and maintain the ecologic balance of the ecosystem. Each population occupies a functional role (niche) within the community. There are a limited number of niches within the community for which populations must compete. More competent populations occupy the niches and displace those less competent. As discussed later, highly structured and spatially organized microbial communities may exhibit properties that are greater than the sum of the component populations. In reality, complex microbial communities have been shown to be endowed with the ability to confront and withstand the challenges imposed by the environment by creating a mosaic of microenvironments that enable the survival and growth of the community members.
Historically, microbiologists dealing with infectious diseases have faced periods of “reductionism” and “holism.” Reductionism is based on the idea that the whole can be understood by examining smaller and smaller pieces of it, that is, all complex systems can be completely understood in terms of their individual components. Through reductionist approaches, individual species are isolated from complex mixed communities and metabolically and genetically studied so as to allow understanding of the community by examining every single constituent. However, it has become quite apparent for the microbiota associated with many human infectious diseases that the whole is very often greater than the simple sum of its parts. This concept has prompted microbiologists to adopt a holistic approach to understand the community behavior associated with pathogenesis of many infectious diseases known to have a polymicrobial etiology. Holism holds that any component cannot be thoroughly understood except in their relation to the whole. The holistic theory has been largely employed in ecology: the interplay of the different parts composing the ecosystem will ultimately determine its properties.
It has been recognized that the biofilm (dental plaque) associated with caries and periodontal diseases represents a sophisticated community that exert functions essential for the biofilm architecture and physiology, with consequent pathogenetic implications. Recent evidence indicates that apical periodontitis can also develop as a result of collaborative activities of a biofilm community established in the root canal system.
Community profiling studies revealed that bacterial composition of the endodontic microbiota differs consistently between individuals suffering from the same disease. This indicates that apical periodontitis has a heterogeneous etiology, where multiple bacterial combinations can play a role in disease causation. Interindividual variability is even more pronounced when different geographic locations are studied. Moreover, community structure differs significantly between different disease forms (e.g., asymptomatic apical periodontitis versus acute apical abscess), suggesting existence of a pattern associated with each form.
Biofilm and Bacterial Interactions
The community-forming ability can be regarded as essential for microbial survival in virtually all environments. Indeed, the majority of microorganisms in nature invariably grow and function as members of metabolically integrated communities, or biofilms. Biofilm can be defined as a sessile multicellular microbial community characterized by cells that are firmly attached to a surface and enmeshed in a self-produced matrix of extracellular polymeric substance (EPS), usually a polysaccharide ( Fig. 14-14 ). The ability to form biofilms has been regarded as a virulence factor, and biofilm infections account for an estimated 65% to 80% of bacterial infections that affect humans in the developed world. Given its importance in varied aspects, there has been a high level of interest in the study of biofilm properties, not only in medical microbiology but also in different sectors of industrial and environmental microbiology.
Bacterial cells in biofilms form microcolonies (±15% by volume) that are embedded and nonrandomly distributed in the EPS matrix (±85% by volume) and separated by water channels. Microcolonies are usually shaped as “towers” or “mushrooms.” Dental biofilms can reach up to 300 or more cell layers in thickness. Individual microcolonies may consist of a single bacterial species but more frequently are composed of several different species in a mixed community.
As the biofilm matures on the surface, extracellular polysaccharides are continually synthesized to form an extracellular matrix that eventually may constitute as much as 85% of the volume of the biofilm. Although the matrix is primarily composed of polysaccharides, it can also contain proteins and nucleic acids. The matrix is not only important physically as part of the scaffold that determines the biofilm structure, but it is also biologically active and can retain nutrients, water, and essential enzymes within the biofilm. The matrix can also protect the biofilm community from exogenous threats and may participate in adherence to the surface.
Community members form distinct populations or microcolonies separated by open water channels that traverse the biofilm matrix and create primitive circulatory systems. Fluid in these channels carries substrate, end products of bacterial metabolism, and signal molecules involved in bacterial interactions. Thus, vital nutrients and communication molecules can diffuse, and wastes can be washed out through these channels.
Microcolonies that form in the biofilm arise from surface colonization by planktonic (unattached) bacterial cells. During the early stages of biofilm formation, bacteria bind to many host proteins and coaggregate with other bacteria. These interactions lead to changes in growth rate, gene expression, and protein production. It has been demonstrated by proteomic techniques or DNA arrays that genes expressed by cells in biofilms differed by 20% to 70% from those expressed by the same cells growing in planktonic culture. Thus, bacteria in biofilms adopt a radically different phenotype compared with their planktonic counterparts. Within biofilms, some bacteria also use sophisticated systems of cell-cell communication (quorum sensing) to coordinate gene expression. Phenotypic heterogeneity in biofilms is also observed as a result of exposure of microcolonies to a variety of gradients (e.g., oxygen tension, pH, osmolarity, type and amounts of nutrients, cell density), which contribute to form diverse microenvironments throughout the biofilm structure.
Biofilm Community Lifestyle
Many naturally occurring biofilms have a highly diverse microbiota. These multispecies biofilms are not merely passive bacterial assemblages that are stuck to surfaces; they are complex biologic systems formed by populations (microcolonies) that are not randomly distributed but are spatially and functionally organized throughout the community. Indeed, populations are strategically positioned for optimal metabolic interaction, and the resultant architecture favors the ecologic role of the community in the ecosystem. The properties displayed by a multispecies biofilm community are mostly dictated by the interactions between populations, which create novel physiologic functions that cannot be usually observed with individual components. As a result, biofilm communities have a collective physiology, responding in concert to environmental challenges.
The biofilm community lifestyle affords a number of advantages to colonizing bacteria, including establishment of a broad habitat range for growth, increased metabolic diversity and efficiency, enhanced possibilities for genetic exchanges and bacterial intercommunications (quorum-sensing systems), and protection from external threats (competing microorganisms, host defenses, antimicrobial agents, and environmental stress).
Biofilm organizations can also result in enhanced pathogenicity. To cause disease, bacteria must adhere to host surfaces, obtain nutrients from the host and multiply, invade tissues, overcome or evade the host defenses, and induce tissue damage. A diverse range of virulence traits are required for these particular stages of the disease process, and it is highly probable that each will require the concerted action of bacteria in a community. Similarly, it is possible that certain species can have more than one role in disease, and different species can perform similar functions. This helps explain why communities with different bacterial composition can be found in different individuals with similar disease. In multispecies communities, a broad spectrum of relationships may arise between the component species, ranging from no effect or reduced pathogenicity to additive or synergistic pathogenic effects. Endodontic abscesses are examples of polymicrobial infections whereby bacterial species that individually have low virulence and are unable to cause disease can do so when in association with others as part of a mixed consortium (pathogenic synergism).
Resistance to Antimicrobial Agents
From a clinical standpoint, biofilm increased resistance to antimicrobial agents is of special concern. Bacteria arranged in biofilms are considered more resistant to antibiotics than the same cells grown in planktonic state. The antibiotic concentration required to kill bacteria in the biofilm is about 100 to 1000 times higher than that needed to kill the same species in planktonic state. Several possible mechanisms are involved with biofilm resistance to antimicrobials.
Biofilm Structure May Restrict Penetration of Antimicrobial Agents
The agent may adsorb to and even inhibit the bacteria at the biofilm surface, but cells deeply located in the biofilm may remain relatively unaffected. The matrix in biofilms can also bind and retain neutralizing enzymes at concentrations that could inactivate the antimicrobial agent.
Altered Growth Rate of Biofilm Bacteria
Many antibiotics can freely penetrate the biofilm matrix, but cells are often still protected. The occurrence of starved bacteria entering the stationary phase in biofilms seems to be a significant factor in the resistance of biofilm populations to antimicrobials. Bacteria grow slowly under conditions of low availability of nutrients in an established biofilm and as a consequence are much less susceptible than faster-dividing cells. Most antibiotics require at least some degree of cellular activity to be effective. Therefore, bacterial cells in stationary phase might represent a general mechanism of antibiotic resistance in the biofilm.
Presence of “Persister” Bacteria
Increased tolerance of some biofilms to antibiotics may be largely due to the presence of a subpopulation of specialized survivor cells known as persisters . It remains unclear whether these bacteria actually represent a distinct phenotype or are simply the most resistant cells within a population.
Apical Periodontitis as a Biofilm-Related Disease
The evidence that apical periodontitis is a disease associated with polymicrobial biofilms comes mostly from in situ morphologic investigations. These studies have observed that bacteria colonizing the root canal system of teeth with primary or posttreatment apical periodontitis usually formed sessile biofilm communities covering the walls of the main canal, apical ramifications, lateral canals, and isthmuses.
Although the concept of apical periodontitis as a biofilm-related disease has been built upon these observations, the prevalence of biofilms and their association with diverse presentations of apical periodontitis were only recently disclosed by Ricucci and Siqueira. These authors evaluated the prevalence of biofilms in untreated teeth with primary apical periodontitis and treated teeth with posttreatment disease and looked for associations between biofilms and clinical/histopathologic conditions. Some of the most important findings of their study are as follows:
- 1.
Intraradicular biofilms were generally observed in the apical segment of approximately 80% of the root canals of teeth with primary or posttreatment apical periodontitis.
- 2.
Morphology of endodontic biofilms differed consistently from individual to individual (e.g., thickness, morphotypes, bacterial cells/extracellular matrix ratio).
- 3.
Dentinal tubules underneath biofilms were often invaded by bacterial cells from the bottom of the biofilm community.
- 4.
Biofilms were also commonly seen covering the walls of apical ramifications, lateral canals, and isthmuses.
- 5.
Bacterial biofilms were more frequent in root canals of teeth with large apical periodontitis lesions. Because it takes time for apical periodontitis to develop and become radiographically visible, one can surmise that large lesions represent a longstanding pathologic process caused by an even “older” intraradicular infection. In longstanding infectious processes, involved bacteria may have had enough time and conditions to adapt themselves to the environment and set a mature and organized biofilm community. The fact that the apical root canal of teeth with large lesions harbors a large number of bacterial cells and species almost always organized in biofilms may help explain the long-held concept that treatment outcome is influenced by lesion size.
- 6.
The prevalences of intraradicular biofilms in teeth associated with apical cysts, abscesses, and granulomas were 95%, 83%, and 69.5%, respectively. Biofilms were significantly associated with epithelialized lesions. Because apical cysts develop as a result of epithelial proliferation in some granulomas, it may be anticipated that the older the apical periodontitis lesion, the greater the probability of it becoming a cyst. Similar to teeth with large lesions, the age of the pathologic process may also help explain the high prevalence of biofilms in association with cysts.
- 7.
Extraradicular biofilms were infrequent; they occurred in only 6% of the cases. Except for one case, they were always associated with intraradicular biofilms. All cases showing an extraradicular biofilm exhibited clinical symptoms. Thus, it seems that extraradicular infections in the form of biofilms or planktonic bacteria are not a common occurrence, are usually dependent on the intraradicular infection, and are more frequent in symptomatic teeth.
- 8.
Bacteria were also seen in the lumen of the main canal, ramifications, and isthmuses as flocs and planktonic cells, either intermixed with necrotic pulp tissue or possibly suspended in a fluid phase. Bacterial flocs are sometimes regarded as “planktonic biofilms” and may originate from growth of cell aggregates/coaggregates in a fluid or they may have detached from biofilms.
Some criteria have been proposed to establish a causal link between biofilms and a given infectious disease :
- 1.
Infecting bacteria are adhered to or associated with a surface.
- 2.
Direct examination of the infected tissue shows bacteria forming clusters or microcolonies encased in an extracellular matrix.
- 3.
The infection is generally confined to a particular site and although dissemination may occur, it is a secondary event.
- 4.
The infection is difficult or impossible to eradicate with antibiotics in spite of the responsible microorganisms being susceptible to killing in the planktonic cell state.
- 5.
Ineffective host clearance is evident, as suggested by the location of bacterial colonies in areas of the host tissue associated with host inflammatory cells. Accumulation of polymorphonuclear neutrophils and macrophages surrounding bacterial aggregates/coaggregates in situ considerably increases the suspicion of biofilm involvement with disease causation.
- 6.
Elimination or significant disruption of the biofilm structure and ecology leads to remission of the disease process.
Based on findings from the Ricucci and Siqueira’s study, apical periodontitis can be considered to fulfill five of the six criteria. Bacterial aggregates/coaggregates are observed adhered to or at least associated with the dentinal root canal walls (criterion 1). Bacterial colonies are often seen encased in an amorphous extracellular matrix (criterion 2). Endodontic biofilms are frequently confined to the root canal system, in only a few cases extending to the external root surface, but dissemination through the lesion never occurred (criterion 3). In the great majority of cases, biofilms are directly faced by an accumulation of inflammatory cells, especially polymorphonuclear neutrophils (criterion 5).
As for criterion 4, it is widely known that intraradicular endodontic infections cannot be effectively treated by systemic antibiotic therapy, even though most endodontic bacteria in the planktonic cell state are susceptible to currently used antibiotics. The lack of efficacy of systemic antibiotics against intraradicular infections is mainly because the drug does not reach endodontic bacteria that are located in an avascular necrotic space. The recognition of biofilms as the main mode of bacterial establishment in the root canal system further strengthens the explanations for the lack of antibiotic effectiveness against endodontic infections. Finally, there is a clear potential for fulfillment of criterion 6, because biofilms are frequently observed in canals of treated teeth with posttreatment apical periodontitis, whereas teeth with a successful outcome show no biofilm infection of the root canal.
Methods for Microbial Identification
The endodontic microbiota has been traditionally investigated by microbiologic culture methods. Culture is the process of propagating microorganisms in the laboratory by providing them with the required nutrients and proper physicochemical conditions, including temperature, moisture, atmosphere, salt concentration, and pH. Essentially, culture analyses involve the following steps: sample collection and transport, dispersion, dilution, cultivation, isolation, and identification. Endodontic samples are collected and transported to the laboratory in a viability-preserving, nonsupportive, anaerobic medium. They are then dispersed by sonication or vortex mixing, diluted, distributed onto various types of agar media, and cultivated under aerobic or anaerobic conditions. After a suitable period of incubation, individual colonies are subcultivated and identified on the basis of multiple phenotype-based aspects, including colony and cellular morphology, gram-staining pattern, oxygen tolerance, comprehensive biochemical characterization, and metabolic end-product analysis by gas-liquid chromatography. The outer cellular membrane protein profile as examined by gel electrophoresis, fluorescence under ultraviolet light, and susceptibility tests to selected antibiotics can be needed for identification of some species. Marketed packaged kits that test for preformed enzymes have also been used for rapid identification of several species.
Culture analyses of endodontic infections have provided a substantial body of information about the etiology of apical periodontitis, composition of the endodontic microbiota in different clinical conditions, effects of treatment procedures in microbial elimination, susceptibilities of endodontic microorganisms to antibiotics, and so on. Advantages and limitations of culture methods are listed in Box 14-1 ; however, some important limitations of culture methods make a comprehensive analysis of the endodontic microbiota difficult to achieve.
| Advantages | Limitations |
|---|---|
|
|
The difficulties in culturing or identifying many microbial species are of special concern. Unfortunately, not all microorganisms can be cultivated under artificial conditions, and this is simply because the nutritional and physiologic needs of most microorganisms are still unknown. Investigations of many aquatic and terrestrial environments using culture-independent methods have revealed that the cultivable members of these systems represent less than 1% of the total extant population. Furthermore, 50% to 80% of bacterial species composing the microbiota associated with diverse human sites, including the oral cavity, represent unknown and still uncultivated bacteria.
That a given species has not been cultivated does not imply that this species will remain indefinitely impossible to cultivate. Myriad obligate anaerobic bacteria were uncultivable in the early 1900s, but further developments in anaerobic culturing techniques have to a large extent helped to solve this problem. It must be assumed that no single method or culture medium is suitable for isolating the vast diversity of microorganisms present in most environments. Because we remain relatively unaware of the requirements for many bacteria to grow, identification methods not based on cultivability are required.
In some situations, even the successful cultivation of a given microorganism does not necessarily mean that this microorganism can be successfully identified. Culture-dependent identification is based on phenotypic traits observed in reference strains, with predictable biochemical and physical properties under optimal growth conditions. However, many phenotype-related factors can lead to difficulties in identification and even to misidentification. As a consequence of all these factors, phenotype-based identification does not always allow an unequivocal identification.
To sidestep the limitations of culturing, tools and procedures based on molecular biology have become available and have substantially improved the ability to achieve a more realistic description of the microbial world without the need for cultivation ( Fig. 14-15 ). Molecular technology has also been applied to reliably identify cultivated bacteria, including strains with ambiguous or aberrant phenotypic behavior, rare isolates, poorly described or uncharacterized bacteria, and newly named species.
Molecular approaches for microbial identification rely on certain genes that contain revealing information about the microbial identity. Of the several genes that have been chosen as targets for bacterial identification, the 16S rRNA gene (or 16S rDNA) has been the most widely used because it is universally distributed among bacteria, is long enough to be highly informative and short enough to be easily sequenced, possesses conserved and variable regions, and affords reliability for inferring phylogenetic relationships. Similarly, the 18S rRNA gene of fungi and other eukaryotes has also been used extensively to identify these organisms.
Data from 16S rRNA gene sequences can be used for accurate and rapid identification of known and unknown bacterial species, using techniques that do not require cultivation. The 16S RNA gene of virtually all bacterial species in a given environment, including still uncultivated and uncharacterized bacteria, can be amplified by polymerase chain reaction (PCR) using broad-range (or universal) primers that are complementary to conserved regions of this gene. Sequencing of the variable regions flanked by the broad-range primers will provide information for accurate bacterial identification. Primers or probes that are complementary to variable regions can also be designed to detect specific target species directly in clinical samples.
Many molecular methods for the study of microorganisms exist; the choice of a particular approach depends on the questions being addressed. Broad-range PCR followed by cloning and sequencing can be used to unravel the breadth of microbial diversity in a given environment. Bacterial community structures can be analyzed via pyrosequencing technology and by fingerprinting techniques such as denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP). Fluorescence in situ hybridization (FISH) can measure the abundance of target species and provide information on their spatial distribution in tissues. Among other applications, DNA-DNA hybridization arrays (checkerboard techniques, DNA microarrays), species-specific single PCR, nested PCR, multiplex PCR, and quantitative real-time PCR can be used to survey large numbers of clinical samples for the presence of target species. Variations in PCR technology can also be used to type microbial strains. As with any other method, molecular methods have their own advantages and limitations ( Box 14-2 ).
| Advantages | Limitations |
|---|---|
|
|
The Five Generations of Endodontic Microbiology Studies
Microbiologic 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. These generations are detailed in Table 14-1 .
| Study Generation | Identification Method | Nature | Description and Findings |
|---|---|---|---|
| First | Culture | Open ended (broad range) | Revealed many cultivable species in association with apical periodontitis |
| Second | Molecular methods (e.g., PCR and its derivatives, original checkerboard assay) | Closed ended (species specific) | Target cultivable bacteria Confirmed and strengthened data from first generation Allowed inclusion of some culture-difficult species in the set of candidate endodontic pathogens |
| Third | Molecular methods (e.g., PCR-cloning-sequencing, T-RFLP) | Open ended (broad range) | Allowed a more comprehensive investigation of the bacterial diversity in endodontic infections Not only cultivable species but also as-yet-uncultivated and uncharacterized bacteria were identified |
| Fourth | Molecular methods (e.g., PCR, microarrays, reverse-capture checkerboard) | Closed ended (species specific) | Target cultivable and as-yet-uncultivated bacteria Large-scale clinical studies to investigate prevalence and association of species/phylotypes with endodontic infections |
| Fifth | Molecular methods (e.g., pyrosequencing) | Open ended (broad range) | Permit a deep-coverage and more comprehensive analysis of the diversity of endodontic infections |
Impact of Molecular Methods in Endodontic Microbiology
Culture studies (first generation) identified a set of species thought to play an important role in the pathogenesis of apical periodontitis. Further, not only have findings from culture-based methods been confirmed, but they have also been significantly supplemented with those from culture-independent molecular biology techniques, which constitute the other four generations of endodontic microbiology studies. Molecular methods have confirmed and strengthened the association of many cultivable bacterial species with apical periodontitis and have also revealed new suspected endodontic pathogens. The list of candidate pathogens has expanded to include culture-difficult species or as-yet-uncultivated bacteria that had never been found in endodontic infections by culturing approaches. The results from molecular studies impact remarkably on the knowledge of bacterial diversity in endodontic infections. More than 400 different bacterial species have already been detected in different types of endodontic infections. Of these, about 45% were exclusively reported by molecular biology studies, compared with 32% detected by culture studies alone. Twenty-three percent of the total bacterial species richness has been detected by application of both culture and molecular studies ( Fig. 14-16 ). As a consequence, it becomes quite evident that the endodontic microbiota has been refined and redefined by molecular methods.
Types of Endodontic Infections
Endodontic infections can be classified according to the anatomic location as intraradicular or extraradicular infection. Intraradicular infection is caused by microorganisms colonizing the root canal system and can be subdivided into three categories according to the time microorganisms entered the root canal system: primary infection, caused by microorganisms that initially invade and colonize the necrotic pulp tissue (initial or “virgin” infection); secondary infection, caused by microorganisms not present in the primary infection but introduced in the root canal at some time after professional intervention (i.e., secondary to intervention); and persistent infection, caused by microorganisms that were members of a primary or secondary infection and in some way resisted intracanal antimicrobial procedures and were able to endure periods of nutrient deprivation in treated canals. Extraradicular infection in turn is characterized by microbial invasion of the inflamed periradicular tissues and is a sequel to the intraradicular infection. Extraradicular infections can be dependent on or independent of the intraradicular infection.
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