CONTINUING EDUCATION

Advances in microbiology and bacteriology

Dr. Navid Saberi presents an overview of microbiology and bacteriology in endodontic treatment

t has been almost 340 years since the Ibirth of microbiology and the discovery Educational aims and objectives of by Robert Hooke and Antonie This clinical article aims to present an overview of the many advances in microbiology van Leeuwenhoek (Gest, 2004) and around and bacteriology, and which bacteria are safe to leave in the root canal after endodontic treatment. 150 years since the dawn of modern bacteriology by the concurrent and rivalry- Expected outcomes driven studies of Robert Koch and Louis Endodontic Practice US subscribers can answer the CE questions on page XX to Pasteur (Ullmann, 2007). earn 2 hours of CE from reading this article. Correctly answering the questions will In the field of endodontology, W.D. Miller demonstrate the reader can: was the first to associate bacteria with pulpal • Realize the many advances in microbiology and bacteriology. • Identify how these remaining bacteria can be detected. disease in 1894. However, even prior to • Recognize which bacteria are safe to remain in the root canals. Miller’s experiments, dental practitioners • Identify how many bacteria is it safe to leave in the root canal after endodontic treatment. were aware of the important role of disinfec- tion and antiseption in endodontic outcomes. In fact, in 1886, Dental Cosmos published therapy is therefore to achieve this goal by of bacterial detection have been associated a report titled “Disinfection and antiseption mechanical instrumentation and biochemical with some drawbacks. by heat.” According to the author, in this irrigation of the root canal systems, which can Culturing has been advocated as a rather procedure “a fine wire heated to redness and be referred to as biomechanical cleansing of simple means of bacterial identification. inserted into the canal to the apical foramen infected root canals. However, up until the late 1960s, culturing … the application repeated according to However, despite being desirable to methods were unable to propagate anaer- circumstances … is all that is necessary” and render the root canals sterile, it is logical and obic bacteria predictably. The routine use of “by his [Dr. G.O. Rogers’] method complete realistic to assume that achieving sterility may anaerobic glove box and sterilized anaerobic disinfection and antiseption are secured” not be feasible in every infected root canal. In culture media has significantly enhanced (Pomeroy, 1886). fact, sterility may not even be required for a accurate identification of these bacteria. In addition, devitalization by means of successful outcome. Actor (2012) reported Furthermore, standardized field decontami- arsenic trioxide and instrumentation with that there are 20 times more bacterial cells nation protocols in the late 1960s reduced modified watch springs were commonplace in human bodies than eukaryotic cells. the possibility of false positive results (Moore, (Grossman, 1982; 1987). These communities live in either a symbiotic 1966; Kantz and Henry, 1974; Ng, et al., It is now well established that bacteria or mutualistic manner and benefit from the 2003). are essential for the development of pulpal existence of one another without causing Although cultivability of all bacteria was and periradicular diseases (Kakehashi, et al., harm. Therefore, at least in theory, it can be first reported in the 1960s, it was not until 1965; Möller, et al., 1981), and the presence assumed that some bacteria in inaccessible the 1990s that scientists fully recognized of bacteria in the root canal space or peri- areas of the root canal systems may be left that not all bacteria were culturable or iden- apical tissue will undermine the success of behind without compromising the overall tifiable (Socransky, et al., 1963; Hugenholtz endodontic treatment (Sjögren, et al., 1997; outcome of the treatment. and Pace, 1996). This meant previous Byström, et al., 1987). In spite of many advances in micro- bacteriological studies based on culturing Therefore, for a successful treatment, all biology and bacteriology, there still remain alone probably discovered only half of the root canals should be identified, thoroughly many unanswered questions. How can we true taxa associated with endodontic infec- disinfected, and rendered bacteria-free prior detect these remaining bacteria? Which tions (Paster, et al., 2001). These uncultivable to obturation. The aim of modern endodontic bacteria are safe to remain in the root canals? bacteria could indeed be the more impor- And, more importantly, how many bacteria tant entities in the pathogenesis of periapical Navid Saberi, BDS, MFDSRCS, MSc, graduated from Manchester is it safe to leave in the root canal after disease. University and worked in the general, salaried, and hospital dental endodontic treatment? The development of polymerase chain services in Scotland for several years. He completed his MSc The aim of this article is to provide reaction (PCR) in the late 1980s and its use with distinction at Glasgow University and became a member of the faculty of dental surgery of the Royal College of Surgeons answers to the above questions. in endodontics by the early 1990s radi- of Edinburgh in 2010. He has limited his practice to endodontics cally changed the dogma that had been and works at several prestigious private practices in London, the Bacterial identification founded on a culture-based understanding South East of England, and the South East of Scotland, including Harley Street Dental Group in London, Elms Lea Dental Practice In order to determine the safe number of endodontic microbiology (Mullis and in Brighton, and Wessex Specialist Dental Centre in Hampshire, of bacteria that may possibly be left in root Faloona, 1987; Pollard, et al., 1989; Spratt, just to name a few. He co-founded the Scottish Endodontic Study canals after endodontic treatment, it is Weightman, and Wade, 1999). Group in 2010 and is an active member of the British Endodontic Society. imperative for researchers to identify these PCR, however, is not without pitfalls. microorganisms. However, most methods This highly sensitive method of nucleic acid

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amplification requires a strict decontami- nation protocol if false positive results are to be avoided (Ng, et al., 2003). In addi- For a successful treatment, tion, Hayden and colleagues (1991) found that PCR amplification methods inherently all root canals should be identified, detect bacteria without specifying whether the microorganisms were dead or alive at thoroughly disinfected, and rendered bacteria-free the time of sampling. This predicament can be overcome by amplifying genomic ribo- prior to obturation. somal ribonucleic acid (RNA), which can only be detected in active and dividing cells. However, bacteriological results based on ribosomal ribonucleic acid (rRNA) amplifica- tion should also be scrutinized for method- of these bacteria could establish an Therefore, a certain combination of ology as a very short half-life of rRNA could (Sundqvist, 1994; Paster, et al., 2006). These bacteria, a favorable environment, nutrient potentially lead to false negative results if the bacteria live in symbiosis with the host within availability, and the host response must be procedure is not carried out swiftly. the oral cavity (Avila, Ojcius, and Yilmaz, the main determining factors in the patho- In spite of all major improvement in 2009). Nonetheless, they are opportunistic genesis of apical periodontitis (Siqueira and bacteriological identification methods, one microorganisms and may cause disease, Rocas 2009a,b; Paster, et al., 2006). major shortcoming still remains unsolved, particularly where the host loses the ability to The main inhabitants of primary and that is access to intraradicular micro- maintain the homeostasis in the ecosystem endodontic infection are gram negative organisms. Even though the main root canal (Actor, 2012). anaerobic rods with different pathogenicities. and associated lateral canals may be acces- As explained earlier, almost half of The most common bacterial involved sible for direct or indirect sampling, most of disease-producing endodontic bacteria are in primary endodontic with their the root canal system, including canal walls, uncultivable (Hugenholtz and Pace, 1996). genera, phyla, and degree of pathogenicity dentinal tubules, isthmuses, fins, and webs, Although with the aid of PCR many uncul- are shown in Table 1 (Siqueira and Rocas may be untouched, inaccessible, or blocked tivable bacteria have been cloned, they are 2009b,c; Siqueira, et al., 2009; Ribeiro, 2011). by debris, hence making accurate sampling yet to be named and meticulously analyzed It is important to mention, however, that an impossible task. In addition, the discovery (Rolph, et al., 2001; Munson, et al., 2002, individual virulence of single species does of biofilms transformed our understanding Siqueira and Rocas, 2005). Therefore, a not directly translate to an overall degree of of bacterial ecology, according to Costerton thorough knowledge of the type, character- pathogenicity of a bacterial colony (Siqueira and colleagues (1994; 1999). Planktonic istics, metabolism, pathogenicity and partic- and Rocas, 2009a,c). Virulence is a direct root canal bacteria seldom sustain peri- ularly interactions of these microorganisms result of bacterial colonization of the root apical disease (Siqueira and Rocas, 2009a). is of utmost importance. This is especially canal as multispecies biofilms, in which A successful endodontic ecosystem may essential if an association between required the entire community as a whole is respon- be composed of complex arrangements numbers of bacteria, host response, and sible for the degree of pathogenecity of the of multispecies bacteria living in symbiotic pathogenesis is to be drawn. disease. This concept will be discussed later. biofilms, in which homeostasis is achieved Siqueira and Rocas found that most Apart from bacteria, other microorgan- by collaboration between the species that bacteria associated with primary endodontic isms such as fungi, archaea, and viruses may are attached firm and deep into root canal infections have been classified under nine also be found within an infected root canal dentinal walls and tubules (Siqueira and phyla with the aid of culture and molecular ecosystem (Peciuliene, et al., 2001; Jiang, Rocas 2009a,b; Avila, et al., 2009). These analysis (2009b). These phyla are: et al., 2009; Vianna, et al., 2009; Sabeti, et biofilms may be pathogenic to the host or • al., 2003). However, in order to focus on the live in symbiosis with the host. Current bacte- • Bacteroidetes main aim of this article, the role of bacteria riological sampling techniques may not be • Spirochaetes will only be discussed here. able to identify all the constituents of these • Proteobacteria Although the bacterial profiles of most biofilms, especially if they are situated in inac- • Fusobacteria primary endodontic infections illustrate vast cessible parts of root canal systems. • Actinobacteria diversity of microorganisms comprising a In order to envisage and analyze different • Synergistes few hundred species, the majority of these possible combinations of bacteria in an • TM7 microorganisms get destroyed and removed endodontic ecosystem and its effect on • SR1 by biomechanical cleansing action of root pathogenicity, host response, and eventual The phyla with the highest species rich- canal treatment procedure or become endodontic outcome, it is prudent to review ness are firmicutes, bacteroidetes, actino- inactivated and die after root canal obtura- the characteristics of root canal microbiota. bacteria, and proteobacteria. These phyla tion due to lack of nutrients and an unfavor- contain around 460 taxa belonging to 100 able ecosystem (Siqueira and Rocas, 2004; Endodontic microbiota genera. 2005; 2009a,b,c; Niazi, et al., 2010). These It is believed that over 300 bacte- However, these bacterial profiles asso- bacteria do not appear in samples taken rial species are capable of colonizing root ciated with apical periodontitis are not the from failed root canal treated cases. Hence, canals containing necrotic pulp, according same in all individuals, which implies hetero- the attention and focus should be drawn to Sundqvist (1994). However, only a fraction geneity of the disease. toward the bacteria involved in secondary

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Table 1: Common bacterial species identified in association with most primary endodontic infections and persistent intracanal infections. In addi- Phylum Genus Morphology Species Pathogenicity tion, their mode of action, characteristics,

1 Firmicutes Dialister Gram negative anaerobic rod D. invisus .++ especially with regard to withstanding biome- D. pneumosintes .++ chanical cleaning of endodontic procedure, Gram negative uncultivated .++ species inter- and intraspecies associations, and their Filifactor Gram positive anaerobic rod F. alcois .+ Peptostreptococcus Gram positive anaerobic coccus P. micros symbiotic affiliations must be analyzed. P. anaerobius Root canal treated teeth have been Gram positive uncultivated species .++ Pseudoramibacter Gram positive anaerobic rod P. alactolyticus .+ shown to harbor only a mean number of one Enterococcus Gram positive facultative coccus E. faecalis .+ Eubacterium Gram positive anaerobic rod .+/++ to six bacterial species per tooth (Siqueria E. saphenum .+/++ and Rocas, 2009b,c). E. nodatum .+/++ E. brachy .+/++ In contrast to primary endodontic infec- E. minitum .+/++ Mogibacterium Gram positive anaerobic rod M. timidum .+ tions, culture-dependent studies demon- M. pumilum .+ strated that persistent and secondary M. neglectum .+ M. vescum .+ endodontic infections were more likely to Streptococcus Gram positive facultative coccus S. mitis .+/++ S. sanguinis .+/++ contain more gram positive bacteria (Chávez S. gordonii .+/++ de Paz, 2004). These included streptococci, S. oralis .+/++ Gram positive anaerobic coccus S. anginosus .++ lactobacilli, staphylococci, enterococcus S. constellatus .++ S. intermedius .++ faecalis, propionibacterium spp, poliana Veilonella Gram negative anaerobic coccus V. parvula .+ micra, and pseudoramibacter alactolyticus, Gram negative uncultivated .+ species to name just a few (Byström and Sundqvist, Lactobacillus Gram positive anaerobic rod L. catenaformis .+ Gram positive facultative rod L. salivarius .+ 1985; Sjögren, et al., 1997; Gomes, et al., L. acidophilus .+ 1996; Peters, et al., 2002; Chávez de Paz, L. paracasei .+ Cantonella Gram negative anaerobic rod C. morbi .+ et al., 2003; 2004; 2005; Chu, et al., 2006). Granulicatella Gram negative facultative G. adiacens .+ coccus However, molecular analysis of persistent Selemomonas Gram negative anaerobic rod S. sputigena .++ endodontic infections has revealed that S. Noxia .++ Gram negative uncultivated .++ almost 42% of the samples contained species Finegoldia Gram positive anaerobic coccus F. magna .++ uncultivated bacteria, which were the domi- Peptoniphilus Gram positive anaerobic coccus P. asaccharolyticus .+ nant taxa (Sakamoto, et al., 2007). The P. lacrimalis .+ Anaerococcus Gram positive anaerobic coccus A. prevotii .+ main bacteria associated with secondary Gemella Gram positive anaerobic coccus G. morbillorum .++ 2 Bacteroidetes Prevotella Gram negative anaerobic rod P. intermedia .++/+++ and persistent endodontic infections are P. nigrescens .++/+++ presented in Table 2 (Siqueira and Rocas, P. tannerae .++/+++ P. multissachariovorax .++/+++ 2005; Niazi, et al., 2010). P. baroniae .++/+++ P. denticola .++/+++ Unfortunately, most outcome, medica- Gram negative uncultivated .++/+++ tion, and irrigation studies that evaluated the species Porphyromonas Gram negative anaerobic rod P. endodontalis .+++ association between bacteria and success P. gingivalis .+++ Tannerella Gram negative anaerobic rod T. forsythia .+++ of root canal treatment have concentrated Capnocytophaga Gram negative facultative rod C. gingivalis .+ only on the presence or total absence of C. ochracea .+ 3 Spirochaetes Treponema Gram negative anaerobic spirilla T. denticola .++/+++ cultivable bacteria. Most of these studies T. parvum .++/+++ T. socranskii .++/+++ have demonstrated that only a negative T. maltophilum .++/+++ culture at the time of obturation will lead T. lecithinolyticum .++/+++ 4 Proteobacteria Campylobacter Gram negative anaerobic rod C. rectus .++ to a successful outcome (Engstrom, et al., C. gracilis .++ 1964; Heling and Shapira, 1978; Byström C. curvus .++ C. showae .++ and Sundqvist, 1985; Sjogren, et al., 1997; Eikenella Gram negative facultative rod E. corrodens .++ Neisseria Gram negative facultative cocci N. mucosa .+ Shuping, et al., 2000; McGurkin-Smith, et N. sicca .+ al., 2005; Fabricius, et al., 2006). However, Aggregatibacter Gram negative anaerobic rod A. aphrophilus .+ 5 Fusobacteria Fusobacterium Gram negative anaerobic rod F. nucleatum .++ there are two major flaws with these studies F. periodonticum .++ Gram negative uncultivated .++ and the conclusions and theories drawn from species them. 6 Actinobacteria Actinomyces Gram positive anaerobic rod A. israelli .+/++ A. gerencseriae .+/++ The first issue is the fact that, as A. meyeri .+/++ A. odontolyticus .+/++ discussed earlier, not all bacteria are culti- Gram positive facultative rod A. naeslundii .+ vable (Hugenholtz and Pace, 1996; Paster, et Corynebacterium Gram positive facultative rod C. matruchotti .+ Propionibacterium Gram positive anaerobic rod P. acnes .++ al., 2001; Sakamoto, et al., 2007). In fact, the P. propionicum .++ Olsenella Gram positive anaerobic rod O. uli .+ majority of bacterial taxa that remain inside O. profusa .+ the root canals and withstand biomechanical Slackia Gram positive anaerobic rod S. exigua .+ Eggerthella Gram positive anaerobic rod E. lenta .+ cleaning procedures were only recently Gram positive anaerobic rod B. dentium .+ Atopobium Gram positive anaerobic rod A. parvulum .+ discovered by means of PCR and are yet A. minutum .+ to be cultivable (Sakamoto, et al., 2007). A. rimae .+ 7 Synergistes Synergistes Gram negative anaerobic rod Gram negative uncultivated Therefore, negative cultures in the previous species studies that illustrated endodontic success 8 TM7 Clone I025 9 SR1 Clone X112 may have, in actual fact, contained bacteria.

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Table 2: Common bacterial species identified in association with failed root The host canal treated teeth (secondary, persistent, and refractory) As explained previously, some of the Phylum Genus Morphology Species major factors that contribute toward the 1 Firmicutes Dialister Gram negative anaerobic rod D. pneumosintes sequelae of apical periodontitis are the D. invisus Gram negative Clone BS095 host and host-bacteria interactions within a uncultivated species homeostatic symbiotic environment (Actor, Filifactor Gram positive anaerobic rod F. alcois Peptostreptococcus Gram positive anaerobic coccus P. micros 2012). It is generally acknowledged that P. stomatis in an event of host defense depression, Pseudoramibacter Gram positive anaerobic rod P. alactolyticus Enterococcus Gram positive facultative coccus E. faecalis opportunistic bacteria that possess essen- Eubacterium Gram positive anaerobic rod E. minitum tial virulence factors may give rise to disease,

E. saburreum according to Actor (2012). Disease initiation E. sulci is carried out by means of exotoxin secre- E. yurii Mogibacterium Gram positive anaerobic rod M. diversum tion via viable bacterial cells, by endotoxins M. neglectum M. timidum arising from byproducts of cell lysis or from Streptococcus Gram positive facultative coccus S. gordonii certain components of bacterial cell wall S. cristatus S. oralis such as lipopolysaccharides, proteins, lipo- S. sanguinis proteins, glycoproteins, carbohydrates, and S. mitis S. mutans lipids, to name just a few (Casadevall and S. sp Oral taxon 071 S. sp C8 Pirofski, 2001; Lawrence, 2005; Actor, 2012). S. australis Moreover, bacterial toxins include entero- S. infantis Gram positive anaerobic coccu s S. anginosus toxins, neurotoxins, cytotoxins, and lysins. S. constellatus These toxins are virulence factors and are Staphylococcus Gram positive facultative coccus S. capare recognized by the host as pathogens and S. warneri Veilonella Gram negative anaerobic coccus V. dispar trigger cytokine release, such as interleukins, Lactobacillus Gram positive facultative rod which leads to the activation of the inflamma- Cantonella Gram negative anaerobic rod Granulicatella Gram negative facultative coccus tion system (Actor, 2012). Therefore, it may Selemomonas Gram negative anaerobic rod be assumed that if endodontic bacteria lack Finegoldia Gram positive anaerobic coccus Gemella Gram positive anaerobic coccus G. haemolysans the ability to produce virulence factors, or 2 Bacteroidetes Prevotella Gram negative anaerobic rod P. intermedia Porphyromonas Gram negative anaerobic rod P. gingivalis more importantly, the host defense mecha- P. endodontalis nism efficiently depresses the formation of Tannerella Gram negative anaerobic rod T. forsythia Capnocytophaga Gram negative facultative rod such factors within the homeostatic symbi- 3 Spirochaetes Treponema Gram negative anaerobic spirilla T. denticola otic environment, at least in theory, apical 4 Proteobacteria Campylobacter Gram negative anaerobic rod C. rectus C. gracilis periodontitis may not ensue. 5 Fusobacteria Fusobacterium Gram negative anaerobic rod F. nucleatum 6 Actinobacteria Actinomyces Gram positive anaerobic rod A. radicidentis The other aspect of the host that is as Propionibacterium Gram positive anaerobic rod P. acnes important as the host defense inflammatory P. propionicum Olsenella Gram positive anaerobic rod O. uli reaction is the dentin and intracanal host O. profusa ecosystem, as bacteria require a suitable Slackia Gram positive anaerobic rod S. exigua 7 Synergistes Synergistes Gram negative anaerobic rod Gram negative Clone BA121 surface to be able to colonize on. The dentin uncultivated species is capable of influencing the ecosystem by regulating the pH, modifying the anaerobic The second issue is the access to the 2005), and a successful outcome may be metabolism of bacteria, providing nutri- bacteria. Not all bacteria are accessible achieved even in the presence of residual ents and suitable attachment surface, and to direct or indirect sampling. Isthmuses, bacteria, other factors must contribute accommodating root filling materials and dentinal tubules, lateral canals, webs, toward the sequelae of apical periodon- sealers. and fins may all harbor bacteria in biofilm titis. These may be the host, the number Unfortunately, studies with regard to the structures. Negative cultures in previous of bacteria, the correct combination of influence of the dentin on intracanal homeo- outcome studies might have failed to demon- bacteria, and/or an interaction between static symbiotic environment and bacterial strate these bacteria. Therefore, previous these elements. interaction are scarce. successful cases and acceptable outcomes Siqueira and Rocas (2009c) found that Bacteria can also directly invade and may have been achieved in the presence of this interaction may lead to a successful damage the host cells by releasing enzymes bacteria. As a result, it may be concluded outcome if all aforementioned elements are in such as collagenases and hyaluronidases that although total sterility is indeed ideal a “homeostatic symbiotic environment.” Any and metabolites such as short-chain fatty and desirable for a successful outcome, breach in this equilibrium may consequently acids. These elements can sometimes exert rendering root canals bacteria-free is neither lead to disease formation. This breach could their mode of action without being detected practical nor achievable in every case. arise as a result of host defense depression, by the host defense mechanism (Hashioka, As most bacteria can be eliminated an increase in the numbers or a develop- 1994; Lawrence, 2005). by adequate biomechanical cleaning of ment of a more favorable environment for the endodontic procedures (Byström and the “essential bacteria” within the commu- Interactions between bacteria Sundqvist, 1985; Shuping, et al., 2000; nity, such as in an event of a rise in nutrient Bacteria are intelligent microorgan- Spratt, et al., 2001; McGurkin-Smith, et al., availability. isms. As explained previously, almost all

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bacteria that inhabit root canals are oral cavity commensals. Although many of these commensal bacteria are opportunistic patho- The aim of modern endodontic therapy is therefore to achieve gens waiting for an “opportune” moment to initiate disease, several bacteria do not this goal by mechanical instrumentation and biochemical inherently possess high virulence but can still cause pathogenicity if accompanied by irrigation of the root canal systems, which can be referred to certain other bacterial species within a biofilm (Casadevall and Pirofski, 2001; Moine and as biomechanical cleansing of infected root canals. Abraham, 2004; Drenkard, 2003). Bacterial virulence acquisition can be performed by means of gene switching and horizontal gene transferring in which valuable genetic material can be transferred between achieve homeostasis, perform better in a (Johnson, Flannagan and Sedgley, 2006; species through extrachromosomal plasmids community as opposed to in isolation, and Khameleelakul, Baumgartner and Pruk- (Lawrence, 2005; Kunin, et al., 2005). This be able to respond to changes as a unit. sakom, 2006). In contrast, species of strep- is a very beneficial mechanism for propa- Apart from proficient communication tococci, probionibacterium propionicum, gating vital genes. Gene transferring can through quorum sensing and genetic trans- capnocytophaga ochracea, and veillonella take place between species; therefore, it fers within and in between species, biofilms parvula have been demonstrated to exhibit will benefit all bacteria within a community improve the survival of the bacteria by nutrient no or negative associations with one another (Lorenz and Wackernagel, 1994; Wang, et trapping, establishing metabolic coopera- (Sundqvist, 1992). Moreover, porphyromonas al., 2002; Chen, et al., 2004; Nallapareddy, tivity, and reducing the risk of desiccation by gingivalis and streptococcus gordonii have et al., 2005). In addition, it is faster than chro- means of extracellular polysaccharide exer- been shown to coexist within a community mosomal gene propagation by means of cell tions and efficient internal water channels but not in the presence of S. mutans (Love division. Furthermore, it enables all species (Costerton, et al., 1994; 1997; 1999). and Jenkinson, 2002; Love, et al., 2000). within a bacterial community to rapidly Not all bacteria are capable of forming In the field of endodontic microbiology, adapt themselves to an adverse change in biofilms. However, once a biofilm is formed many studies have been conducted in the ecosystem. Adaptation to changes in by the interaction of one or several species, order to identify the bacteria responsible the pH, nutrition availability, lack of oxygen, other species can join the community as for the pathogenicity of apical periodontitis. and developing resistance to medicaments long as they possess the specific charac- However, not even one high-quality research are a few examples (Hayes, 2003; Martinez teristics mentioned above (Costerton, et could be found on the issue of the relation of and Baquero, 2002; Socransky and Haffajee, al., 1999; Cowan, Taylor, and Doyle, 1987; bacterial load, with regard to the numbers, 2000). For this precise interaction to occur, Al-Hashimi and Levine, 1989; Handley, to pathogenicity. bacteria need to exist in a cohesive, suit- Carter, and Fielding, 1984). In root canals, It is logical to assume that total sterility able, and protective environment. This E. faecalis can form different biofilms based of root canals, despite being desirable, is setting facilitates the communication and on the changes in the ecosystem and survive seldom achievable. Furthermore, sterility transfer of information and materials between in difficult conditions (Gentry-Weeks, et al., may not even be essential for the treatment different bacteria and improves their chance 1999; Capiaux, et al., 2000; Fidgor, Davies, outcome. Hence, identifying bacterial load of survival. This cohesive, suitable, and and Sundqvist, 2003). threshold for the development of apical peri- protective setting is referred to as the biofilm. Siren and colleagues (1997) reported that odontitis could be of utmost importance. fusobacterium nucleatum is capable of co- The number of colony-forming units Biofilm, community, and colony- existing with enterococcus faecalis within the (CFU) in infected root canals has been esti- forming units same community by means of coaggrega- mated to be between 103 and 108 (Siqueira According to Costerton and colleagues tion. Fusobacterium nucleatum may in fact be and Rocas, 2005; Vianna, et al., 2006), and (1994), a biofilm is “a mode of microbial an important link between different species a direct association has been established growth where dynamic communities of inter- in endodontic microbiota. Fusobacterium between the radiographic dimensions of peri- acting sessile cells are irreversibly attached nucleatum and many streptococci have apical lesions and the number of CFUs. This to a solid substratum, as well as each other, been revealed to coaggregate by bridging number is probably somewhere between and are embedded in a self-made matrix (Kolenbrander, Andersen, and Moore, 1989; 102 and 103 in secondary and persistent of extracellular polymeric substances.” It is Lancy, et al., 1983). Furthermore, positive infections with lower number of species important to state that while bacteria create associations have been identified between involved per root canals. However, there is no the biofilms to improve their interaction and fusobacterium nucleatum and poliana micra, evidence on the bacterial load (CFU) required survival, they inherently require a host and a porphyromonas endodontalis, selenomonas for the induction of periapical disease. This surface — in this case, the root canal dentin sputigena, and campylobacter rectus in teeth lack of evidence may be a result of three — to colonize. This is yet another example with apical periodontitis (Sundqvist, 1992). main problems facing research in this field. of the importance of the host in the sequelae Other community-forming species appear The first major problem associated with of apical periodontitis. to be streptococcus anginosus, peptostrep- analyzing the required CFU is the sensi- Furthermore, Costerton and colleagues tococcus anaerobius and prevotella oralis tivity of the tests. The sensitivity of culture- (1994; 1999) reported that the bacteria and prevotella, streptococcus and fuso- dependent investigation is at best around within biofilms must be able to self-organize, bacterium in acute endodontic infections 103 cells per milliliter, according to Zambon

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and Haraszthy (1995). Siqueria and Rocas It is important to point out that it is not such as fungi and archaea in the ecosystem showed that PCR can potentially reduce this being suggested here that leaving bacteria should also be further investigated. number to 1 cell per milliliter (2003). However, in root canals should be actively practiced. In the future, endodontic treatment may at least for the time being, no study has Meticulous root canal treatment with thor- move toward active biologic therapeutics managed to achieve this number within the ough biomechanical cleansing and use of with the aid of site-specific and highly selec- field of endodontics. medicaments or sampling, where neces- tive probiotics that can provide a cascade of Realistic and achievable sensitivity for the sary, must be carried out in every case. Our desirable biofilms across the root canals, and PCR is estimated to be 10 cells per milliliter. aim must always be the elimination of the disturb all pathogenic activity of the micro- This lack of sensitivity hampers the accurate bacteria or the reduction of the numbers biota that are capable of initiating apical estimation of bacterial numbers required to to as low as biologically and anatomically periodontitis. establish apical periodontitis (Siqueira and possible. Hopefully, if this number is below In addition, the development of advanced Rocas, 2003). the disease initiation threshold and stays at methods of real-time detection of bacteria The second problem, as explained this level, success will ensue. presence will be extremely valuable. A before, is the anatomy of most root canals, definitive sealing of the system, including its which makes direct, accurate, and predict- Conclusion extremities, will then prevent reinfection, and able sampling almost impossible in most Although over a century has passed is of utmost importance. EP cases. As a result, occurrence of false nega- since what might once have been referred tive samples will inherently lead to incorrect to as the “dark ages of endodontics,” and estimation of bacterial numbers. new materials, methods, and procedures REFERENCES The third problem is the bacterial inter- have been developed, the principles of root 1. Actor JK. Immunology and Microbiology. 2nd ed. Phila- action within a biofilm. Task allocation and canal treatment have somehow remained the delphia, PA: Elsevier; 2012. responsibility sharing among different bacte- same. These principles are the removal of the 2. Al-Hashimi I, Levine MJ. Characterization of in vivo salivary-derived enamel pellicle. Arch Oral Biol. rial species, together with genetic information necrotic pulp tissue and infected dentin from 1989;34(4):289-295. transfer between bacteria, makes identifica- the root canal system and filling the space. 3. Avila M, Ojcius DM, Yilmaz O. The oral micro- biota: living with a permanent guest. DNA Cell Biol. tion and counting of the real culprits a very It is now well established that bacteria 2009;28(8):405-411. difficult task. are essential for the development of pulpal 4. Bystrom A, Happonen RP, Sjogren U, Sundqvist G. Healing of periapical lesions of pulpless teeth after Furthermore, heterogeneity of the bacte- and periradicular diseases, and the presence endodontic treatment with controlled asepsis. Endod rial taxa in different individuals makes labo- of bacteria in the root canal space or peri- Dent Traumatol. 1987;3(2):58-63. ratory extrapolation of the numbers invalid. apical tissue will undermine the success of 5. Bystrom A, Sundqvist G. The antibacterial action of sodium hypochlorite and EDTA in 60 cases of endodontic In addition, even correctly identified and endodontic treatment. therapy. Int Endod J. 1985;18(1):35-40. counted bacteria may not necessarily be part Therefore, for a successful treatment, the 6. Capiaux H, Giard JC, Lemarinier S, Auffray Y. Character- ization and analysis of a new gene involved in glucose of the “essential bacteria” responsible for the canals should be thoroughly disinfected and starvation response in Enterococcus faecalis. Int J Food pathogenesis of apical periodontitis. In fact, rendered bacteria-free prior to obturation. Microbiol. 2000;55(1-3):99-102. 7. Casadevall A, Pirofski L. Host-pathogen interaction: the the unidentified bacteria may be as impor- However, it can be stated that total sterility attributes of virulence. J Infect Dis. 2001;184(3):337-334. tant, if not more important, in the pathogen- of root canals, despite being desirable, is 8. Chávez de Paz LE. Gram-positive organisms in esis of the disease than the identified taxa. seldom achievable. Furthermore, sterility may endodontic infections. Endod Topics. 2004;9(1):79-96. 9. Chávez de Paz LE, Dahlén G, Molander A, Möller A, Despite the importance of bacterial not even be essential for successful outcome Bergenholtz G. Bacteria recovered from teeth with apical periodontitis after antimicrobial endodontic treatment. Int load, the exact estimation of CFU may not of the treatment. Hence, identifying bacterial Endod J. 2003;36(7):500-508. be a crucial factor in managing endodontic load threshold for the development of apical 10. Chávez de Paz LE, Molander A, Dahlén G. Gram- infections if we consider the presence and periodontitis could be of utmost importance. positive rods prevailing in teeth with apical periodon- titis undergoing root canal treatment. Int Endod J. activity of the bacteria within a homeostatic Unfortunately, the identification of necessary 2004;37(9):579-587. symbiotic environment. The host defense, CFUs for the initiation of apical periodontitis 11. Chávez de Paz L, Svensäter G, Dahlén G, Bergenholtz G. Streptococci from root canals in teeth with apical peri- root canal dentin and intracanal microbiota poses several technological and biological odontitis receiving endodontic treatment. Oral Surg Oral may be able to live in symbiosis. issues. Med Oral Pathol Oral Radiol Endod. 2005;100(2):232-241. 12. Chen T, Hosogi Y, Nishikawa K, Abbey K, Fleischmann To be able to address this issue, more It is therefore not possible to estimate this RD, Walling J, Duncan M. Comparative whole-genome analysis of virulent and avirulent strains of Porphyromonas studies will be required to focus on the number with certainty. However, it can be gingivalis. J Bacteriol. 2004;186(16):5473-5479. following: concluded that due to heterogeneity of the 13. Chu FC, Leung WK, Tsang PC, Chow TW, Samaranayake • Interactions between the endodontic endodontic microbiota and the complexity LP. Identification of cultivable microorganisms from root canals with apical periodontitis following two-visit microbiota in secondary root canal of the endodontic ecosystem, a synergistic endodontic treatment with antibiotics/steroid or calcium infections, identification of the essen- collaboration of several elements are respon- hydroxide dressings. J Endod. 2006;32(1):17-23. 14. Costerton JW, Lewandowski Z. The biofilm lifestyle. Adv tial bacteria, their necessary numbers sible for a successful endodontic outcome. Dent Res. 1997;11(1):1. to initiate apical periodontitis, and These elements are all part of a homeo- 15. Costerton JW, Lewandowski Z, DeBeer D, Caldwell D, Korber D, James G. Biofilms, the customized their effect on endodontic outcomes static symbiotic environment and include the microniche. Journal of Bacteriology. J Bacteriol. • Microbiota and host/dentin symbi- host, the bacteria (numbers and essential 1994;176(8):2137-2142. 16. Costerton JW, Stewart PS, Greenberg EP. Bacte- osis and endodontic outcomes species), the ecosystem, and the interaction rial biofilm: a common cause of persistent infections. • Development of a live CFU measure- between these elements. Science. 1999;284(5418):1318-1322. ment device with high sensitivity More research is needed to analyze the 17. Cowan MM, Taylor KG, Doyle RJ. Energetics of the initial phase of adhesion of Streptococcus sanguis to hydroxyl- • Development of a standard labora- effect of these elements, both individually apatite. J Bacteriol. 1987;169(7):2995-3000. tory “homeostatic symbiotic environ- and as a unit, on endodontic outcomes. In 18. Drenkard E. 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19. Engstrom B, Hard AF, Segerstad L, Ramstrom G, Frostell of dentinal tubules by Porphyromonas gingivalis and tool for identification of endodontic pathogens. J Dent. G. Correlation of positive cultures with prognosis for root Streptococcus gordonii depends upon binding speci- 2003;31(5):333-339. canal treatment. Odontol Revy. 1964;15:257-270. ficity of streptococcal antigen I/II adhesin. Infect Immun. 68. Siqueira JF Jr, Rôças IN. Polymerase chain reaction- 2000;68(3):1359-1365. 20. Fabricius L, Dahlén G, Sundqvist G, Happonen RP, Möller based analysis of microorganisms associated with failed AJ. Influence of residual bacteria on periapical tissues 45. Martínez JL, Baquero F. Interactions among strate- endodontic treatment. Oral Surg Oral Med Oral Pathol healing after chemomechanical treatment and root filling gies associated with bacterial infection: pathogenicity, Oral Radiol Endod. 2004;97(1):85-94. of experimentally infected monkey teeth. 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