Journal of Dental Research http://jdr.sagepub.com Diversity of Endodontic Microbiota Revisited J.F. Siqueira, Jr. and I.N. Rôças J DENT RES 2009; 88; 969 DOI: 10.1177/0022034509346549 The online version of this article can be found at: http://jdr.sagepub.com/cgi/content/abstract/88/11/969 Published by: http://www.sagepublications.com On behalf of: International and American Associations for Dental Research Additional services and information for Journal of Dental Research can be found at: Email Alerts: http://jdr.sagepub.com/cgi/alerts Subscriptions: http://jdr.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav Downloaded from http://jdr.sagepub.com at UNIV OF PENNSYLVANIA on January 7, 2010 crItIcAL reVIews IN orAL bIoLoGy & MedIcINe J.F. Siqueira Jr.* and I.N. Rôças diversity of endodontic Department of Endodontics and Molecular Microbiology, Microbiota revisited Faculty of Dentistry, Estácio de Sá University, Av. Alfredo Baltazar da Silveira, 580/cobertura, Recreio, Rio de Janeiro, RJ, Brazil 22790-710; *corresponding author, jf_siqueira@ yahoo.com or [email protected] J Dent Res 88(11):969–981, 2009 AbstrAct Although fungi, archaea, and viruses contribute to the INtrodUctIoN microbial diversity in endodontic infections, bacteria are the most common micro-organisms occurring in essentially, endodontic infection is the infection of the dental root canal sys- these infections. Datasets from culture and molecular tem and the major etiologic agent of apical periodontitis (Siqueira, 2008). studies, integrated here for the first time, showed Although chemical and physical factors can induce periradicular inflamma- that over 460 unique bacterial taxa belonging to tion, a large body of scientific evidence clearly indicates that micro-organisms 100 genera and 9 phyla have been identified in are essential to the progression and perpetuation of the different forms of different types of endodontic infections. The phyla apical periodontitis (Kakehashi et al., 1965; Sundqvist, 1976; Möller et al., with the highest species richness were Firmicutes, 1981). For endodontic infection to develop, the root canal must be devoid of Bacteroidetes, Actinobacteria, and Proteobacteria. vital pulp tissue and its defenses, as a consequence of either pulp necrosis (as Diversity varies significantly according to the type of a sequel to caries, trauma, periodontal disease, or iatrogenic operative proce- infection. Overall, more taxa have been disclosed by dures) or pulp removal for treatment. The borderline between the infecting molecular studies than by culture. Many cultivable microbiota and the host defenses is often located intraradicularly, i.e., short of and as-yet-uncultivated phylotypes have emerged as or at the apical foramen. In some cases, however, micro-organisms may reach candidate pathogens based on detection in several the periradicular tissues, and the borderline is then situated extraradicularly, studies and/or high prevalence. Now that a com- i.e., beyond the boundaries of the apical foramen. prehensive inventory of the endodontic microbial Because apical periodontitis is an infectious disease, the rationale for taxa has been established, future research should endodontic treatment is inarguably to eradicate the occurring infection and/or focus on the association with different disease condi- to prevent micro-organisms from infecting or re-infecting the root canal or the tions, functional roles in the community, and suscep- periradicular tissues. The cardinal principle of any healthcare profession is the tibility to antimicrobial treatment procedures. thorough understanding of the disease etiology and pathogenesis, which pro- vides a framework for effective prevention and treatment. In this context, understanding and defining the endodontic microbiota associated with diffe- Key words: endodontic microbiology, molecu- rent forms of disease are the basis for an endodontic practice of high quality lar biology methods, culture, taxonomy. and founded on a solid scientific basis. Such knowledge has the potential to contribute to the development of more effective preventive and therapeutic protocols. Furthermore, establishing an inventory of endodontic bacteria may also help future investigations of potential sources of pathogenic species for diseases in other human sites. Traditionally, endodontic infections have been studied by means of culture approaches. Such studies have resulted in the establishment of a set of species thought to play an important role in the pathogenesis of apical periodontitis. More recently, not only have findings from culture-based methods been con- firmed, but they have also been significantly supplemented with those from culture-independent molecular diagnostic techniques. Molecular methods have confirmed and strengthened the association of many cultivable bacterial species with apical periodontitis and have also revealed suspected new DOI: 10.1177/0022034509346549 endodontic pathogens (Siqueira and Rôças, 2005b). The list of candidate pathogens has expanded to include culture-difficult species or even as-yet- Received March 28, 2008; Last revision October 1, 2008; uncultivated bacteria that had never been previously found in endodontic Accepted November 4, 2008; Updated July 3, 2009 infections by culturing approaches. As a consequence, the endodontic micro- A supplemental appendix to this article is published elec- biota has been refined and redefined by molecular methods (Siqueira and tronically only at http://jdr.sagepub.com/supplemental. Rôças, 2005b). 969 Downloaded from http://jdr.sagepub.com at UNIV OF PENNSYLVANIA on January 7, 2010 970 Siqueira and Rôças J Dent Res 88(11) 2009 Because knowledge of the endodontic infection associated with characterization of over 400 species (Rajilić-Stojanović et al., different clinical conditions originates from culture-dependent and 2007). Application of molecular biology techniques revealed culture-independent molecular biology studies, the most com- that the microbiota in the gut is significantly more complex prehensive view of the diversity of the endodontic microbiota than previously anticipated, since only a fraction of the bacte- requires integration of the data from both types of studies. This ria encountered can currently be cultivated (Suau et al., 1999; review focuses on the diversity of the microbiota in different types Eckburg et al., 2005). Another major outcome of the revolu- of endodontic infections and is mostly based on cataloguing the tion brought about by molecular methods was the recognition microbial taxa already identified in culture and molecular studies. that the composition of the microbiota is subject-specific and Overall, data from 128 studies published up to March 2008, were dominated by as-yet-uncultivated and uncharacterized phylo- used for compilation (listed in the Appendix). Culture studies inclu- types (Hold et al., 2002; Eckburg et al., 2005; Hayashi et al., ded in this compilation are restricted to those performed from the 2005). The integration of data from culture and molecular mid-1970s to 2008, when reliable methods for the cultivation and studies reveals that more than 1000 different taxa compose the identification of fastidious anaerobic bacteria were introduced and microbial diversity in the human gastrointestinal tract (Rajilić- adopted in endodontic microbiology research. Species names Stojanović et al., 2007). were considered valid when listed on the DSMZ Bacterial The oral cavity is another good example of the impact Nomenclature Web site (http://www.dsmz.de/microorganisms/ of culture-independent molecular biology methods on the bacterial_nomenclature.php) or mentioned in recent articles knowledge of the microbial diversity. Data from culture and published in the International Journal of Systematic and molecular studies have collectively revealed that almost 800 Evolutionary Microbiology. distinct bacterial taxa may be able to live in the human oral cavity, though not all of them are present in the same indivi- MoLecULAr stUdIes ANd dual at the same time (Paster et al., 2006). Indeed, any particu- lar individual can harbor about 100-200 of these 800 taxa of tHe UNcULtIVAted MAJorIty oral bacteria, indicating that there is a substantial diversity Culture methods have been highly successful over the past 100 among different people (Paster et al., 2006). Taken as a years, nurturing the expectation that most micro-organisms whole, bacteria detected from the oral cavity fall into 13 sepa- can be grown in the laboratory. It is now widely recognized rate phyla, namely, Firmicutes, Bacteroidetes, Actinobacteria, that culture techniques can strongly bias and underestimate the Proteobacteria, Spirochaetes, Fusobacteria, Synergistes (in - diversity of microbial populations (Hugenholtz, 2002; Relman, c luding phylotypes previously assigned to the phylum 2002). Only a small fraction of the bacteria present in most Deferribacteres), SR1 (originating from the OP11 division), microbial ecosystems is amenable to propagation ex vivo TM7, Chloroflexi, Deinococcus, Acidobacteria, and (Handelsman, 2004; Fredricks and Marrazzo, 2005). Less than Cyanobacteria (Paster et al., 2001, 2002; Kazor et al., 2003; 1% of bacteria have been estimated to be cultivated from envi- de Lillo et al., 2006; Aas et al., 2007). This number may be ronments such as sea water, lakes, and soil (Amann et al., even higher, since a recent study with DNA microarray tech- 1995; Gewin, 2006). To sidestep the limitations of culture, nology suggested that members of 4 other phyla (Aquificae, tools and procedures based on molecular biology have become Nitrospira, Planctomycetes, and Thermomicrobia) may have available and have been substantially improved to achieve a oral representatives, even though none has been identified more realistic description of the microbial world without the (Huyghe et al., 2008). A recent study with pyrosequencing, need for cultivation. The most significant contributions of a high-throughput molecular approach that allows for exten- culture-independent molecular biology methods to medical sive sequencing of microbial populations, revealed about microbiology relate to the identification of previously un known 5600 and 10,000 species-level phylotypes representing 22 human pathogens (Fredricks and Relman, 1996; Relman, phyla in saliva and plaque, respectively (Keijser et al., 1999; Kellam and Weiss, 2001; Lawson, 2004) and the disco- 2008). The estimated number of oral phylotypes is about very of a far broader diversity of the human microbiota asso- 20,000, which is considerably higher when compared with ciated with different human sites. Studies based on the 16S that reported in previous culture and clone library studies. rRNA gene approach have re-vealed that about 60% of the It is worth pointing out that both culture and molecular bacterial species in the oral cavity (Paster et al., 2001; Aas approaches have their own limitations in portrayals of microbial et al., 2005; Kumar et al., 2005; de Lillo et al., 2006), 50% on diversity. Limitations of culture have been extensively recog- the skin (Dekio et al., 2005), 38% in the esophagus (Pei et al., nized, and many of them have been successfully overcome by 2004), 50% in the stomach (Bik et al., 2006), 45% in the molecular technology (Fredricks and Relman, 1999; Hugenholtz, vagina (Fredricks et al., 2005), and about 80% in the gut (Suau 2002; Siqueira and Rôças, 2005a). However, molecular methods et al., 1999; Eckburg et al., 2005) represent as-yet-uncultiva- can also give a biased picture of bacterial diversity, and their ted and uncharacterized bacteria. limitations in this regard are mostly related to different levels of The study of the human gastrointestinal microbiota is effectiveness of cell lysis for DNA extraction and PCR-related a formidable example of the revolution fomented by molec- biases (von Wintzingerode et al., 1997; Siqueira and Rôças, ular studies with regard to the study of the human micro- 2005a; Rajilić-Stojanović et al., 2007). biota in health and disease (Furrie, 2006). Culture-dependent It has been claimed that the high sensitivity of molecular studies of the gastrointestinal microbiota have resulted in the methods allows for the detection of species present in very low Downloaded from http://jdr.sagepub.com at UNIV OF PENNSYLVANIA on January 7, 2010 J Dent Res 88(11) 2009 Diversity of Endodontic Microbiota 971 numbers and, consequently, with possibly no clinical importance. checkerboard, microarrays) in large-scale clinical studies to However, in microbial ecology, it has been demonstrated that some investigate prevalence and association with disease (Rôças and of these low-abundant bacteria might still serve as keystone species Siqueira, 2005b, 2008; Siqueira and Rôças, 2005c, 2009a). within complex mixed consortia (Sogin et al., 2006). Moreover, some of the low-abundant species may be a result of historical dIVersIty oF tHe eNdodoNtIc ecological changes, in the sense that they may have been dominant MIcrobIotA—oVerALL FINdINGs in the past or have the potential to become dominant in the future in response to shifts in environmental conditions that favor ‘Microbiota’ is arguably the best collective term for micro- their growth. Thus, low-abundant populations may eventually organisms, since widely used terms such as ‘microbial flora’ become dominant in response to environmental changes (Sogin and ‘microflora’ perpetuate an outdated classification of et al., 2006). Furthermore, species that are low-abundant in one micro-organisms as plants (Dethlefsen et al., 2006). ‘Diversity’ individual can dominate the community in another individual. is a function of both the number of species present in a com- Therefore, at least from an ecological perspective, all species in a munity (species richness) and the relative abundance of those mixed community should be successfully detected and identified species in the system (species evenness). The highest diver- (Siqueira and Rôças, 2009b). sity occurs in communities with many different species pres- A broad range of molecular biology techniques has been used ent (richness) in relatively equal abundance (evenness) for diverse purposes in microbial ecology research (Siqueira and (Huston, 1994). The richness and evenness of microbial com- Rôças, 2005a; Spiegelman et al., 2005). In endodontic micro- munities are the result of selective pressures that shape diver- biology research, the vast majority of molecular studies have sity within communities. This review discusses the diversity addressed the issue of species composition in different types of of the endodontic microbiota, with the main focus on the endodontic infections. In spite of the great advantages of these aspect of species richness or composition. The integration of methods, at this time, there is no available technique that can data from culture-dependent and culture-independent methods provide a comprehensive list of all bacterial species in a sample. for microbial identification in endodontic infections is presen- As a consequence, a variety of techniques has been used to offer ted here for the first time. a better picture of the endodontic bacterial communities. Representatives of the domains Archaea and Eukarya have been The chronology of the study of the diversity of the endo- occasionally reported to occur in endodontic infections. Archaea dontic microbiology can be didactly divided into phases based diversity is restricted to an as-yet-uncultivated Methanobravibacter on different strategic approaches. Early studies of the endo- oralis-like phylotype (Vianna et al., 2006a; Vickerman et al., 2007). dontic microbiota were conducted with broad-range culture Except for a couple of molecular studies targeting Candida albi- methods (Bergenholtz, 1974; Kantz and Henry, 1974; Wittgow cans (Baumgartner et al., 2000; Siqueira and Rôças, 2004a), the and Sabiston, 1975; Sundqvist, 1976; Baumgartner and Falkler, diversity of Eukarya in infected canals has been assessed exclusi- 1991; Sundqvist, 1992). These were followed by a generation vely by culture-dependent methods. Six Candida, one Geotrichum, of studies that used molecular detection methods, such as one Rhodotorula, and one Saccharomyces species have been isola- species-specific PCR and the original checkerboard DNA- ted from root canals (see Appendix for references). DNA hybridization assay, to target cultivable bacteria previously Although a few representatives of the domains Eukarya and isolated from infected canals or from other diseased oral sites Archaea have been found in endodontic infections, and reports (Conrads et al., 1997; Jung et al., 2000; Machado de Oliveira indicate the occurrence of herpesviruses (cytomegalovirus et al., 2000; Rupf et al., 2000; Siqueira et al., 2000a,b, 2001b). and Epstein-Barr virus) in apical periodontitis lesions (Sabeti These methods allowed for the inclusion of some culture-diffi- et al., 2003), the domain Bacteria is far more dominant and diverse cult species in the set of putative endodontic pathogens. The in endodontic infections. Integrated datasets from culture and adoption of 16S rRNA gene clone library analysis facilitated molecular studies published as of March 2008, showed that 468 an even more comprehensive broad-range investigation of unique bacterial taxa have been found in endodontic infections bacterial communities in endodontic infections (Munson et al., associated with different clinical conditions. The vast majority of 2002; Saito et al., 2006; Sakamoto et al., 2006, 2008). By these bacterial taxa fall into 100 genera, while 22 taxa remained this approach, not only cultivable species but also as-yet- undefined and were assigned to family or even to phylum level. uncultivated and uncharacterized bacteria have been identi- Prevotella (39 taxa), Eubacterium (27 taxa), Streptococcus (26 fied. Studies with 16S rRNA gene clone library analysis have taxa), and Lactobacillus (21 taxa) are the most represented genera. revealed that 40-55% of the bacterial taxa found in primary All taxa already reported in endodontic infections and the respec- endodontic infections have not been cultivated and validly tive studies that detected them are listed in the Appendix. named (Munson et al., 2002; Sakamoto et al., 2006, 2007). At the broader phylogenetic level, all the detected bacterial While technical difficulties and high cost can make it difficult taxa have been found to belong to 9 of the 13 phyla that to analyze a large number of samples by the clone library have oral representatives, namely, Firmicutes, Bacteroidetes, method, cataloguing bacterial species in the oral cavity by Actinobacteria, Proteobacteria, Fusobacteria, Spirochaetes, clone libraries provides 16S rRNA gene sequence data that Synergistes, TM7, and SR1 (Table 1). The most common can be used to design primers or oligonucleotide probes to representatives from each of these phyla are shown in Fig. 1. target both cultivable and as-yet-uncultivated bacteria in infec- Members of Acidobacteria, a phylum sporadically found in the ted root canals. Primers and probes can be used in PCR oral cavity, were not detected by group-specific PCR (unpub- and DNA-DNA hybridization assays (e.g., reverse-capture lished data from the authors’ laboratory). Downloaded from http://jdr.sagepub.com at UNIV OF PENNSYLVANIA on January 7, 2010 972 Siqueira and Rôças J Dent Res 88(11) 2009 Common representative species/phylotypes Dialister spp., Filifactor alocis, Parvimonas micra, Pseudoramibacter alactolyticus, Enterococcus faecalis, Firmicutes Eubacterium spp., Mogibacterium spp., Streptococcus spp., Lachnospiraceae spp.,Veillonella parvula, Lactobacillus spp., Catonella morbi, Gemella morbillorum, Selenomonas spp., Peptostreptococcus spp. Actinobacteria Olsenella uli, Actinomyces spp., Propionibacterium acnes, Propionibacterium propionicum, Slackia exigua Synergistes Clone BA121, clone W090 Spirochaetes Treponema denticola, Treponema socranskii, Treponema maltophilum,Treponema parvum Fusobacteria Fusobacterium nucleatum Proteobacteria Eikenella corrodens, Campylobacter rectus, Campylobacter gracilis TM7 Clone I025 SR1 Clone X112 Bacteroidetes Tannerella forsythia, Porphyromonas endodontalis, Porphyromonas gingivalis,Prevotella spp., clone X083 0.05 Figure 1. Bacterial phyla that have representatives in endodontic infections. On the right, example species or phylotypes for each phylum are presented. It is important to point out that the numbers of bacterial taxa be introduced in the clinical setting during sample collection reported refer exclusively to richness within each phylum and (usually oral contaminants) or in the laboratory during specimen have no relationship to prevalence and importance in disease handling (non-oral contaminants). Also, “single-study” taxa causation. For instance, the phyla Fusobacteria, Spirochaetes, were rarely very prevalent in the individual study that reported and Synergistes have few representative species/phylotypes their occurrence. In contrast, as-yet-uncultivated phylotypes or in endodontic infections, but some of them, including culture-difficult species detected in only one molecular study Fusobacterium nucleatum, Treponema denticola, and Synergistes may not necessarily be interpreted as irrelevant because their clone BA121, respectively, are among the most commonly occurrence in a single study may reflect the fact that, thus far, detected taxa in primary intraradicular infections. only a few broad-range molecular studies have been published, Of the recovered taxa, 204 (44%) were isolated or detected and the microbiota have been investigated in only a limited in only one study. This may indicate that they are subject- number of individuals. specific and then contribute to the high level of interindividual The analysis of the bacteria detected in endodontic infections variation in bacterial community profiles, or they may be revealed that 210 distinct bacterial taxa were reported in molec- contaminants or species poorly identified. Contaminants may ular biology studies alone. This figure corresponds to 45% of all taxa already found in endodontic infections, as opposed to 151 table 1. Overall Findings of Bacterial Species/Phylotype (taxa) taxa (32%) detected by culture studies alone. One hundred Richness in the Different Types of Endodontic Infections seven taxa, 23% of the total bacterial species richness, were detected by the application of both culture-dependent and As-Yet- Taxa Detected Taxa Detected culture-independent techniques (Fig. 2). Uncultivated by Molecular by Culture Culture-dependent and culture-independent molecular Phyla Taxa Phylotypes Studies Studies studies provide a somewhat different description of the diver- Firmicutes 220 81 146 124 sity of the endodontic microbiota, as indicated by the degree Bacteroidetes 73 28 47 48 of overlapping findings (Fig. 2). However, both approaches Actinobacteria 69 21 43 42 show unequivocally that Firmicutes are by far the most Proteobacteria 65 17 44 34 diverse group, even though the community structure of this Fusobacteria 15 6 11 9 Spirochaetes 14 4 14 0 group has been largely underestimated in the reports from Synergistes 10 10 10 1 culture-dependent studies. Representatives of 4 phyla— TM7 1 1 1 0 Spirochaetes, Synergistes, TM7, and SR1—have been found SR1 1 1 1 0 almost exclusively in endodontic infections by molecular Downloaded from http://jdr.sagepub.com at UNIV OF PENNSYLVANIA on January 7, 2010 J Dent Res 88(11) 2009 Diversity of Endodontic Microbiota 973 biology methods. While the latter two OVERALL 210 107 151 have only one representative each, both of which have been detected in low prev alence, members of the two former Actinobacteria 27 16 26 have been as frequent as, or even more frequent than, most representatives of Bacteroidetes 25 22 26 the other, more diverse, phyla. Firmicutes 96 50 74 Molecular studies have allowed for the Fusobacteria 6 5 4 detection of 169 phylotypes representing Proteobacteria 31 13 21 bacteria that have not yet been cultivated and/or fully characterized. Most of these Spirochaetes 14 phylotypes belong to the phyla Firmicutes, Synergistes 9 1 Bacteroidetes, and Actinobacteria. As-yet- TM7 1 uncultivated and/or uncharacterized phylotypes can be important pathogens SR1 1 that have been overlooked by culture- dependent studies. Taxa detected in molecular studies Molecular methods have succeeded Taxa detected in both molecular and culture studies over culture methods in the detection Taxa detected in culture studies of not only as-yet-uncultivated and uncharacterized phylotypes, but also of Figure 2. Distribution of bacterial species/phylotypes found in endodontic infections according cultivable and validly named species, to the detection method. Data are given overall and for the 9 phyla that have endodontic since about 40 taxa included in this representatives. category have been exclusively detected by molecular approaches. These bacteria either represent fasti- types of infection, mainly primary infections. In spite of dious species that were not recovered (probably because speci- having been found in samples from every type of infection, fic culture media were not used), or they are species that were Enterococcus faecalis is encountered more frequently in trea- successfully cultivated but inadequately identified, because ted root canals of teeth evincing post-treatment disease methods improper for definite identification were used or the (Appendix). All of these ubiquitous bacteria are cultivable and isolate presented ambiguous phenotypic behavior (Siqueira et validly named species. However, it is worth pointing out that al., 2007c). Indeed, application of the 16S rRNA gene-sequen- this list is biased by the fact that, for some types of infection, cing approach for the identification of culture isolates has there is no or only one molecular study using broad-range demonstrated that some phylotypes previously supposed to be analysis. Thus, some taxa found in primary infections in even “uncultivable” can actually be cultivated by conventional anae- high prevalence may have been deprived of this “status” robic techniques with ordinary growth media (Munson et al., because of the lack of a body of molecular studies investiga- 2002; Siqueira et al., 2007c). Most of these previously “unculti- ting other disease categories. vable” bacteria remain uncharacterized, and a valid species name is pending. PrIMAry INtrArAdIcULAr INFectIoNs The strength of molecular methods to unravel the bacterial diversity and its superiority over culture approaches in this Primary intraradicular infection is caused by micro-organisms regard is in clear agreement with studies in other areas (Amann that initially invade and colonize the necrotic pulp tissue. It is et al., 1995; Rappe and Giovannoni, 2003; Janssen, 2006; characterized by a mixed consortium conspicuously dominated Rajilić-Stojanović et al., 2007). In endodontic microbiology by anaerobic bacteria and composed of 10 to 30 species per research, it is quite remarkable that molecular studies performed canal (Munson et al., 2002; Siqueira et al., 2004b; Siqueira and over the past 10 years have revealed 317 distinct bacterial taxa Rôças, 2005b). Total bacterial counts vary from 103 to 108 cells in infected canals, while culture studies over the past 30 years per infected canal (Sundqvist, 1976; Vianna et al., 2006b; revealed 258 taxa. Sakamoto et al., 2007; Siqueira et al., 2007d). Bacterial taxa for which there is moderate-to-strong evi- Bacterial profiles of the endodontic microbiota vary from indi- dence of involvement with causation of apical periodontitis are vidual to individual (Siqueira et al., 2004b; Sakamoto et al., 2006), shown in Fig. 3. Selection of these taxa was based on their i.e., each individual harbors a unique endodontic microbiota in detection in several independent studies and moderate-to-high terms of species richness and abundance. This indicates that pri- prevalence in more than one study. Thus, the taxa shown in mary apical periodontitis has a heterogeneous etiology, where no Fig. 3 can be regarded as the main candidate endodontic patho- single species can be considered as the main endodontic pathogen, gens associated with different types of infection. One will and multiple bacterial combinations play a role in disease causa- notice that there are some ubiquitous endodontic taxa that have tion. This is consistent with the high numbers of different taxa that been found by at least one study in every type of endodontic have been detected in primary infections. infection. Most of these “cosmopolitan” species have been Collectively, culture and molecular methods have allowed 391 found to be very prevalent in association with some specific bacterial, 4 fungal, and one archaeal taxa to be detected in primary Downloaded from http://jdr.sagepub.com at UNIV OF PENNSYLVANIA on January 7, 2010 974 Siqueira and Rôças J Dent Res 88(11) 2009 infections. As-yet-uncultivated and/or uncharacterized bacteria have been repre- 100 Peptostreptococcus anaerobius sented by 136 taxa. Molecular methods 98 Peptostreptococcus stomatis Filifactor alocis have identified 271 taxa, while 216 taxa 61 62 Eubacterium nodatum have been isolated by culture. 93 Mogibacterium timidum Bacterial species/phylotypes detec- Pseudoramibacter alactolyticus ted in primary infections fall into the 9 54 Anaerococcus prevotii phyla mentioned above and 82 genera. 92 Parvimonas micra Gemella morbillorum Seventeen taxa have not been assigned Enterococcus faecalis to a genus, only to family or phylum. 100 100 Streptococcus constellatus The highest species richness was ob- 98 Streptococcus intermedius 99 served for the Firmicutes, followed by Streptococcus anginosus Bacteroidetes and Actinobacteria 100 Streptococcus mitis 54 100 Streptococcus oralis (Table 2). Of the 391 taxa detected, 261 85 Streptococcus sanguinis were unique to primary infections, i.e., Catonella morbi they have not been detected in samples Lactobacillus catenaformis from other types of infections. Selenomonas sputigena 70 Diverse groups of Gram-negative Veillonella parvula 100 and Gram-positive bacteria have been Dialister invisus 100 100 Dialister pneumosintes identified in primary infections. Black- Fusobacterium nucleatum pigmented Gram-negative anaerobic 97 Atopobium parvulum rods are named after their ability to form 100 Olsenella uli brown to black colonies on blood agar Eggerthella lenta plates and have been classified into two 100 Propionibacterium acnes 100 Propionibacterium propionicum genera—Prevotella (containing saccha- 100 Actinomyces meyeri rolytic species) and Porphyromonas 100 Actinomyces odontolyticus (containing asaccharolytic species). The 100 Actinomyces israelii genus Prevotella also includes some Actinomyces naeslundii 100 bile-sensitive non-pigmented species. 99 Actinomyces viscosus 100 Synergistesoral clone W090 Prevotella species, especially P. interme- Synergistesoral clone BA121 dia, P. nigrescens, P. tannerae, P. baro- Treponema socranskii niae, and P. denticola, have been among Treponema denticola 100 the most frequently detected taxa in pri- 57 Treponema maltophilum mary infections (see Appendix for refe- Eikenella corrodens rences). Of the Porphyromonas species, Campylobacter gracilis 68 100 Campylobacter rectus P. endodontalis and P. gingivalis have 86 Porphyromona sendodontalis been consistently encountered in endo- 100 Porphyromonas gingivalis dontic infections, and a role in the etiol- Tannerela forsythia ogy of acute abscesses is suspected Bacteroidetesoral clone X083 100 (van Winkelhoff et al., 1985; Haapasalo Prevotella tannerae 99 99 Prevotella baroniae et al., 1986; Sundqvist et al., 1989; 76 Prevotella buccae Siqueira et al., 2001a; Seol et al., 2006). 75 Prevotella loescheii An important periodontal patho- 80 Prevotella oralis gen, Tannerella forsythia (formerly 100 97 Prevotella denticola Bacteroides forsythus), a fastidious Prevotella melaninogenica 83 Prevotella intermedia Gram-negative obligate anaerobe that 100 Prevotella nigrescens had never been detected in root canals 0.05 by culture, was for the first time Figure 3. Phylogenetic tree based on 16S rRNA gene comparisons, showing several candidate endo- reported to occur in primary endo- dontic pathogens and the clinical conditions with which they have been associated. Distribution of dontic infections in a study with bacterial species/phylotypes among the different types of infections is shown by the columns of boxes species-specific PCR (Conrads et al., to the right of the tree. Different darknesses of the box indicate strength of association based on 1997). Subsequent studies with diffe- prevalence data and the number of studies in which the species was detected, i.e., clear box means rent molecular biology approaches not detected, while black box means detected by several studies and/or in high prevalence. The refe- have confirmed that T. forsythia is a rence bar indicates 5% sequence divergence. Pi, primary infections; Fs, filling stage; Rc, retreatment cases; Ex, extraradicular infections. common member of the microbiota Downloaded from http://jdr.sagepub.com at UNIV OF PENNSYLVANIA on January 7, 2010 J Dent Res 88(11) 2009 Diversity of Endodontic Microbiota 975 associated with different types of primary infections, including table 2. Bacterial Species/Phylotype (taxa) Richness in Primary Endodontic abscesses (Jung et al., 2000; Siqueira et al., 2000b, 2001b; Infections Fouad et al., 2002; Siqueira and Rôças, 2003c). As-Yet- Taxa Detected Taxa Detected Dialister species are asaccharolytic obligately anaerobic Uncultivated by Molecular by Culture Gram-negative coccobacilli that represent another example of Phyla Taxa Phylotypes Studies Studies bacteria that have been consistently detected in endodontic Firmicutes 184 69 131 98 infections only after the advent of molecular biology techniques. Bacteroidetes 69 24 42 48 D. pneumosintes and the recently described D. invisus have been Actinobacteria 54 11 31 39 frequently present in the microbiota associated with asympto- Proteobacteria 44 11 32 21 matic and symptomatic primary infections (Appendix). Fusobacteria 14 5 9 9 Fusobacterium nucleatum is an anaerobic spindle-shaped Spirochaetes 14 4 14 0 rod that is one of the most commonly encountered Gram- Synergistes 10 10 10 1 negative species in endodontic infections. F. nucleatum has been TM7 1 1 1 0 frequently isolated from or detected in primarily infected root SR1 1 1 1 0 canals as well as in endodontic abscesses (Appendix). Spirochetes are highly motile spiral-shaped bacteria that been isolated from or detected in about one-third of the primarily have been frequently observed by microscopy in samples taken infected canals, and its prevalence in symptomatic infections has from endodontic infections. Nonetheless, they had never been also been relatively high (Sundqvist, 1992; Weiger et al., 1995; identified to the species level. The application of molecular Gomes et al., 1996; Khemaleelakul et al., 2002; Siqueira diagnostic methods to the identification of spirochetes has et al., 2003; Chu et al., 2005). Members of the Streptococcus angi- demonstrated that occurrence of these spiral bacteria in endo- nosus group have been reported to be the most prevalent strepto- dontic infections has been overlooked by technical hurdles of cocci, but S. oralis, S. mitis, and S. sanguinis can also often be culture techniques. All of the currently cultivable and named recovered/detected (Sundqvist, 1992; Siqueira et al., 2002). oral Treponema species have been disclosed by molecular stud- Campylobacter species, including C. rectus and C. gracilis, ies of primary endodontic infections (Appendix). The most pre- are Gram-negative anaerobic rods that have been detected in dominant species in primary infections are T. denticola and T. primary endodontic infections, but in low-to-moderate preva- socranskii (Siqueira et al., 2000b; Baumgartner et al., 2003; lence values (Sundqvist, 1992; Le Goff et al., 1997; Siqueira Rôças et al., 2003; Siqueira and Rôças, 2004b). and Rôças, 2003b). Catonella morbi, a saccharolytic obligately Even though anaerobic Gram-negative bacteria have been anaerobic Gram-negative rod associated with marginal perio- found to be the most common micro-organisms in primary infec- dontitis, has been found in about one-fourth of the cases of pri- tions, several Gram-positive rods have also been frequent mem- mary endodontic infections (Siqueira and Rôças, 2006). Other bers of the endodontic microbial consortium. Of these, bacteria detected in low-to-moderate frequencies in primary Pseudoramibacter alactolyticus has been frequently isolated infections include: Veillonella parvula, Eikenella corrodens, from or detected in samples from endodontic infections, in preva- Neisseria mucosa, Centipeda periodontii, Granulicatella adia- lence values as high as those of the most commonly found Gram- cens, Gemella morbillorum, Capnocytophaga species, and anae- negative species (Sundqvist, 1992; Siqueira and Rôças, 2003d; robic lactobacilli (Appendix). Siqueira et al., 2004a). Filifactor alocis is an obligately anaerobic Investigations of the diversity of the microbiota of primary rod that has been detected by molecular studies in about one-half infections by broad-range PCR and 16S rRNA gene clone of the samples from primary infections (Siqueira and Rôças, library analysis have demonstrated that it is far more complex 2003a; Gomes et al., 2006). Actinomyces species, which have than previously reported by culture studies. Noteworthy is the been associated with failure of the endodontic treatment by cau- common occurrence of as-yet-uncultivated bacteria—about sing apical actinomycosis, have been found in about 10% of 40-55% of the endodontic microbiota is composed of as-yet- infected canals (Sundqvist, 1992; Siqueira et al., 2002). uncultivated phylotypes (Munson et al., 2002; Sakamoto et al., Propionibacterium propionicum, another species that can partici- 2006, 2007). A molecular study investigating primary infections pate in apical actinomycosis, has also been commonly detected in revealed that 55% and 40% of the taxa detected in association samples from primary infections (Siqueira and Rôças, 2003e). with chronic apical periodontitis and acute apical abscesses Olsenella species consist of small non-motile Gram-positive obli- were as-yet-uncultivated phylotypes, respectively (Sakamoto gately anaerobic rods, which represent another example of bacte- et al., 2006). As for their abundance in these infections, as-yet- ria that have been detected in endodontic infections only after the uncultivated phylotypes corresponded to 38% and 30% of the introduction of molecular biology methods (Fouad et al., 2002; clones sequenced from samples of chronic apical periodontitis Munson et al., 2002). Among the Olsenella clade, O. uli has been and abscesses, respectively (Sakamoto et al., 2006). the most commonly found species in endodontic infections As mentioned above, uncultivated phylotypes have been repor- (Chávez de Paz et al., 2004; Rôças and Siqueira, 2005a). ted for all phyla with endodontic representatives. Several of these Gram-positive cocci, specifically peptostreptococci and strepto- phylotypes can be candidate endodontic pathogens based on asso- cocci, are frequently present in primary endodontic infections. ciation data. For instance, oral Synergistes clones BA121, E3_33, Parvimonas (formerly Peptostreptococcus or Micromonas) micra BH017, and W090, which had been originally assigned to the is an asaccharolytic anaerobic Gram-positive small coccus that has Flexistipes or Deferribacteres group (Paster et al., 2001; Godon Downloaded from http://jdr.sagepub.com at UNIV OF PENNSYLVANIA on January 7, 2010 976 Siqueira and Rôças J Dent Res 88(11) 2009 et al., 2005), have been commonly detected in samples from secondary infections that developed by lack of a bacteria-tight asymptomatic and symptomatic endodontic infections (Rôças and coronal seal. Taxa detected at the filling stage, but not at the time Siqueira, 2005b; Siqueira and Rôças, 2005c; Siqueira et al., 2005; of re-treatment, may not be able to endure the conditions within Vianna et al., 2007). The great majority of Synergistes bacteria obturated canals. Although all this discussion sounds logical and remain uncultivated, and this can be the primary reason for the fact interesting, it is largely speculative, because the data belong to that their presence in endodontic infections has been overlooked by separate cross-sectional studies, and no strong evidence can be culture studies. Similarly, clones I025 and X112 from the TM7 and taken in this regard. SR1 candidate phyla, respectively, which so far have no cultivable representatives, have also been detected in primary infections bActerIA At tHe root-cANAL-FILLING (Rôças and Siqueira, 2005a, 2008; Siqueira and Rôças, 2005c). stAGe (Post-treAtMeNt sAMPLes) Uncultivated phylotypes related to several genera have been dis- closed, mainly Dialister, Prevotella, Peptostreptococcus, Solobact- Root canal samples can be taken at the time of treatment to assess erium, Olsenella, Selenomonas, Eubacterium, Megasphaera, the antimicrobial efficacy of treatment protocols and/or check the and Veillonella, as well as phylotypes related to the family bacteriologic conditions of the canal before filling procedures. Lachnospiraceae (Rolph et al., 2001; Munson et al., 2002; Saito Samples positive for bacterial growth after chemomechanical prep- et al., 2006; Sakamoto et al., 2006; Vickerman et al., 2007). One aration, followed (or not) by intracanal medication, have been study (Sakamoto et al., 2006) found some uncultivated phylotypes shown to harbor an average of 1 to 5 bacterial species, at counts among the most prevalent bacteria in primary infections, including reaching up to 102 to 105 cells per canal (Byström and Sundqvist, Lachnospiraceae oral clone 55A-34, Megasphaera oral clone 1985; Sjögren et al., 1997; Vianna et al., 2006b; Sakamoto et al., CS025, and Veillonella oral clone BP1-85. Prevotella oral clone 2007; Siqueira et al., 2007a,b). These numbers indicate that, even PUS9.180, Eubacterium oral clone BP1-89, and Lachnospiraceae if total bacterial elimination does not occur, at least a substantial oral clone MCE7_60 were exclusively detected in symptomatic reduction in species diversity is attained after treatment. samples (Sakamoto et al., 2006). Uncultivated phylotypes are pre- Culture and molecular methods have allowed 103 bacterial viously unrecognized and overlooked bacteria that may play a role and 6 fungal taxa to be detected in samples taken after chemo- in the pathogenesis of different forms of apical periodontitis. mechanical preparation and/or intracanal medication. Molecular methods have detected 26 taxa, while 88 taxa were isolated in PersIsteNt/secoNdAry culture studies. Forty-one taxa were found in only one study. Bacterial species/phylotypes detected in post-treatment samples INtrArAdIcULAr INFectIoNs belong to 5 phyla and 41 genera. Three taxa have been assigned to Secondary infection is caused by micro-organisms that were not taxonomic levels above genus. The highest species richness was present in the primary infection, but were introduced in the root observed for the Firmicutes, followed by Proteobacteria and canal at some time after professional intervention (so called because Actinobacteria (Table 3). it is secondary to intervention) (Siqueira, 2002). Persistent infection Gram-negative bacteria, which are common members of pri- is caused by micro-organisms that were members of a primary or mary infections, are usually eliminated after treatment procedures secondary infection and that, in some way, resisted intracanal anti- (Chávez de Paz, 2005). Exceptions may include some anaerobic microbial procedures and endured periods of nutrient deprivation in rods, such as F. nucleatum, Prevotella species, and C. rectus, which treated canals (Siqueira, 2002). are among the species found in post-instrumentation/medication Persistent or secondary intraradicular infections are the major samples (Appendix). However, most studies have clearly revealed causes of several clinical problems, including endodontic treatment that, when bacteria resist treatment procedures, Gram-positive failure, which is characterized by persistence or appearance of api- bacteria are more frequently present. They include streptococci cal periodontitis after treatment (Siqueira, 2008). Studies have (S. mitis, S. gordonii, S. anginosus, S. sanguinis, and S. oralis), attempted to identify the micro-organisms found at the root-canal- P. micra, Actinomyces species (A. israelii and A. odontolyticus), filling stage, which may jeopardize the treatment outcome, and the Propionibacterium species (P. acnes and P. propionicum), P. alac- micro-organisms in root-canal-treated teeth with apical periodonti- tolyticus, lactobacilli (L. paracasei and L. acidophilus), E. faecalis, tis, which are arguably the cause of failure. and O. uli (Appendix). Other Gram-positive bacteria, including Analysis of integrated data from culture and molecular stud- Bifidobacterium species, Eubacterium species, and staphylococci, ies revealed that 51 bacterial and 2 fungal taxa have been detec- can also be found, but in lower frequencies (Sjögren et al., 1997; ted in samples taken at the time of filling and also at the time of Chávez de Paz, 2004; Chu et al., 2006). re-treatment. The following taxa have been detected by several With the recent findings showing as-yet-uncultivated bacteria as studies: Propionibacterium acnes, P. propionicum, Actinomyces constituents of a significant proportion of the endodontic micro- naeslundii, Actinomyces odontolyticus, P. intermedia, biota (Munson et al., 2002; Sakamoto et al., 2006), studies on the Anaerococcus prevotii, Eggerthela lenta, E. faecalis, G. mor- effects of intracanal antimicrobial procedures should also focus on billorum, P. micra, P. alactolyticus, S. anginosus group, S. mitis, these bacteria. Thus far, the only broad-range molecular study F. nucleatum, and C. albicans. Theoretically, taxa found at both to investigate samples from this condition used a 16S rRNA the time of filling and during re-treatment may be involved in gene clone library analysis to identify the bacteria persisting after persistent infections. Likewise, taxa that were found only at the chemomechanical preparation, with 2.5% NaOCl as irrigant and time of re-treatment, but not at the time of filling, may represent intracanal medication with a calcium hydroxide paste (Sakamoto Downloaded from http://jdr.sagepub.com at UNIV OF PENNSYLVANIA on January 7, 2010 J Dent Res 88(11) 2009 Diversity of Endodontic Microbiota 977 table 3. Bacterial Species/Phylotype (taxa) Richness in Samples Taken table 4. Bacterial Species/Phylotype (taxa) Richness in Persistent/ at the Time of Root Canal Filling Secondary Infections Related to Treatment Failure As-Yet- Taxa Detected Taxa Detected As-Yet- Taxa Detected Uncultivated by Molecular by Culture Uncultivated Taxa Detected by by Culture Phyla Taxa Phylotypes Studies Studies Phyla Taxa Phylotypes Molecular Studies Studies Firmicutes 45 2 10 40 Firmicutes 76 21 50 40 Proteobacteria 20 3 3 17 Actinobacteria 28 11 21 10 Actinobacteria 19 2 9 15 Proteobacteria 26 5 17 11 Bacteroidetes 16 3 3 13 Bacteroidetes 22 6 16 9 Fusobacteria 3 0 1 3 Fusobacteria 4 2 3 2 Spirochaetes 1 0 1 0 et al., 2007). Eleven, 4, and 5 taxa were detected in initial (S1), Synergistes 1 1 1 0 post-instrumentation (S2), and post-medication (S3) samples, re- spectively. No taxon found in post-treatment samples was shown to Rôças et al., 2004a,b; Siqueira and Rôças, 2004a; Sedgley et al., dominate the initial (primary) infection (baseline). Streptococcus 2006; Zoletti et al., 2006). Root-canal-treated teeth are about 9 times species were detected in all post-treatment samples and were also more likely to harbor E. faecalis than cases of primary infec- the most dominant taxa in these samples, except for a S2 sample tions (Rôças et al., 2004a). Other bacteria found in re-treatment in which Solobacterium clone K010 corresponded to 56% of cases include streptococci and some fastidious anaerobic bacterial the clones sequenced. Forty-two percent of the taxa found in post- species—P. alactolyticus, P. propionicum, F. alocis, D. pneumosin- treatment samples were as-yet-uncultivated bacteria, indicating that tes, and D. invisus (see Appendix). previously uncharacterized bacteria may also persist after treat- In a study using 16S rRNA gene clone library analysis, Sakamoto ment, and their role in the long-term treatment outcome requires et al. (2008) found a mean of 10 taxa per treated root canal, ranging elucidation. from 1 to 26. As-yet-uncultivated phylotypes corresponded to 55% of the taxa detected. Only 11 taxa were found in more than one MIcrobIotA IN root-cANAL-treAted case: Bacteroidetes oral clone X083, an uncultured Saprospiraceae clone, Prevotella oris, P. baroniae, Pseudomonas aeruginosa, teetH (re-treAtMeNt sAMPLes) Streptococcus mutans, Synergistes clone BA121, Dialister Root canal samples can be taken from teeth that were treated months clone 9N-1, E. faecalis, Flavobacteriaceae genomospecies C1, and to years previously and need endodontic re-treatment because of the Peptostreptococcus clone FG014. A high interindividual variability emergence or persistence of disease. The microbiota in these cases in the composition of the microbiota was clearly evident, and, along also exhibit a decreased diversity in comparison with that with the findings from a bacterial community profiling technique of primary infections. Canals apparently well-treated contain from (Rôças et al., 2004c), suggests that distinct bacterial combinations 1 to 5 species, while the number of species in canals with inadequate can play a role in treatment failures. Bacterial abundance treatment can reach 30, which is very similar to that in untreated also varied from sample to sample, and some uncultivated canals (Sundqvist et al., 1998; Pinheiro et al., 2003; Rôças et al., phylotypes were the most dominant taxa in about one-half of the 2004c; Siqueira and Rôças, 2004a). A single treated canal associated cases examined. In fact, uncultivated phylotypes made up a signifi- with post-treatment disease can harbor a density of 103 to 107 bacte- cant fraction of the microbiota in root-canal-treated teeth, since they rial cells (Peciuliene et al., 2001; Sedgley et al., 2006). comprised about 50% of the total number of clones sequenced Integrated datasets from culture and molecular methods have (Sakamoto et al., 2008). revealed 158 bacterial and 3 fungal taxa in samples taken at the Fungi are only occasionally found in primary infections, but time of root canal re-treatment of teeth evincing apical perio- Candida species have been detected in root-canal-treated teeth dontitis lesions. Forty-six as-yet-uncultivated phylotypes have in up to 18% of the cases (Cheung and Ho, 2001; Peciuliene been detected by molecular studies. Molecular methods have et al., 2001; Egan et al., 2002). C. albicans is by far the most detected 109 taxa, while culture studies isolated 72 taxa. A great commonly detected fungal species in re-treatment cases. many taxa (106) have been reported by only one study. Bacterial species/phylotypes detected in re-treatment sam- eXtrArAdIcULAr INFectIoNs ples belong to 7 phyla and 58 genera. Nine taxa have been assigned to the family or phylum level. The highest species Extraradicular infection is characterized by microbial invasion richness was again observed for the Firmicutes, followed by of and proliferation in the inflamed periradicular tissues, and is Actinobacteria and Proteobacteria (Table 4). Molecular studies derived from intraradicular infection (Tronstad et al., 1987; have allowed members of the Spirochaetes and Synergistes Siqueira, 2002; Tronstad and Sunde, 2003). Extraradicular phyla to be detected in these cases. infection in cases of chronic apical periodontitis represents one Culture-dependent and culture-independent studies have re- of the greatest controversies in endodontics (Bergenholtz and vealed that E. faecalis is the most frequent species in root-canal- Spangberg, 2004). The infection is conceivably limited to a treated teeth, with prevalence values reaching up to 90% of the cases small proportion of cases, and, when present, it can be depen- (Molander et al., 1998; Sundqvist et al., 1998; Pinheiro et al., 2003; dent on or independent of intraradicular infection (Siqueira, Downloaded from http://jdr.sagepub.com at UNIV OF PENNSYLVANIA on January 7, 2010