Associations of endodontic symptoms and signs with particular combinations of specific bacteria

Significant associations have been reported between (a) specific bacterial species isolated from root canals and (b) between individual bacterial species and endodontic symptoms and signs. The prime objective of this study was to determine whether particular combinations of specific bacteria are associated with individual endodontic symptoms and signs. Seventy root canals were investigated microbiologically taking care to maintain the viability of obligate anaerobes, which accounted for 64% of the total species isolated, including Peptostreptococcus micros, Prevotella melaninogenica, Prevotella oralis, Eubacterium aerofaciens, Eubacterium lentum, Fusobacterium nucleatum, Prevotella buccae and Prevotella intermedia. Significant associations were found between individual clinical features and the following pairs of species: (a) pain (37 cases) and Peptostreptococcus spp./Prevotella spp., Peptostreptococcus spp./Prevotella melaninogenica, Pstr. micros/Prev. melaninogenica (all P < 0.01); (b) swelling (23 cases) and Pstr. micros/Prevotella spp. (P < 0.01); (c)‘Wet’canal (57 cases) and Prevotella spp./Eubacterium spp. (P < 0.01), Peptostreptococcus spp./Eubacterium spp. (P < 0.05). Thus data from this investigation suggests that statistically significant associations exist between individual endodontic symptoms and signs and particular combinations of specific bacteria.     

Bacterial examination of endodontic infections by clonal analysis in concert with denaturing high-performance liquid chromatography

BACKGROUND/AIMS: The aim of this study was to examine the diversity of bacterial species in the infected root canals of teeth associated with endodontic abscesses by cloning and sequencing techniques in concert with denaturing high-performance liquid chromatography. METHODS: Samples collected from five infected root canals were subjected to polymerase chain reaction (PCR) with universal 16S ribosomal DNA primers. Products of these PCRs were cloned and sequenced. Denaturing high-performance liquid chromatography (DHPLC) was used as a screening method to reduce the number of clones necessary for DNA sequencing. RESULTS: All samples were positive for the presence of bacteria and a range of 7-13 different bacteria were found per root canal sample. In total, 48 different oral clones were detected among the five root canal samples. Olsenella profusa was the only species present in all samples. Porphyromonas gingivalis, Dialister pneumosintes, Dialister invisus, Lachnospiraceae oral clone, Staphylococcus aureus, Pseudoramibacter alactolyticus, Peptostreptococcus micros and Enterococcus faecalis were found in two of the five samples. The majority of the taxa were present in only one sample, for example Tannerella forsythia, Shuttleworthia satelles and Filifactor alocis. Some facultative anaerobes that are frequently isolated from endodontic infections such as E. faecalis, Streptococcus anginosus and Lactobacillus spp. were also found in this study. CONCLUSION: Clonal analysis of the microflora associated with endodontic infections revealed a wide diversity of oral species.     

Microorganisms from canals of root-filled teeth with periapical lesions

AIM: The objective of the present study was to identify the microbial flora within root canals of teeth with failed root-canal treatment and to determine the association of the various species with clinical features. METHODOLOGY: Sixty root-filled teeth with persisting periapical lesions were selected for this study. During nonsurgical endodontic re-treatment, the root-filling material was removed and the canals were sampled. Microbial sampling, isolation and species determination were performed using advanced microbiological techniques for anaerobic species. The association of microbiological findings with clinical features was investigated. RESULTS: Microorganisms were recovered from 51 teeth. In most cases, one or two strains per canal were found. Of the microbial species isolated, 57.4% were facultative anaerobic species and 83.3% Gram-positive microorganisms. Enterococcus faecalis was the most frequently recovered bacterial species. Obligate anaerobes accounted for 42.6% of the species and the most frequently isolated genera was Peptostreptococcus. which was associated with clinical symptoms (P < 0.01). Significant associations were also observed between: (a) pain or history of pain and polymicrobial infections or anaerobes (P < 0.05): (b) tenderness to percussion and Prevotella intermedia/P. nigrescens (P < 0.05); (c) sinus and Streptococcus spp. (P < 0.001) or Actinomyces spp. (P < 0.01); (d) coronally unsealed teeth and Streptococcus spp. or Candida spp. (both with P < 0.01). CONCLUSION: The microbial flora in canals after failure of root-canal treatment were limited to a small number of predominantly Gram-positive microbial species. Facultative anaerobes, especially E. faecalis, were the most commonly isolated microorganisms, however, polymicrobial infections and obligate anaerobes were frequently found in canals of symptomatic root-filled teeth.     

Variations in the susceptibilities of components of the endodontic microflora to biomechanical procedures

The role of the endodontic micoflora in pulpal disease and in endodontic treatment failures is well established. Thus the need for effective microbial control is one of the important justifications for biomechanical procedures. However the efficacy of this stage of treatment is dependent upon the vulnerability of the involved species, which may not be uniform. The aim of this study was to investigate variations in the susceptibilities of members of the root canal microflora to routine biomechanical procedures. Forty-two root canals were investigated microbiologically. Samples were collected before and after instrumentation and the bacterial findings were compared. In 15 cases of'primary'root canal therapy, despite changes in the population size, no significant change in the species composition of the microflora was observed. However in 27 cases secondary'treatment, a decrease in the number of isolations of Peptostreptococcus spp. was found ( P = 0.008). When all 42 cases were considered together, significant decreases were found between first and second samples for anaerobes ( P = 0.0117) and for Grampositive species ( P = 0.008), especially Peptostreptococcus spp. ( P = 0.02). It was therefore concluded that certain species are more resistant to the biomechanical procedures than others.     

Associations between microbial species in dental root canal infections

The existence of commensal or antagonistic relationships between microorganisms in the root canals of teeth with apical periodontitis was investigated. Samples were taken from 65 infected human root canals and were analysed according to species, frequency of occurrence and proportion of the total isolated flora. The most frequent species were Fusobacterium nucleatum, Prevotella intermedia, Peptostreptococcus micros, Peptostreptococcus anaerobius, Eubacterium alactolyticum, Eubacterium lentum and Wolinella recta. An odds ratio system was used to calculate positive or negative associations between the isolated bacteria. Strong positive associations were found between F. nucleatum and P. micros, Porphyromonas endodontalis, Selenomonas sputigena and W. recta. There was also a positive association between P. intermedia and P. micros, P. anaerobius and the eubacteria. In general, species of streptococci, Propionibacterium propionica, Capnocytophaga ochracea and Veillonella parvula showed no or negative associations with the other bacteria. The results are consistent with the concept of a special and selective environment occurring in the root canal that is due, in part, to the cooperative as well as antagonistic nature of the relationships between bacteria in the root canal.     

Microorganisms isolated from root canals presenting necrotic pulp and their drug susceptibility in vitro

The knowledge about causative agents involved in endodontic infections is increasing, especially due to the improvement of culture techniques for anaerobic bacteria, showing that these microorganisms are predominant in this pathology. In this study, 31 canals with pulp necrosis were microbiologically analyzed before and after manipulation. Obligate and facultative anaerobes, microaerophilic bacteria and yeasts were recovered from 24, 14, 5 and 2 clinical specimens, respectively. The most frequent genera were Prevotella, Fusobacterium, Lactobacillus, Streptococcus, Clostridium and Peptostreptococcus for bacteria and Candida and Saccharomyces for yeasts. Strong positive associations, using an odds ratio system, were found between Clostridium and Prevotella and between Peptostreptococcus and Fusobacterium. Even after the instrumentation and the use of Ca(OH)2, facultative anaerobes were detected in two root canals and yeasts in three. Microorganisms were isolated from seven canals at the end of the endodontic treatment: facultative anaerobes from five and yeasts from one. The microbiological evaluation of root canals with pulp necrosis suggests the presence of polymicrobial infections, mainly involving obligate anaerobes, and shows that the infection may persist after treatment.     

Combinations of bacterial species in endodontic infections

AIM: This study was undertaken to investigate combinations of bacteria found in root-canal infections of teeth with periapical bone destruction without clinical signs and symptoms. METHODOLOGY: Endodontic samples from 58 root canals were cultured anaerobically and microorganisms were counted and identified. Eighty-one combinations of microorganisms were found and tested for a symbiotic relationship using the Fisher's exact test and Odds ratio calculation. RESULTS: All samples contained microorganisms with a median CFU mL(-1) of 8x10(4) per sample. Strict anaerobic species accounted for 87% of the microflora. The most prevalent bacteria were Prevotella intermedia, Peptostreptococcus micros and Actinomyces odontolyticus, present in 33, 29 and 19%, respectively, of the cultured canals. A significant relationship (P<0.05) and an Odds ratio >2 were found between P. intermedia and P. micros, P. intermedia and P. oralis, A. odontolyticus and P. micros, Bifidobacterium spp. and Veillonella spp. Conclusions: These results indicate that endodontic pathogens do not occur at random but are found in specific combinations.     

Association of specific bacteria with some endodontic signs and symptoms

Although a wide range of bacterial species has been isolated from infected dental root canals it remains necessary to determine whether any particular group of such bacteria is associated with specific endodontic symptoms and clinical signs. In this study 30 root canals were examined microbiologically; of these, 14 were associated with pain, 20 with tenderness to percussion, 23 presented with wet root canals, seven with swelling, five with purulent exudate and four with a sinus. Clinical and microbiological correlation was observed particularly with regard to pain where anaerobes were isolated from 93% of painful canals and only from 53% of painfree canals. The former yielded means of 2.5 anaerobic species and 1.6 facultative species per canal, compared with 1.5 and 2.4 in painfree canals. Prevotella spp. were isolated from 64.2% of painful canals and 12.5% of painless canals (P<0.01) similarly, peptostreptococci were isolated from 71.4% of painful and 31.3% of painless canals (P<0.05). It was concluded that a significant association exists between pain and the presence of Prevotella and Peptostreptococcus spp. in dental root canals.     

The microbial flora from root canals and periodontal pockets of non-vital teeth associated with advanced periodontitis

Microflora from root canals and periodontal pockets of periodontally affected teeth were compared in order to elucidate the as yet unknown relationship between pulpal and periodontal disease. Caries-free teeth affected with advanced periodontitis and diagnosed as clinically dead by electric pulp testing were selected. The root canals and periodontal pockets were sampled, and the bacterial flora examined by both culture and interference microscopy. The results indicated that the aerobe/anaerobe ratio in the periodontal pocket was 0.23, while it was 0.0022 in the root canal, the large predominance of obligate anaerobes reflecting the anaerobic environment found in the root canal. Morphological classification obtained from interference microscopy showed similar proportions of morphotypes in the two sites. Results of anaerobic culture demonstrated a significantly higher rate of detection of facultative Streptococcus bacteria in the periodontal pocket than in the root canal. The predominant bacterial species common to both regions were Streptococcus, Peptostreptococcus, Eubacterium, Bacteroides, and Fusobacterium for obligate anaerobes. As for facultative anaerobes, Actinomyces and Streptococcus were detected predominantly in the periodontal pocket. The occurrence of micro-organisms common to both sites in this study suggests that the periodontal pocket may be a possible source of root canal infections.     

Bacterial examination of endodontic infections by clonal analysis in concert with denaturing high‐performance liquid chromatography

Background/aims: The aim of this study was to examine the diversity of bacterial species in the infected root canals of teeth associated with endodontic abscesses by cloning and sequencing techniques in concert with denaturing high‐performance liquid chromatography. Methods: Samples collected from five infected root canals were subjected to polymerase chain reaction (PCR) with universal 16S ribosomal DNA primers. Products of these PCRs were cloned and sequenced. Denaturing high‐performance liquid chromatography (DHPLC) was used as a screening method to reduce the number of clones necessary for DNA sequencing. Results: All samples were positive for the presence of bacteria and a range of 7–13 different bacteria were found per root canal sample. In total, 48 different oral clones were detected among the five root canal samples. Olsenella profusa was the only species present in all samples. Porphyromonas gingivalis, Dialister pneumosintes, Dialister invisus, Lachnospiraceae oral clone, Staphylococcus aureus, Pseudoramibacter alactolyticus, Peptostreptococcus micros and Enterococcus faecalis were found in two of the five samples. The majority of the taxa were present in only one sample, for example Tannerella forsythia, Shuttleworthia satelles and Filifactor alocis. Some facultative anaerobes that are frequently isolated from endodontic infections such as E. faecalis, Streptococcus anginosus and Lactobacillus spp. were also found in this study. Conclusion: Clonal analysis of the microflora associated with endodontic infections revealed a wide diversity of oral species.     

慢性根尖周炎瘘管发生与根管内特殊细菌定植相关性的初步研究

目的:检测和比较有瘘型与无瘘型根尖周病感染根管内优势菌丛的分布特点.方法:选择60位慢性根尖周炎患者共64颗患牙,在对每个患牙进行根管治疗的过程中,髓腔预备及根管初预备后,用无菌纸尖吸取根管内容物进行采样,置入无菌EP管,于10 min内送至-20℃冰箱保存.对根管内样本进行genomic DNA提取,使用27种选取的由基因公司提供的感染根管内优势菌的引物对上述64例样本进行多重模板扩增,根据扩增产物的有无判断各样本中含有的菌种类型.结果:瘘管的发生与Prevotella nigrescens(变黑普氏菌)、Porphyromonas spp.(卟啉单胞菌)、Peptostreptococcus stomatis(口炎消化链球菌)、革兰氏阴性球菌、Capnocytophaga ochracea(黄褐二氧化碳嗜纤维菌)、Corynebacterium matruchotii(马氏棒杆菌)、Hafnia spp.(哈夫尼菌属)及Eubacterium spp.(真杆菌属)的感染有关.结论:慢性根尖周炎瘘管发生与根管内分离到的Prevotella nigrescens、Porphyromonas spp.、Peptostreptococcus stomatis、革兰氏阴性球菌、Capnocytophaga ochracea、Corynebacterium matruchotii、Hafnia spp.及Eubacterium spp.的特异性定植有关.     

Identical clonal types of Porphyromonas gingivalis or Prevotella nigrescens recovered from infected root canals and subgingival plaque

Clinical samples from 10 infected root canals and from subgingival plaque in 10 patients were screened by anaerobic culture for black-pigmented anaerobes. A total of 17 Porphyromonas gingivalis and 9 Prevotella nigrescens were obtained from four patients and were identified by species-specific polymerase chain reaction (PCR) amplification. The arbitrarily primed PCR reaction used to examine the genetic diversity of the isolates revealed that the P. gingivalis or P. nigrescens simultaneously present in the root canal system and in subgingival plaque of all four patients were genotypically indistinguishable. These data indicate that the endodontium and the periodontium can be colonized by the same clonal types of black-pigmented anaerobes.     

Antibacterial action of photoactivated disinfection {PAD} used on endodontic bacteria in planktonic suspension and in artificial and human root canals

OBJECTIVES: To measure antibacterial action of photoactivated disinfection (PAD) on endodontic bacteria in planktonic suspension and root canals. Methods: Four bacteria, Fusobacterium nucleatum,Peptostreptococcus micros, Prevotella intermedia and Streptococcus intermedius, were tested in suspension. After mixing equal volumes of Tolonium chloride and bacterial suspension for 60s, each 200 microL of concentration (>10(6)cfu mL(-1)) was irradiated with light at 633+/-2 nm. Each energy dose/Tolonium chloride concentration combination was tested eight times, with controls. Prepared root canals in Training Blocs and extracted human teeth were inoculated with S. intermedius followed by 10 mg L(-1) Tolonium chloride or saline. Bacteria in canals were sampled before and after light irradiation. Student t-test assessed significance of changes in viable bacteria produced by treatment of either light or Tolonium chloride alone and light/Tolonium chloride combinations. RESULTS: In suspension, reductions in bacteria were highly significant (P<0.01) for light/Tolonium chloride combinations compared to light or Tolonium chloride alone. Maximum mean log reductions of 1.14 (P. intermedia), 2.48 (P. micros), 2.81 (F. nucleatum) and 6.73 (S. intermedius) were at 4.8 J/20 mg L(-1). Antibacterial action was increased by energy dose increase (not always significantly), but not by Tolonium chloride concentration. In control canals mean log reductions of 0.42 (Blocs) and 0.38 (teeth) from initial levels were not significant. PAD mean log reductions of 2.40 (Blocs) and 2.01 (teeth) were highly significant. Changes for PAD/energy dose combinations were not significant. CONCLUSION: PAD killed endodontic bacteria at statistically significant levels compared to controls. Kills varied with bacterial species.     

Elevated blood pressure is not related to saliva flow in patients with Sjögren's syndrome

OBJECTIVE: The extent to which Fusobacterium nucleatum is recovered from root canals of teeth that present with an interappointment flare-up following endodontic instrumentation was investigated. STUDY DESIGN: Included in the study were 28 patients that sought emergency treatment after initiation of root canal therapy. Only non-painful teeth that had been treated because of a necrotic pulp and periapical inflammatory lesion were studied. Root canal samples for bacterial analysis were taken, transported to a bacteriological laboratory, and processed for a semiquantitative assessment of bacterial isolates. Bacterial findings were correlated with self-assessed pain intensity as recorded by means of a Visual Analogue Scale. Clinical presentation of swelling and presence of exudate in the treated root canals were also linked. RESULTS: Bacteria were recovered from all teeth examined. Gram-negative anaerobic coccoid rods (Prevotella species and Porphyromonas species) were frequent isolates. All teeth in patients who were reported to be in severe pain (Visual Analogue Scale > or = 6) displayed F nucleatum. Nine out of 10 of these teeth also had swelling and exudate in the root canals. Samples from the remaining patients that had teeth with less pain score showed a variable bacterial recovery. None of these teeth displayed F nucleatum. CONCLUSION: F nucleatum appears to be associated with the development of the most severe forms of interappointment endodontic flare-ups.     

Fungi in therapy-resistant apical periodontitis

The occurrence of yeasts in 967 microbiological endodontic samples taken from root canals in persistent endodontic infections was studied. The sampling was done by general practitioners in various parts of Finland from root canal infections which did not respond favourably to standard conservative therapy. The samples were cultivated aerobically on a non-selective enriched horse blood agar medium, on TSBV agar medium in 5% CO₂ and anaerobically on horse blood agar medium. Micro-organisms were found in 692 of the samples while 275 showed no growth. Forty-eight fungi were isolated from 47 samples which is 7% of the culture-positive samples. Twenty yeast strains were identified further by their colony morphology, growth and cellular characteristics and patterns of carbohydrate assimilation. All isolates except one belonged to the genus Candida. Candida albicans was the most common species. C. glabrata was found together with C. albicans in one sample. C. guilliermondii, C. inconspicua and Geotrichum candidum were each isolated once. Yeasts were found in pure culture in six samples and together with bacteria in 41 samples. In all the samples except two, the accompanying facultative bacteria were Gram positive. The most frequent of them were α- and non-haemolytic Streptococcus species which were found in 31 samples. Anaerobic bacteria were isolated together with yeasts from 12 root canals. They included both Gram positive species such as Peptostreptococcus micros and Gram negative species such as Fusobacterium nucleatum . The regular isolation of yeasts, also in pure culture, indicates that yeasts may have an important role in cases of apical periodontitis persisting after conventional treatment.     

Microorganisms in closed periapical lesions

The purpose of this study was to investigate the microorganisms of strictly selected closed periapical lesions associated with both refractory endodontic therapy and pulpal calcification. Definitive criteria were established that assured complete clinical isolation of the periapical lesion from the oral and periodontal environment. A total of 13 criteria-referenced lesions were selected from 70 patients with endodontic surgical indications. A well controlled culturing method was used in all cases and samples were taken by one clinician at three separate sites during each surgery. Samples taken at the surgical window and within the body of the lesion served as controls, whilst a third sample was taken at the apex. In all 13 cases, samples taken from the apex yielded microorganisms comprising 63.6% obligate anaerobes and 36.4% facultative anaerobes. Prevalence of the isolated species was 31.8% for Actinomyces sp., 22.7%, Propionibacterium sp., 18.2% Streptococcus sp., 13.6% Staphlyococcus sp., 4.6% Porphyromonas gingivalis , 4.6% Peptostreptococcus micros and 4.6% Gram-negative enterics. The results of this investigation indicate that closed periapical lesions associated with calcified teeth or those resistant to root canal treatment harbour bacteria. The inability to eradicate all root canal microorganisms during root canal treatment, along with anatomical factors, may allow further bacterial colonization of the root apex and surrounding periapical tissues, and consequently prevent healing.     

Bacteria recovered from teeth with apical periodontitis after antimicrobial endodontic treatment

Chávez de Paz LE, Dahlén G, Molander A, Möller Å, Bergenholtz G . Bacteria recovered from teeth with apical periodontitis after antimicrobial endodontic treatment. International Endodontic Journal, 36 , 500–508, 2003. Aim To determine whether there is a pattern for certain bacteria to remain after chemo‐mechanical treatment of root canals in teeth with apical periodontitis. Methodology Consecutive root‐canal samples of 200 teeth receiving root‐canal treatment, referred from general practitioners and endodontic specialists for analyses of cultivable microbes, were studied prospectively. To be included, samples had to be taken at a treatment session subsequent to the one at which endodontic therapy was initiated. All samples were from teeth that either presented with clinical or radiographic evidence of apical periodontitis or both. Bacteriological findings were linked to clinical and radiographic parameters including status of the root canal prior to treatment, namely, vital pulp, necrotic pulp or root filled. Results A total of 248 strains were isolated from 107 teeth giving bacterial growth. Gram‐positives predominated (85%). Lactobacillus spp. (22%), nonmutans streptococci (18%), and Enterococcus spp. (12%) were the most common isolates. Gram‐negative anaerobes were relatively sporadic. Large radiographic bone lesions, persistent pain and use of intracanal calcium hydroxide dressing correlated with bacterial presence (P < 0.05). Conclusions Once established, nonmutans streptococci, enterococci and lactobacilli appear to survive commonly following root‐canal treatment of teeth with clinical and radiographical signs of apical periodontitis.     

The microbiota of periodontal pockets with different depths in therapy-resistant periodontitis

This study presents the composition of the cultivable microbiota colonising periodontal pockets of different depths among 2 patient-groups classified as non-responsive (NR-group; 11 participants) or responsive (R-group; 10 participants) to periodontal treatment. Microbiological samples from three types of pocket (< 4 mm deep A-samples; 4-5 mm B-samples; > 5 mm C-samples) were analysed by cultural methods for putative periodontitis pathogens, microbial groups constituting > or = 5% of the total cultivable flora and opportunistic pathogens. Actinomyces naeslundii, A. israelii, Bacteroides forsythus, Fusobacterium spp, Porphyromonas gingivalis, Prevotella intermedia, Peptostreptococcus micros, anaerobic streptococci and facultative anaerobic streptococci were most prevalent. Actinobacillus actinomycetemcomitans, Staphylococcus aureus, enteric rods and yeasts were less prevalent. The periodontitis pathogens Bacteroides forsythus, Fusobacterium spp, Porphyromonas gingivalis, Prevotella intermedia and Peptostreptococcus micros constituted together (on average) < or = 23% of the viable counts in the A- and B-samples of both patient groups and in the C-samples of the R-group. In the C-samples of the NR-group their mean counts were 45%. Correlations were found between smoking habits and the five pathogens in the C-samples and in pooled pocket depth samples. The results show that groups of periodontopathogens should be considered a causal factor in therapy-resistant periodontitis. Further, smoking and deep pockets can enhance a shift in the balance of the subgingival microflora predisposing a site to disease and a susceptible host may be the pre-requisite to therapy-resistant periodontitis.     

Reactivity of IgG from explant cultures of periapical lesions with implicated microorganisms

The presence of IgG in periapical inflammatory lesions suggests that immune responses participate in the disease process. The purpose of this investigation was to study the reactivity of IgG from the supernatant fluids of explant cultures of periapical lesions with microorganisms implicated in infections of endodontic origin. Ninety periapical lesions that had been contiguous with the apex of a root were removed and maintained in explant cultures. A dot-enzyme-linked immunosorbent assay (dot-ELISA) was used to demonstrate the presence of IgG in the supernatant fluids of the explant cultures reactive with a panel of microorganisms associated with infections of endodontic origin. The percentages of reactivity by dot-ELISA follow: Bacteroides intermedius (84%), B. buccae (12%), Porphyromonas (Bacteroides) gingivalis (50%), P. endodontalis (58%), P. asaccharolyticus (17%), Peptostreptococcus micros (44%), P. anaerobius (26%), Eubacterium alactolyticum (34%), Fusobacterium nucleatum (14%), and Actinomyces israelii (6%). At least one of the three species of B. intermedius, P. gingivalis, or P. endodontalis tested gave a positive dot-ELISA with 89% of the supernatant fluids from explant cultures of periapical lesions. A lack of cross reactivity of IgG in supernatant fluids from explants of periapical lesions was demonstrated for the four strains of black-pigmented Bacteroides/Porphyromonas by dot-ELISA.     

Cultivable bacteria in infected root canals as identified by 16S rRNA gene sequencing

INTRODUCTION: Traditionally, cultivable bacteria isolated from infected root canals have been identified by phenotype-based methods. Because 16S ribosomal RNA (rRNA) gene sequencing has emerged as a more accurate and reliable tool for bacterial identification, the present study applied this approach to identify bacterial isolates recovered from the root canals of teeth with chronic apical periodontitis. METHODS: Anaerobic techniques were used for culturing; identification of the isolates was carried out by polymerase chain reaction amplification and sequencing of the V5-V8 region of the 16S rRNA gene. Bacteria were found in all samples. The mean number of taxa per canal was 3.1, ranging from 2 to 8. The median number of cultivable bacterial cells in the root canals was 4.2 x 10(5), ranging from 2.8 x 10(3) to 3.3 x 10(7). Eighty-seven strains belonging to 52 bacterial taxa were identified. The most prevalent taxa were Fusobacterium nucleatum, Porphyromonas gingivalis, Pseudoramibacter alactolyticus, Micromonas micros and streptococci. The following bacterial phyla were represented in this study: Firmicutes (22 taxa, 46% of the identified isolates), Actinobacteria (14 taxa, 25.3% of the isolates), Bacteroidetes (eight taxa, 13.8% of the isolates), Fusobacteria (three taxa, 9.2% of the isolates) and Proteobacteria (five taxa, 5.7% of the isolates). Some of the isolates represented unnamed species not previously cultivated and characterized. In conclusion, our findings using a combined anaerobic culture-molecular identification approach confirmed the polymicrobial nature of primary endodontic infections with dominance of anaerobic bacteria. Notably several bacteria that are difficult or impossible to identify by phenotypic means were identified, including previously uncultivated taxa, cultivated-but-not-yet-characterized taxa and newly named species.