Association of Enterococcus faecalis with different forms of periradicular diseases

Data from culture studies have revealed that Enterococcus faecalis is occasionally isolated from primary endodontic infections but frequently recovered from treatment failures. This molecular study was undertaken to investigate the prevalence of E. faecalis in endodontic infections and to determine whether this species is associated with particular forms of periradicular diseases. Samples were taken from cases of untreated teeth with asymptomatic chronic periradicular lesions, acute apical periodontitis, or acute periradicular abscesses, and from root-filled teeth associated with asymptomatic chronic periradicular lesions. DNA was extracted from the samples, and a 16S rDNA-based nested polymerase chain reaction assay was used to identify E. faecalis. This species occurred in seven of 21 root canals associated with asymptomatic chronic periradicular lesions, in one of 10 root canals associated with acute apical periodontitis, and in one of 19 pus samples aspirated from acute periradicular abscesses. Statistical analysis showed that E. faecalis was significantly more associated with asymptomatic cases than with symptomatic ones. E. faecalis was detected in 20 of 30 cases of persistent endodontic infections associated with root-filled teeth. When comparing the frequencies of this species in 30 cases of persistent infections with 50 cases of primary infections, statistical analysis demonstrated that E. faecalis was strongly associated with persistent infections. The average odds of detecting E. faecalis in cases of persistent infections associated with treatment failure were 9.1. The results of this study indicated that E. faecalis is significantly more associated with asymptomatic cases of primary endodontic infections than with symptomatic ones. Furthermore, E. faecalis was much more likely to be found in cases of failed endodontic therapy than in primary infections.     

Polymerase chain reaction identification of microorganisms in previously root-filled teeth in a South Korean population

A large body of evidence indicates that microorganisms are the primary causative agents of endodontic treatment failures. This study intended to assess the occurrence of nine putative endodontic pathogens in root-filled teeth associated with periradicular lesions in a South Korean population using a culture-independent molecular approach. Fourteen root-filled teeth with persistent periradicular diseases were selected for retreatment. After removal of the root canal filling, the canals were sampled, and a polymerase chain reaction assay using taxon-specific oligonucleotide primers was used for microbial detection. Bacteria were present in all cases, as revealed by amplification using ubiquitous 16S rDNA primers. The most frequently detected taxon was Enterococcus faecalis (64%), followed by Streptococcus spp. (21%) and Tannerella forsythensis (14%). The results of this study using a highly sensitive identification method are concurrent with those from other geographical locations using diverse identification methods in that E. faecalis is the main species found in cases of root-filled teeth associated with periradicular lesions.     

Microbial analysis of canals of root-filled teeth with periapical lesions using polymerase chain reaction

The objective of the present study was to investigate the presence of nine bacterial species in root-filled teeth associated with periapical lesions using a polymerase chain reaction analysis and to correlate these species with clinical features of the cases. DNA was extracted from 45 canal samples of root-filled teeth with periapical lesions. A PCR assay using species-specific primers of 16S rDNA and the downstream intergenic spacer region was used for microbial detection. Enterococcus faecalis was the most prevalent species, detected in 77.8% of the study teeth, followed by Peptostreptococcus micros, detected in 51.1%. Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia, and Prevotella nigrescens were detected in 35.6%, 22.2%, 11.1%, and 11.1% of the sampled teeth, respectively. Moreover, PCR detected Filifactor alocis in 26.7%, Treponema denticola in 24.4%, and Tannerella forsythia in 4.4% of the samples. T. denticola and P. micros were statistically associated with tenderness to percussion (p < 0.05). P. nigrescens was associated with the presence of spontaneous pain and abscess (p < 0.05). P. endodontalis and P. nigrescens were associated with purulent exudates (p < 0.05). Synergistic relationship was also observed between some species. The results of this study indicated that E. faecalis was the most frequently identified test species by PCR in teeth with failing endodontic treatment.     

Pseudoramibacter alactolyticus in primary endodontic infections

A nested polymerase chain reaction (PCR)-based method was used to directly survey samples taken from primary endodontic infections for the occurrence of Pseudoramibacter alactolyticus. Identification by nested PCR was performed in root-canal samples from teeth associated with asymptomatic periradicular lesions or acute apical periodontitis, and in pus samples from acute periradicular abscesses. DNA was extracted from the samples and initially amplified using universal 16S rDNA primers. A second round of amplification used the first PCR products to detect a specific fragment of P. alactolyticus 16S rDNA. P. alactolyticus was detected in 76% of root-canal samples from teeth showing asymptomatic periradicular lesions, in 60% of samples taken from root canals associated with acute apical periodontitis, and in 32% of pus samples aspirated from acute periradicular abscesses. No significant association of this species with clinical symptoms was observed (p > 0.01). In general, P. alactolyticus occurred in 56% of samples taken from infections of endodontic origin. The high prevalence of P. alactolyticus in infections of endodontic origin as detected by nested PCR in this study, and its apparent pathogenicity, particularly in mixed infections, indicate that this bacterial species is a candidate endodontic pathogen that can participate in the etiology of different forms of periradicular diseases.     

Evaluation of root canal microorganisms isolated from teeth with endodontic failure and their antimicrobial susceptibility

Studies of the microbiota from the canals of teeth with failure of endodontic therapy have revealed that it differs markedly from that of untreated necrotic dental pulps. This study aimed to evaluate the microbiota of 30 root-filled teeth with persisting periapical lesions and to test the antibiotic susceptibility of the most prevalent species. Microbial samples, isolation and speciation were done using advanced microbiologic techniques for anaerobic species. A total of 55 bacterial species were isolated, 80% were gram-positives and 58% facultative anaerobic microorganisms. The bacterial genera most frequently recovered were Enterococcus, Streptococcus, Peptostreptococcus and Actinomyces. Antibiotic sensitivity of Enterococcus faecalis and Peptostreptococcus spp. was accomplished with the E-test system. All species studied were susceptible to benzylpenicillin, amoxicillin, amoxicillin combined with clavulanate. However, 20% of the E.faecalis strains were resistant to erythromycin and 60% to azithromycin. It was concluded that microbial flora in canals after endodontic failure comprised predominantly facultative anaerobes and gram-positive species. E.faecalis was the species most frequently isolated and showed erythromycin and azithromycin resistance among the isolates.     

Actinomyces species,streptococci, and Enterococcus faecalis in primary root canal infections

The purpose of this study was to evaluate the prevalence of Actinomyces species, streptococci, and Enterococcus faecalis in primary root canal infections by using a molecular genetic method. Samples were obtained from 53 infected teeth, of which 27 cases were diagnosed as acute periradicular abscesses. DNA was extracted to evaluate the occurrence of 13 bacterial species by using whole genomic DNA probes and checkerboard DNA-DNA hybridization. Polymerase chain reaction using an ubiquitous bacterial primer was undertaken to check the presence of bacterial DNA in clinical samples. All root canal samples contained bacteria as demonstrated by polymerase chain reaction. The checkerboard DNA-DNA hybridization assay allowed the detection of streptococci in 22.6% of the samples, Actinomyces species in 9.4%, and E. faecalis in 7.5%. The most prevalent species were members of the Streptococcus anginosus group. With regard to the asymptomatic lesions, the most prevalent species were S. intermedius (11.5% of the cases), E. faecalis (11.5%), and S. anginosus (7.7%). S. constellatus was the most prevalent species in pus samples (25.9% of the cases). The other most prevalent species in abscessed teeth were A. gerencseriae (14.8%), S. gordonii (11.1%), S. intermedius (11.1%), A. israelii (7.4%), S. anginosus (7.4%), and S. sanguis (7.4%). S. constellatus was the only species positively associated with acute periradicular abscess (p < 0.01).     

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.     

Bacteroides forsythus in primary endodontic infections as detected by nested PCR

The purpose of this study was to investigate the prevalence of Bacteroides forsythus in primary endodontic infections using a species-specific nested polymerase chain reaction assay. Samples were collected from 50 teeth having carious lesions, necrotic pulps, and different forms of periradicular diseases. DNA extracted from the samples was initially amplified using universal 16S rDNA primers. A second round of amplification used the first polymerase chain reaction products to detect a specific fragment of B. forsythus 16S rDNA. B. forsythus was detected in 13 of 22 asymptomatic cases (59.1%), 4 of 10 root canals associated with acute apical periodontitis (40%), and 9 of 18 cases diagnosed as acute periradicular abscesses (50%). There was no relationship between the presence of B. forsythus and the occurrence of symptoms. In general, this bacterial species was detected in 26 of 50 samples of endodontic infections (52%). The findings of this study support the assertion that this bacterial species is associated with infections of endodontic origin and suggest that B. forsythus may be involved in the pathogenesis of different forms of periradicular lesions.     

Detection and eradication of microorganisms in root-filled teeth associated with periradicular lesions: an in vivo study

This study determined the presence of microorganisms by culture and polymerase chain reaction in asymptomatic root-filled teeth with periradicular lesions. Furthermore, a disinfecting regimen using sodium hypochlorite (NaOCl), ethylenediaminetetraacetic acid (EDTA), chlorhexidine digluconate (CHX) irrigation, and calcium hydroxide (Ca(OH)(2)) dressing was assessed. After removal of the root-filling material, specimens of 20 cases undergoing retreatment were sampled. Moreover, the canals were sampled after each step of the disinfecting regimen. Prevalence of microorganisms was 60% by culture and 65% by polymerase chain reaction. In four of those samples (31%), DNA of Enterococcus faecalis was found. After further root canal preparation and irrigation using NaOCl and EDTA, microorganisms could be detected in none of the teeth. Thus, CHX and Ca(OH)(2) could not show further disinfection. In contrast, microorganisms were found in two teeth after the interappointment dressing. It may be concluded that proper root canal preparation and irrigation using NaOCl and EDTA are sufficient for decontamination of the root canal system during endodontic retreatment.     

Application of denaturing gradient gel electrophoresis (DGGE) to the analysis of endodontic infections

The recent expanding use of cultivation-independent techniques for bacterial identification is reliant on the lack of knowledge of the conditions under which most bacteria are growing in their natural habitat and the difficulty to develop culture media that accurately reproduce these conditions. A molecular method that has been recently used in several areas to examine the bacterial diversity living in diverse environments is the denaturing gradient gel electrophoresis (DGGE). In DGGE, polymerase chain reaction (PCR)-generated DNA fragments of the same length but with different base-pair sequences can be separated. Separation is based on electrophorectic mobility of a partially melted double-strand DNA molecule in polyacrylamide gels, which is decreased when compared with that of the completely helical form of the molecule. Molecules with different sequences may have a different melting behavior and will therefore stop migrating at different positions in the gel. Application of the PCR-DGGE method in endodontic research has revealed that there are significant differences in the predominant bacterial composition between asymptomatic and symptomatic cases. This suggests that the structure of the bacterial community can play a role in the development of symptoms. In addition, new bacterial phylotypes have been disclosed in primary endodontic infections. PCR-DGGE has also confirmed that intra-radicular infections are a common finding in root-filled teeth associated with persistent periradicular lesions. The microbiota in failed cases significantly vary from teeth to teeth, with a mean number of species far higher than previously shown by culturing approaches. Application of the PCR-DGGE technique in endodontic microbiology research has the potential to shed light on several aspects of the different types of endodontic infection as well as on the effects of treatment procedures with regard to infection control.     

Nested PCR detection of Centipeda periodontii in primary endodontic infections

In recent years, molecular genetic methodologies have provided significant additional knowledge about components of the microbiota associated with infections of endodontic origin. Following this research line, the purpose of the present study was to investigate the prevalence of Centipeda periodontii in primary endodontic infections using a species-specific nested PCR assay. Samples were collected from fifty teeth having carious lesions, necrotic pulps, and different forms of periradicular diseases. DNA extracted from the samples was initially amplified using universal 16S rDNA primers, and a second round of amplification used the first PCR products to detect a specific fragment of C. periodontii 16S rDNA. This species was detected in 3 (13%) of 23 asymptomatic cases, in 1 (14%) of 7 cases diagnosed as acute apical periodontitis, and in 3 (15%) of 20 pus samples aspirated from acute periradicular abscesses. There was no significant association between C. periodontii and the presence of clinical symptoms. Overall, C. periodontii was detected in 14% of the cases of endodontic infections. This is probably the hitherto first study to detect C. periodontii in primary endodontic infections. The specific role played by this bacterial species in infections of endodontic origin awaits further clarification.     

Phenotypic and genotypic identification of enterococci isolated from canals of root-filled teeth with periapical lesions

The objectives of the present study were to identify enterococcal species isolated from the canals of root-filled teeth with periapical lesions using biochemical and molecular techniques, and to investigate the genetic diversity of the isolates. Twenty-two Enterococcus strains, isolated from the canals of root-filled teeth with persisting periapical lesions, were identified to species level using rapid ID 32 STREP galleries and partial 16S rDNA sequencing. To subtype the strains, genomic DNA from the isolates was analyzed by pulsed field gel electrophoresis (PFGE) after digestion with SmaI. Intragenic regions of two genes, ace and salA, were sequenced for further differentiation of the isolates. All strains were identified as Enterococcus faecalis by both commercial kit and partial 16S rDNA sequencing. PFGE with SmaI of 22 isolates demonstrated 18 macrorestriction profiles, whereas 13 distinct genotypes were identified after analysis of the ace and salA composite sequences. Most of the isolates from distinct patients had different PFGE profiles. Moreover, in two cases, different E. faecalis strains were found in different root-filled teeth from the same mouth. E. faecalis was the only enterococcal species isolated from the canals of root-filled teeth with periapical lesions. Genetic heterogeneity was observed among the E. faecalis isolates following PFGE and sequence-based typing method. Furthermore, the genetic diversity within root canal strains was similar to previous reports regarding E. faecalis isolates from different clinical and geographic origins.     

Detection of Filifactor alocis in endodontic infections associated with different forms of periradicular diseases

Recent molecular studies have expanded the list of suspected endodontic pathogens. The aim of this study was to evaluate the occurrence of Filifactor alocis in primary endodontic infections associated with different forms of periradicular diseases. Identification by nested polymerase chain reaction was performed in root canal samples from teeth associated with either asymptomatic periradicular lesions or acute apical periodontitis. Samples were also taken by aspiration of purulent exudate associated with acute apical abscesses. DNA extracted from the samples was initially amplified using universal 16S rDNA primers followed by a second round of amplification using the first PCR products to detect a specific fragment of F. alocis 16S rDNA. F. alocis was detected in 12/21 (57.1%) root canal samples from teeth showing asymptomatic periradicular lesions and in 3/10 (30%) samples taken from root canals associated with acute apical periodontitis. This species occurred in 8/19 (42.1%) pus aspirates obtained from abscessed teeth. In general, F. alocis was detected in 23/50 (46%) samples taken from endodontic infections. These findings suggest that F. alocis is involved in the etiology of different forms of periradicular diseases and has the potential to be an endodontic pathogen.     

Detection of bacteria in endodontic samples by polymerase chain reaction assays and association with defined clinical signs in Italian patients

BACKGROUND/AIMS: The presence of selected bacteria (Enterococcus faecalis, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis, Treponema denticola) in infected root canals was studied using polymerase chain reaction (PCR) assays, and the association of bacteria with clinical signs of endodontic disease was assessed. The null hypothesis, that no difference could be observed between clinical signs of apical periodontitis and a specific bacterial strain, was tested. METHODS: Microbial samples were obtained from 62 teeth in 54 patients with endodontic disease. For each tooth, clinical data including patient symptoms were collected. Teeth were categorized by diagnosis as having acute apical periodontitis (AAP, teeth with clinical symptoms but no periapical radiolucency, n=22), chronic apical periodontitis (CAP, teeth with radiolucency but no clinical symptoms, n=15) or exacerbated apical periodontitis (EAP, teeth with symptoms and radiolucency, n=25). Seventy-one percent of cases were primary endodontic infections, and 29% were recurrent ('secondary') endodontic infections (failing cases). PCR assays were used to detect the presence of the selected bacteria. RESULTS: T. denticola and E. faecalis were each detected in 15 of 62 samples (24%), P. gingivalis in 8 samples (13%), P. intermedia in 5 samples (8%), and T. forsythensis in 4 samples (7%). T. denticola was detected in 56% of teeth with EAP. E. faecalis was found in 60% of teeth with CAP and in 72% of teeth with secondary infection. Statistical analysis demonstrated an association of CAP and secondary endodontic infection with the presence of E. faecalis. (P<0.01). EAP was associated with the presence of T. denticola (P<0.01). CONCLUSION: T. denticola was associated with symptomatic endodontic disease in the presence of apical bone resorption. E. faecalis was associated with treatment failures. We suggest that these species may play critical roles in endodontic pathology.     

Detection of Porphyromonas endodontalis in infected root canals by 16S rRNA gene-directed polymerase chain reaction

Porphyromonas endodontalis has been isolated from the endodontic infections mainly in symptomatic teeth. This study evaluated the occurrence of P. endodontalis in both symptomatic and asymptomatic endodontic infections using 16S rRNA gene-directed polymerase chain reaction. P. endodontalis was detected in 39.5% of the cases (17 of 43 teeth). It was present in 4 of the 6 cases with acute periradicular abscess (66.7%) and in 13 of the 37 other cases (35.1%). The presence of P. endodontalis was associated with an asymptomatic periradicular lesion in 6 cases (25%) and in 10 teeth with tenderness to percussion (52.6%). P. endodontalis was also found in one asymptomatic case without evidence of periradicular pathosis. Our results indicated that, although P. endodontalis is commonly detected in symptomatic cases, it can be present in asymptomatic root canal infections. Further studies should determine if this bacterial species is really an important endodontopathogen.     

Isolation of yeasts and enteric bacteria in root-filled teeth with chronic apical periodontitis

AIMS: The aim of this study was to determine the occurrence and role of yeasts, enteric gram-negative rods and Enterococcus species in root-filled teeth with chronic apical periodontitis, and the antimicrobial effect of iodine potassium iodide (IKI) irrigation. METHODOLOGY: Forty symptom-free root-filled teeth with chronic apical periodontitis were included in the study. The patients were divided into two groups. In group A the canals were filled with calcium hydroxide for 10-14 days after cleaning and shaping; in group B the canals were irrigated with IKI for 5 min after cleaning and shaping followed by a permanent root filling. Microbiological samples were taken from the canals before and after the chemomechanical preparation and after iodine irrigation (group B). RESULTS: Microbes were isolated from 33 of 40 teeth in the initial sampling. Yeasts were isolated from six teeth, three of them together with E. faecalis. Enteric rods (Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis) were present in three teeth and E. faecalis was isolated from 21 of the 33 culture positive teeth, 11 in pure culture. Growth was detected in 10 teeth of the second samples. Six of the 10 cases were E. faecalis, with five being a pure culture. All third samples (after IKI) except one were negative. The number of microbial cells per sample did not correlate with lesion size. Two flare-ups were recorded, both in teeth with a mixed infection. CONCLUSION: The high prevalence of enteric bacteria and yeasts in root-filled teeth with chronic apical periodontitis was established. IKI improved the antimicrobial effect of the treatment.     

Detection of Treponema denticola in endodontic infections by 16S rRNA gene-directed polymerase chain reaction

A 16S rDNA-based polymerase chain reaction (PCR) method was used to detect the occurrence of Treponema denticola in root canal infections. Samples were collected from 21 single-root teeth having carious lesions, necrotic pulps and radiographic evidences of periradicular bone loss. DNA extracted from the samples was amplified using the PCR assay, which yielded specific fragment of T. denticola 16S rDNA. T. denticola was detected in 11 of 21 cases (52.4%), regardless of the presence or absence of symptoms. Since this spirochete was found in a relatively high percentage of the endodontic infections examined and because it is a pathogenic microorganism involved in periodontal diseases, there are reasons to believe that T. denticola can also participate in the pathogenesis of periradicular lesions of endodontic origin.     

Molecular detection of Enterococcus species in root canals of therapy-resistant endodontic infections

OBJECTIVES: The objectives were to identify Enterococcus spp in nonhealing endodontic cases using PCR amplification and molecular sequencing, and to determine if the prevalence of enterococci is increased in diabetic patients. STUDY DESIGN: Specimens from 40 cases undergoing retreatment were incubated in prereduced thioglycollate broth at 37 degrees C. Extracted DNA had PCR amplification using primers that target the tuf gene of 14 Enterococcus spp. PCR products were directly sequenced and identified phylogenetically. RESULTS: Three cases were eliminated because the patients were on antibiotics or the tooth did not have a periradicular radiolucency. The remaining 37 specimens included 6 from diabetic patients. Eight specimens were positive for Enterococcus spp. Of these, 6 (19%) were from nondiabetic and 2 (33%) from diabetic patients (odds ratio = 2.1; chi squared, P = .45). Phylogenetically, all sequences from positive specimens matched E faecalis V583 (AE016947). Conclusion E faecalis was the only enterococcal species detected, with an overall prevalence of 22%.     

根管内粪肠球菌感染的研究进展

粪肠球菌（E.faecalis）在治疗失败根管内的检出率较高,是根管持续感染和再感染的重要微生物之一。粪肠球菌在治疗前后根管内的感染特点不同,并且对抗菌药物有较强耐药性。目前的根管清理和消毒方法难以将定植于根管中的粪肠球菌彻底清除。本文就有关根管内粪肠球菌的感染特点及其对抗菌剂的敏感性研究进展作一综述。     

Intraradicular bacteria and fungi in root-filled, asymptomatic human teeth with therapy-resistant periapical lesions: a long-term light and electron microscopic follow-up study

Light and electron microscopy were used to analyze nine therapy-resistant and asymptomatic human periapical lesions, which were removed as block biopsies during surgical treatment of the affected teeth. The cases that required surgery represented about 10% of all of the cases which received endodontic treatment and root fillings during the period 1977 to 1984. These cases revealed periapical lesions when they were examined 4 to 10 yr after treatment. The biopsies were processed for correlated light and electron microscopy. Six of the nine biopsies revealed the presence of microorganisms in the apical root canal. Four contained one or more species of bacteria and two revealed yeasts. Of the four cases in which bacteria were found, only in one biopsy could they be found by light microscope. In the other three specimens, the bacterial presence could be confirmed only after repeated electron microscopic examination of the apical root canal by serial step-cutting technique. Among the three cases in which no microorganisms could be encountered, one showed histopathological features of a foreign body giant cell granuloma. These findings suggest that in the majority of root-filled human teeth with therapy-resistant periapical lesions, microorganisms may persist and may play a significant role in endodontic treatment failures. In certain instances such lesions may also be sustained by foreign body giant cell type of tissue responses at the periapex of root-filled teeth.