Ecological moments that determine the selection of a post-treatment root canal community. Environmental disturbances such as mechanical instrumentation, irrigation with antimicrobials and inter-appointment medication cause a simplification of the original root canal microbiota. Further disturbances such as lack of nutrients and interactions with the host’s immune cells lead to the formation of a resilient microbiota.
3.2 Root Canal Biofilms
The biofilm concept recognizes biofilm formation as a key mechanism linked to microbial survival, and its application in endodontics has led to the understanding of their involvement in the pathogenesis of endodontic infections [1–3]. In general, biofilm formation reflects an essential mechanism of microbial adaptation to environmental conditions. Bacteria in biofilms are surrounded by a matrix of bacterial exopolysaccharides and exogenous substances (polysaccharides, proteins, mineral crystals, extracellular DNA) [10, 11] that protect them from the host’s immune defences. Antibodies and phagocytes have difficulties to penetrate into the biofilm and may even undergo deactivation whilst inside the matrix [10, 11]. Bacteria in biofilms are also less susceptible to the action of antibiotics, which may contribute to the development of chronic infections and relapses [12, 13].
Several studies have described the presence of biofilms formed in infected root canals [14–16]. Biofilm structures have been reported to be formed alongside the canal walls, inside dentinal tubules, apical deltas and periapical areas [1–3]. The presence of these microbial structures has been associated with different clinical states including post-treatment endodontic infections [14–16].
Of importance is to understand the biological basis of biofilm formation as it is possible that various microbial genetic regulatory pathways involved may also play a crucial role in mechanisms of resistance to host immune defences and antimicrobial treatment . Notwithstanding the characterization of biofilms in infected root canals, the mechanisms behind their formation in root canals have not been well established. As most of the species found in root canals are also found in the oral cavity, it is reasonable to speculate that the formation of microbial biofilms in root canals may have similar mechanisms as oral biofilms. Figure 3.2 depicts the main events occurring during the formation of a biofilm.
Schematic depiction of the temporal sequence of biofilm formation . (a) Clean surfaces are coated with environmental molecules. (b) Pioneer microorganisms adhere to the conditioned surface, utilizing different cell-surface interactions. (c) Incorporation of secondary colonizers by adhesion to the pioneers by utilizing different engaging adhesins. (d) The production of extracellular polymeric substance (matrix) results in the formation of mature biofilms where intermicrobial signalling and intergeneric co-aggregation leads to the development of complex communities
3.2.1 Initial Adherence to Surfaces
In the oral ecosystem, the deposition of salivary components provides a set of receptor molecules which are primary recognized by the early colonizers, such as streptococci and actinomyces . In root canals of teeth, the presence of plasma constituents, which increase exponentially due to inflammatory transudation, may form the active conditioning film paving the way for subsequent microbial colonization . Plasma constituents, such as plasminogen, may endow with primary receptors for adhesion on root canal surfaces . This previous hypothesis is supported by the fact that several oral species have an affinity to bind to plasminogen via very specific lysine-dependent mechanisms. Among the most common plasminogen-specific binding receptors in oral species are enolase and GAPDH.
The conditioning film may not only influence the initial adhesion of colonizing cells, but it will also influence the production of signalling molecules that control cell physiology and resistance to antimicrobials. In a recent study, it was found that biofilms formed by root canal bacteria on surfaces preconditioned with collagen showed irregular architectures, which apparently also influenced their responsiveness to the exposure with antimicrobials . Biofilms formed on collagen-coated surfaces by Streptococcus gordonii, E. faecalis and Lactobacillus paracasei showed a much higher resistance to NaOCl than those biofilms formed on non-coated surfaces. Interestingly, it was found that the levels of dehydrogenase and esterase activities of biofilm cells which adhered to collagen-coated surfaces were very low, a finding which may partially explain their high resistance to antimicrobials. The metabolic downregulation of biofilm cells on surfaces coated with collagen may give some indications as to how the surface condition may influence bacterial physiology and consequently resistance to antimicrobials.
3.2.2 Secondary Colonizers
Secondary colonizers co-aggregate to adhering cells after the first colonizers have irreversibly adhered to the surfaces . The newcomers will form close metabolic relationships with the adhered cells, developing microenvironments for the establishment of bacteria with special requirements such as obligate anaerobes . Bacteria with plenty of receptors that are recognized by many other organisms, such as fusobacteria, play a key role in forming a link between primary colonizing species and later colonizing pathogens . In infected root canals, the presence of fusobacteria has been widely reported and has been linked with the occurrence of cases with most severe inflammatory symptoms . In such cases, fusobacteria were found in combination with highly proteolytic organisms, e.g. Prevotella and Porphyromonas. Hence, it is likely that the surface receptors from fusobacteria promote the colonization of these proteolytic pathogens in root canals. A similar case is seen in microbiological screening of sites of periodontal inflammation, where fusobacteria appear just before the pathogenic “red” complex consisting of Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia .
The presence of E. faecalis in post-treatment infected root canals has received much attention since this is an organism that shows, among other interesting capacities, high tolerance to alkaline pH [21–23]. Although the majority of these observations have been made in vitro, its high tolerance to alkaline has been clinically linked to a potential resistance to treatment with inter-appointment dressings containing calcium hydroxide [5, 7]. However, the origin of E. faecalis in infected root canals has remained highly controversial because this organism is not commonly found in untreated necrotic pulps and has been until recently considered a ‘transient’ microorganism in the oral flora . E. faecalis has been isolated from teeth presenting post-treatment infections with a prevalence of 24% and 70% in studies utilizing traditional culture-based techniques [4–7, 25, 26] and between 66 and 77% when molecular methods were applied [27, 28]. In a recent series of studies , it was determined that E. faecalis is not likely to be derived from the endogenous commensal flora of the gastrointestinal tract and that even the chances for nosocomial transmission during a root canal treatment from contaminated high-touch surfaces in dental operatory were slight. It was stated, however, that E. faecalis in root canal infections are most likely food-borne since strains from root canals and food items shared common genotypic patterns .
3.2.3 Growth and Maturation
During growth and maturation of the biofilm, the concentration of chemical signals produced by metabolism provokes a range of phenotypic differentiations among the species forming microbial communities [13, 30]. These different phenotypes trigger molecular responses that are generated as chemical signals corresponding to secondary metabolites, also known as quorum sensing . The quorum sensing of microbial cells in biofilms recognizes the proximity of cells reaching a critical number in a limited space in the environment and that ultimately results in the autoinduction and synthesis of the extracellular matrix . The biofilm matrix is mainly composed of polysaccharides, proteins, nucleic acids and lipids and is a key feature to the maturation of biofilm formation. The matrix will constitute the backbone of the biofilm’s three-dimensional structure and will allow the free circulation of metabolites and wastes among cells and microcolonies. The structure cohesiveness conferred by the matrix permits that the biofilm community to respond like a mass and behave as a group .
The composition of the matrix varies depending on the bacterial species, the environmental conditions and the metabolites available. The presence of high levels of nutrients can lead to very dense biofilms. For instance, in oral biofilms the presence of high levels of sucrose in the media yields very dense and large biofilms . This phenomenon was explained to be due to the ability of many oral bacteria to synthesize dextrans (including the insoluble 1,3-α-D-glucan mutan) and levans using sucrose as a substrate. It has also been observed that mixed biofilms grown on limited nutrients that are then switched to a rich medium change considerably in their structural appearance .
3.3 Extra-radicular Colonization
Contrary to the traditional view of extra-radicular tissues being always free of bacteria, compelling clinical evidence now exists on bacteria forming biofilms on extra-radicular surfaces [33–36]. Although most of the studies are described as case reports, it is reasonable to conclude from the available information that the formation of extra-radicular biofilms occurs with relative frequency.
Although still unclear, the formation of extra-radicular biofilms seems to be a consequence of massive infection of the root canal system associated with prolonged exposure of the canal space to the oral environment . Of interest is, however, that most cases presenting extra-radicular biofilms are associated with sinus tracts which may indicate inclusion of oral fluids during biofilm formation. The latter hypothesis is sustained with the finding of calculus-like extra-radicular biofilms [33, 34, 36]. Figure 3.3 shows a case of maxillary right central and lateral incisors with deficient root canal treatments and presenting calculus-like material covering the apexes of roots . Upon clinical inspection, an open fistula was detected in the apical area of teeth 11 and 12. Both teeth were sensitive to percussion, and the apical mucosa was sensitive to palpation. Radiographic examination showed a large periapical lesion with a thick layer of radiopaque material covering both root tips. Treatment included orthograde retreatment followed by apical surgery. As it is visualized in the clinical photograph, during surgery both root tips presented a calculus-like material covering the root surfaces. Examination by scanning electron microscopy (SEM) of the resected specimens confirmed the presence of mineralized biofilms in the apexes, where cells were embedded in mineralized matrix in a very similar fashion as supra- or sub-gingival calculus. One of the possible explanations for the occurrence of these mineralized structures is the long-standing sinus tract which may have allowed passage of fluids from the oral environment, including minerals and salts that could form these solid mineralized masses.
Case presenting extra-radicular biofilm formation in the form of calculus. (a) Preoperative radiograph shows extensive calcifications on the apex of 12 and 11. (b) Post-operative radiograph taken after orthograde root canal retreatment. (c) Postsurgical radiograph. (d) Clinical photograph during surgical procedure shows the bone defect and a dark calculus-like structure covering the apex of teeth 12 and 11. (e) Representative scanning electron microscopy (SEM) micrograph showing clusters of cells forming extra-radicular mineralized biofilm structures. Case is published in 
3.4 Host-Microbe Interactions
The presence of biofilms in root-filled teeth leads to a chronic inflammatory reaction in the periapex which is characterized by the proliferation of macrophages, lymphocytes and plasma cells [37, 38]. Chronic apical lesions become encapsulated in collagenous connective tissue which is stimulated by the upregulation of connective tissue growth factors (TGF-β) [37, 38]. In this chronic phase which can remain symptomless for long periods of time, activated T cells produce cytokines that downregulate the output of pro-inflammatory cytokines (IL-1, IL-6 and TNF-α), leading to the suppression of osteoclastic activity and reduced bone resorption [37, 38]. Upon a secondary invasion of microorganisms, the lesion can spontaneously turn into an acute inflammatory reaction by rapid recruitment of PMNs , a feature that is characterized by a rapid restitution of apical bone resorption. And the previous silent clinical situation may suddenly turn into a symptomatic phase.
The chronic inflammatory lesion associated to failed-root canal treatments is in many cases associated to the presence of well-developed fibrous capsules consisting of dense collagenous fibres that are firmly attached to the root surface [37, 38]. These chronic lesions, also known as granulomas , do not normally harbour microorganisms but only in special cases: (a) acute inflammatory phase , (b) periapical actinomycosis [40–42], (c) transient contamination during root canal instrumentation [43, 44] and (d) infected periapical cysts with cavities open to the root canal . The main function of the apical granuloma is thus to contain and encapsulate the advancement of the infection. In the lumen of the granuloma, macrophages, including blood-derived macrophages, epithelioid cells and multinucleated giant are aimed to kill bacteria. However, complete eradication of bacteria does not always occur. As it has been described in few case reports [40–42], species of Actinomyces and Propionibacterium (formerly Arachnia) have been found forming clusters within the lumen of the granulation mass. Although the mechanisms behind clustering formation are not clear, it may seem that clustering occurs as a microbial strategy to persist within the granuloma and perhaps to reactivate and escape under special circumstances.
The survival of Mycobacterium tuberculosis in granulomatous tissues is a good example for understanding the mechanisms behind bacterial survival within a granuloma. It has been proposed that the unfavourable conditions inside the granuloma, such as nutrient limitation and low oxygen tension, trigger the metabolic downshift of M. tuberculosis into dormancy . Of critical interest is, however, that under specific circumstances M. tuberculosis re-establishes its metabolic and replicative activity by the activation of a complex cascade of enzymes regulated by resuscitation-promoting factors (Rpf) . Although it has not been established if Rpf orthologs are present in Actinomyces or other endodontic pathogens, the reactivation from a dormant state seems to be an interesting hypothesis to clarify the occurrence of exacerbations of chronic infections. This hypothesis was tested in an experimental study, where biofilm cultures of S. anginosus and L. salivarius were forced to enter a state of dormancy by exposing them to nutrient deprivation . Dormant cells were then forced to reactivate by exposure to fresh nutrients, but even after 96 h the cells remained metabolically inactive. This observation highlights the null physiological response of dormant cells even in the presence of fresh nutrients, which may act as a mechanism to resist further disturbances.
3.5 Resistance vs. Tolerance
The increased survival rate of bacteria is one of the fundamental causes of endodontic treatment failure and because chronic infections present as a complicated challenge [1–3]. In order to understand the mechanisms by which bacteria survive, it is important to differentiate two main concepts: resistance and tolerance. As it is illustrated in Fig. 3.4, resistance comprises the mechanisms that are specifically exerted by bacteria in the presence of antimicrobials and that are aimed to inactivate them. Common resistance mechanisms include physical prevention of the antimicrobials from reaching its target (e.g. low diffusion through the biofilm matrix), alteration of the target such that it is no longer recognized by the antimicrobial (e.g. modification of cell receptors) and inactivation of the antibiotic properties to obstruct its ability to interact with its target . Tolerance is fundamentally different as it does not affect the ability of the antimicrobial to interact with its target. Although the molecular events that lead to antimicrobial tolerance in bacteria are not yet clear, the mechanisms that are involved seem to be mainly controlled by phenotypic adaptive processes (e.g. metabolic downregulation or adaptation) . Phenotypic tolerance is elicited as a result of environmental factors (such as nutrient deprivation and pH changes) that affect antimicrobial-induced killing, whereas genotypic tolerance can arise from specific genetic changes within the tolerant bacteria .
Schematic illustrating the differences between microbial resistance and tolerance. (a) Cell death is normally expected after treatment of a bacterial population with an antimicrobial. (b) Resistance is regulated by mechanisms that are specifically exerted by bacteria to restrain the interaction of antimicrobials with cells. (c) Tolerance comprises mechanisms of phenotypic adaptation in the presence of antimicrobials upon interaction with the cells
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