Molecular identification and quantification of bacteria from endodontic infections using real-time polymerase chain reaction

Introduction:  It was the aim of the present study to evaluate root canal samples for the presence and numbers of specific species as well as for total bacterial load in teeth with chronic apical periodontitis using quantitative real-time polymerase chain reaction (PCR).
Methods:  Forty adult patients with one radiographically documented periapical lesion were included. Twenty teeth presented with primary infections and 20 with secondary infections, requiring retreatment. After removal of necrotic pulp tissue or root canal filling, a first bacterial sample was obtained. Following chemo-mechanical root canal preparation a second sample was taken and a third sample was obtained after 14 days of intracanal dressing with calcium hydroxide. Analysis by real-time PCR enabled the quantification of total bacterial counts and of nine selected species.
Results:  Root canals with primary infections harbored significantly more bacteria (by total bacterial count) than teeth with secondary infections ( P  < 0.05). Mean total bacterial count in the retreatment group was 2.1 × 10⁶ and was significantly reduced following root canal preparation (3.6 × 10⁴) and intracanal dressing (1.4 × 10⁵). Corresponding values for primary infections were: 4.6 × 10⁷, 3.6 × 10⁴, and 6.9 × 10⁴. The numbers of the selected bacteria and their detection frequency were also significantly reduced.
Conclusion:  Root canals with primary infections contained a higher bacterial load. Chemo-mechanical root canal preparation reduced bacterial counts by at least 95%.     

Multiplex polymerase chain reaction detection of black-pigmented bacteria in infections of endodontic origin

The purpose of this study was to detect the presence of Porphyromonas endodontalis, P. gingivalis, Prevotella intermedia, P. nigrescens, and P. tannerae from clinical samples using multiplex polymerase chain reactions (PCR). Two different multiplex PCR protocols were used (one for the two Porphyromonas species and the other for the three Prevotella species), each one using a primer pair specific for each target species. The results were compared to those of the conventional culture procedures. Microbial samples were taken aseptically from 40 infected root canals and abscesses from patients. Samples were cultured in an anaerobic condition for conventional identification using a Rapid ID 32 A kit. Multiplex PCR was processed using the DNA extracted from each sample. At least one of the five species of black-pigmented bacteria (BPB) were detected in 65% (26 of 40) of the samples using multiplex PCR, and in 15% (6 of 40) using the conventional culture procedures. Multiplex PCR was more rapid, sensitive, specific, and effective in detecting BPB than the conventional culture procedures.     

Peptostreptococcus micros in primary endodontic infections as detected by 16S rDNA-based polymerase chain reaction

A 16S rDNA-based polymerase chain reaction (PCR) method was used to detect Peptostreptococcus micros in primary root canal infections. Samples were collected from 50 teeth having carious lesions, necrotic pulps, and different forms of periradicular diseases. DNA extracted from the samples was amplified using the PCR assay, which yielded a specific fragment of P. micros 16S rDNA. P. micros was detected in 6 of 22 root canals associated with asymptomatic chronic periradicular lesions (27.3%), 2 of 8 teeth with acute apical periodontitis (25%), and 6 of 20 cases of acute periradicular abscess (30%). In general, P. micros was found in 14 of 50 cases (28%). There was no correlation between the presence of P. micros and the occurrence of symptoms. Findings suggested that P. micros can be involved in the pathogenesis of different forms of periradicular lesions.     

Molecular identification and quantification of bacteria from endodontic infections using real‐time polymerase chain reaction

Introduction: It was the aim of the present study to evaluate root canal samples for the presence and numbers of specific species as well as for total bacterial load in teeth with chronic apical periodontitis using quantitative real‐time polymerase chain reaction (PCR). Methods: Forty adult patients with one radiographically documented periapical lesion were included. Twenty teeth presented with primary infections and 20 with secondary infections, requiring retreatment. After removal of necrotic pulp tissue or root canal filling, a first bacterial sample was obtained. Following chemo‐mechanical root canal preparation a second sample was taken and a third sample was obtained after 14 days of intracanal dressing with calcium hydroxide. Analysis by real‐time PCR enabled the quantification of total bacterial counts and of nine selected species. Results: Root canals with primary infections harbored significantly more bacteria (by total bacterial count) than teeth with secondary infections (P < 0.05). Mean total bacterial count in the retreatment group was 2.1 × 10⁶ and was significantly reduced following root canal preparation (3.6 × 10⁴) and intracanal dressing (1.4 × 10⁵). Corresponding values for primary infections were: 4.6 × 10⁷, 3.6 × 10⁴, and 6.9 × 10⁴. The numbers of the selected bacteria and their detection frequency were also significantly reduced. Conclusion: Root canals with primary infections contained a higher bacterial load. Chemo‐mechanical root canal preparation reduced bacterial counts by at least 95%.     

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.     

Novel subgingival bacterial phylotypes detected using multiple universal polymerase chain reaction primer sets

INTRODUCTION: Molecular ecological analysis based on 16S rRNA gene sequence analysis is well established for the characterisation of complex bacterial communities. However, 'universal' PCR primers can introduce biases into the analysis of the species composition of clone libraries because of mismatches between the primer and target organism sequences. In this study, three universal primer sets were compared for the analysis of the microflora in subgingival plaque. METHODS: Three subgingival plaque samples were collected from two subjects with localised severe chronic periodontitis. Half of each sample was cultured while DNA was extracted from the remaining half and 16S rDNA amplified with universal primer pairs 27F, 1525R (A); 27F, 1492R (B) and 530F, 1525R (C). Amplified genes were cloned, sequenced and identified by comparison with 16S rRNA databases. RESULTS: 137 taxa were identified among 177 isolates and 417 clones sequenced. Of these, 86 were detected only by the molecular technique whereas 26 were found only by culture. Sequences from 81 taxa did not correspond to those of named species and of these, 38 were not represented in the nucleotide databases. 16S RNA genes for these 38 taxa were sequenced and deposited with GenBank. CONCLUSION: The use of three sets of universal primers allowed the identification of 38 novel bacterial phylotypes. There were marked differences in the composition of the libraries generated by the different primer sets. A combination of molecular and cultural techniques is recommended to maximise the coverage of detection of bacterial taxa in oral samples.     

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.     

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.     

The effect of antibiotics and endodontic antimicrobials on the polymerase chain reaction

The effectiveness of endodontic antimicrobial treatment could be determined using sensitive molecular methods. The purpose of this study was to determine if antibiotics or endodontic reagents interfere with the ability of PCR to detect Enterococcus faecalis in vitro. Amoxicillin (25 mg/ml), clindamycin (15 mg/ml), tetracycline (25 mg/ml), doxycycline (10 mg/ml), calcium hydroxide, 1% buffered sodium hypochlorite (NaOCl1), 3% and 6% unbuffered NaOCl (NaOCl3 and NaOCl6), 2% chlorhexidine (CHX), 5% tincture iodine (TI), 2% iodine potassium iodide (IKI), chloroform (CF), 70% ethyl alcohol, 5% sodium thiosulphate, 5% citric acid or saline were added to 10 or 10 cells/ml E. faecalis ATCC 19433 for 1 h (1 wk for Ca(OH)2). Using PCR, all specimens were positive except for NaOCl3 and NaOCl6. PCR with Ca(OH)2 was positive with 10 cells/ml but negative with 10 cells/ml. The following reagents yielded negative culturing results: all antibiotics, Ca(OH)2, CHX, IKI, TI, NaOCl3, NaOCl6, and CF. BacLight nuclear staining revealed the presence of viable cells in all PCR positive, culture negative combinations, except for those with CF. Therefore, in the presence of threshold values of bacterial concentrations, all reagents tested except for NaOCl3 and NaOCl6 do not interfere with the detection of E. faecalis using PCR.     

Quantification of bacteria in oral samples by competitive polymerase chain reaction.

Information about the total amount of bacteria in oral samples contributes to assessment of an individual's risk of contracting dental caries or developing periodontitis and the prediction of that individual's clinical course. Since existing techniques are often time-consuming and expensive, it seemed attractive to look for alternative methods for the quantification of eubacteria. With their high specificity and sensitivity, polymerase chain-reaction (PCR) techniques have the potential of supplying fast and reliable results. We developed a method of competitive PCR for the quantification of eubacteria. We designed forward and reverse PCR primers which bind to highly conserved sequences of the bacterial 16S rRNA gene. A homologous competitor was synthesized with Escherichia coli 16S rDNA as a template, with the reverse primer and a hybrid primer which binds 67 bases downstream to the forward primer and carries the forward primer sequence at its 5' end. Specificity controls with 30 different bacterial species, 5 Archaea, 3 fungi, human astrocytoma cells, and rat hepatoblastoma cells were carried out. Results were positive for all eubacteria and negative for all other cells tested. Calibration curves were obtained by co-amplification of known amounts of E. coli cells in the presence of the homologous competitor. The developed method was successfully applied to assessment of the accumulation of bacteria during an oral hygiene cessation experiment. The competitive PCR method proved to be a reliable and fast method for the quantification of bacterial DNA and cultured eubacteria, as well as of bacteria in biological samples. It may find further applications not only in periodontology and cariology but also in other fields of medical microbiology.     

Detection of periodontal bacteria in atheromatous plaque by nested polymerase chain reaction

Background: In recent years, increasing evidence regarding the potential association between periodontal diseases and cardiovascular diseases has been identified. The available evidence underlines the importance of detecting periodontal pathogens on atheromatous plaque as the first step in demonstrating the causal relationship between the two conditions. The main aim of this investigation is to detect periodontitis‐associated bacteria from carotid artery atheromatous plaque from patients who received an endarterectomy using strict sample procurement and laboratory procedures. Methods: Atheromatous plaque from endarterectomies from carotid arteries were scraped and homogenized, and bacterial DNA was extracted. To obtain a representative concentration of amplicons, two amplifications of the bacterial 16S ribosomal‐RNA gene were carried out for each sample with universal eubacteria primers by a polymerase chain reaction (PCR). A nested PCR with specific primers for the target bacteria was performed next. Statistical tests included the χ² test. Results: Forty‐two atheromatous plaque were analyzed. All of them were positive for ≥1 target bacterial species. The bacterial species most commonly found was Porphyromonas gingivalis (78.57%; 33 of 42), followed by Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) (66.67%; 28 of 42), Tannerella forsythia (previously T. forsythensis) (61.90%; 26 of 42), Eikenella corrodens (54.76%; 23 of 42), Fusobacterium nucleatum (50.00%; 21 of 42), and Campylobacter rectus (9.52%; four of 42). The simultaneous presence of various bacterial species within the same specimen was a common observation. Conclusion: Within the limitations of this study, the presence of DNA from periodontitis‐associated bacteria in carotid artery atheromatous plaque retrieved by endarterectomy is confirmed.     

Comparative analysis of putative periodontopathic bacteria by multiplex polymerase chain reaction

Background and Objective:  The polymerase chain reaction (PCR) has been applied for the rapid and specific detection of periodontopathic bacteria in subgingival plaque and is potentially of clinical benefit in the diagnosis and treatment of periodontitis subjects. However, several technical points need to be modified before the conventional PCR detection system can be used by clinicians.
Material and Methods:  To develop a PCR-based technique more applicable for clinical use than conventional PCR, we established a multiplex PCR for five putative periodontopathic ( Treponema denticola , Porphyromonas gingivalis , Aggregatibacter actinomycetemcomitans , Prevotella intermedia and Tannerella forsythia ) and two nonperiodontopathic ( Streptococcus sanguinis and Streptococcus salivarius ) species of bacteria using whole-plaque suspension as templates, and detected bacteria in subgingival plaque taken from 85 subjects at the supportive periodontal therapy stage after active periodontal treatments.
Results:  Among putative periodontopathic bacteria, the detection frequency of T. denticola and P. gingivalis was elevated in parallel with higher probing pocket depth and clinical attachment loss, and had 4.2–14.1 times increasing odds of the clinical parameters tested. Detection of any of the five species of putative periodontopathic bacteria markedly increased the odds ratio of a higher probing pocket depth, clinical attachment loss and bleeding on probing.
Conclusion:  The multiplex PCR system developed in this study enabled the detection of all the bacteria under investigation in one reaction tube in a less time- and labor-intensive manner than conventional PCR. These results support the potential clinical use of multiplex PCR for detecting periodontopathic bacteria and for evaluating therapeutic strategies and predicting the prognosis for each subject.     

Real-time polymerase chain reaction for detection and quantification of bacteria in periodontal patients

BACKGROUND: Accurate laboratory tests for the detection and quantification of periodontopathogens in subgingival plaque samples of periodontal disease patients are becoming essential to study the pathogenesis of this polymicrobial condition. We used a real-time polymerase chain reaction (PCR) assay for the quantification of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Dialister pneumosintes, Campylobacter rectus, and Micromonas micros as well as total eubacteria in subgingival plaque samples from individuals with periodontitis. METHODS: Eighty-three subgingival samples from periodontally diseased patients and 43 samples from periodontally healthy subjects were tested and the results of bacterial quantification were correlated to clinical parameters. Quantification was performed with specific 16S rRNA target sequences with double fluorescence labeled probes and serial dilutions of plasmid standards by real-time PCR. RESULTS: Results showed that patients as well as healthy subjects were positive for the presence of target periodontopathogens; however, median values were higher in samples from periodontitis subjects. In addition, a positive association was observed between colonization at high levels by P. gingivalis and M. micros and the presence of deep periodontal pockets. CONCLUSION: Real-time PCR provides a reliable high-throughput method for quantification of periodontopathogens and may be useful for understanding the complex etiology observed in periodontal diseases.     

Association of black-pigmented bacteria with endodontic infections.

Black-pigmented bacteria (BPB) have been associated with endodontic infections. The purpose of this study was to evaluate further the presence of BPB with the clinical signs and symptoms associated with endodontic infections. Microbial samples were collected from the root canals of 40 intact teeth with necrotic pulps and apical periodontitis. Conventional laboratory methods were used for identification of the strains of BPB isolated in pure culture. In addition, the polymerase chain reaction and specific primers for 16S r-RNA genes were used to differentiate Prevotella nigrescens from Prevotella intermedia. Twenty-two (55%) samples were positive for the growth of BPB. Of those, 11 of 22 (50%) were identified as P. nigrescens, 8 of 22 (36%) were P. intermedia, 2 of 22 (9%) were Porphyromonas gingivalis, and 1 of 22 (5%) was Prevotella melaninogenica. Sixteen of the 22 root canals positive for the growth of BPB were associated with purulent drainage either from the root canal or an associated sinus tract. Statistical analysis did not show a significant relationship for the presence of BPB with clinical signs and symptoms.     

Taxonomic changes of bacteria associated with endodontic infections

There have been major recent reorganizations among bacterial taxa as a result of phylogenetic taxonomic approaches. As a consequence, old species have been renamed and novel species have been proposed. The introduction of molecular technology for microbial identification has also allowed the detection of microbial taxa never previously found in endodontic infections. Therefore, the list of putative endodontic pathogens is frequently changing and expanding. The purpose of this review is twofold: to cover the taxonomic changes that the major putative endodontic pathogens have undergone in the recent years and to compile data from studies regarding the detection of known or novel bacterial species that had been only recently reported to occur in endodontic infections.