Black-pigmented anaerobic rods in closed periapical lesions

AIM: This study determined the frequency of Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens in 20 closed periapical lesions associated with symptomatic and asymptomatic refractory endodontic disease. METHODOLOGY: To deliniate possible oral sources of P. endodontalis, the presence of the organism was assessed in selected subgingival sites and saliva in the same study patients. Periapical samples were obtained by paper points during surgical endodontic procedures using methods designed to minimize contamination by non-endodontic microorganisms. Subgingival plaque samples were obtained by paper points from three periodontal pockets and from the pocket of the tooth associated with the closed periapical lesion. Unstimulated saliva was collected from the surface of the soft palate. Bacterial identification was performed using a species-specific polymerase chain reaction (PCR) detection method. RESULTS: P. endodontalis was not identified in any periapical lesion, even though subgingival samples from eight patients (40%) revealed the P. endodontalis-specific amplicon. P. gingivalis occurred in one periapical lesion that was associated with moderate pain. P. nigrescens, P. endodontalis and P. intermedia were not detected in any periapical lesion studied. CONCLUSIONS: Black-pigmented anaerobic rods appear to be infrequent inhabitants of the closed periapical lesion.     

Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia and Prevotella nigrescens in endodontic lesions detected by culture and by PCR

he aim of this study was to investigate the presence of four black-pigmented bacteria, Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia and Prevotella nigrescens, in endodontic infections by culture and polymerase chain reaction (PCR) analyses. Microbial samples were obtained from 50 teeth with untreated necrotic pulps (primary infection) and from 50 teeth with failing endodontic treatment (secondary infection). Microbiological strict anaerobic techniques were used for serial dilution, plating, incubation, and identification. For PCR detection, the samples were analyzed using species-specific primers of 16S rDNA and the downstream intergenic spacer region. Culture and PCR detected the test species in 13/100 and 50/100 of the study teeth, respectively. The organisms were cultured from 11/50 (22%) of primarily infected root canal samples and from 2/50 (4%) of secondary root canal samples. PCR detection identified the target species in 32/50 (64%) and 18/50 (36%) of primary and secondary infections, respectively. P. gingivalis was rarely isolated by culture methods (1%), but was the most frequently identified test species by PCR (38%). Similarly, P. endodontalis was not recovered by culture from any tooth studied, but was detected by PCR in 25% of the sampled teeth. PCR-based identification also showed higher detection rates of P. intermedia (33%) and P. nigrescens (22%) than culture (13%). In conclusion, P. gingivalis, P. endodontalis, P. intermedia, and P. nigrescens were identified more frequently in teeth with necrotic pulp than in teeth with failing endodontic treatment. Also, a higher frequency of black-pigmented species was detected by PCR than by culture.     

Detection of putative oral pathogens in acute periradicular abscesses by 16S rDNA-directed polymerase chain reaction

A 16S rDNA-directed polymerase chain reaction method was used to assess the occurrence of four black-pigmented anaerobic rods, Treponema denticola, and Actinobacillus actinomycetemcomitans in acute periradicular abscesses. Pus was collected by aspiration from 10 cases diagnosed as acute abscesses of endodontic origin. DNA was extracted from the samples and analyzed using a polymerase chain reaction-based identification assay. The method allowed detecting black-pigmented anaerobes in 80% of the examined abscesses. Porphyromonas endodontalis was found in 70%, T. denticola in 50%, Porphyromonas gingivalis in 40%, and Prevotella intermedia in 10% of the cases. P. gingivalis was always found associated with P. endodontalis. Prevotella nigrescens and A. actinomycetemcomitans were not found in any pus sample. The high prevalence of P. endodontalis, T. denticola, and P. gingivalis suggests that they can play an important role in the etiology of acute periradicular abscesses.     

Molecular detection of black-pigmented bacteria in infections of endodontic origin

A 16S rDNA-directed polymerase chain reaction method was used to assess the occurrence of four black-pigmented anaerobic rods in root canal infections. Samples were obtained from 54 infected teeth. Ten cases were diagnosed as acute periradicular abscesses. DNA was extracted from the samples and analyzed using a polymerase chain reaction-based identification assay. The method allowed detection of black-pigmented bacteria anaerobes in 59.3% of the examined teeth. Twelve cases yielded more than one black-pigmented species. In general Porphyromonas endodontalis was found in 42.6%, Porphyromonas gingivalis in 27.8%, Prevotella nigrescens in 7.4%, and Prevotella intermedia in 5.6% of the cases. P. endodontalis was found in 70% of the pus samples, P. gingivalis in 40%, and P. intermedia in 10%. P. gingivalis was always found associated with P. endodontalis in abscessed teeth. P. nigrescens was not found in any pus sample. The high prevalence of P. endodontalis and P. gingivalis suggests that they can play an important role in the pathogenesis of periradicular diseases.     

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.     

Assessment of periradicular microbiota by DNA-DNA hybridization

In the present study the "checkerboard" DNA-DNA hybridization technique was used to identify bacteria in periapical endodontic lesions of asymptomatic teeth. Thirty-four patients with root-filled teeth and apical periodontitis were divided into two groups, each containing 17 patients. In Group 1, a marginal incision was performed during surgery to expose the lesion, and in Group 2, a submarginal incision was applied. The gingiva and mucosa were swabbed with an 0.2% chlorhexidine gluconate solution prior to surgery. Bacterial DNA was identified in all samples from the two groups using 40 different whole genomic probes. The mean number (+/- SD) of species detected was 33.7 +/- 3.3 in Group 1 and 21.3 +/- 6.3 in Group 2 (P < 0.001). The majority of the probe-detected bacteria were present in more lesions from Group 1 than from Group 2. The differences were most notable for Campylobacter gracilis, Porphyromonas endodontalis, Propionibacterium acnes, Capnocytophaga gingivalis, Fusobacterium nucleatum ssp. nucleatum, Fusobacterium nucleatum ssp. polymorphum, Prevotella intermedia, Treponema denticola, Streptococcus constellatus and Actinomyces naeslundii I. Bacterial species such as Actinobacillus actinomycetemcomitans and Bacteroides forsythus were detected in more than 60% of the lesions from both groups. Also, P. endodontalis was abundant in periapical tissue. The data supported the idea that following a marginal incision, bacteria from the periodontal pocket might reach the underlying tissues by surgeon-released bacteremia. The study provided solid evidence that bacteria invade the periapical tissue of asymptomatic teeth with apical periodontitis. The detection of much more bacteria with the "checkerboard" DNA-DNA hybridization method than has previously been recovered by anaerobic culture indicated that the endodontic (and periodontal) microfloras should be redefined using molecular methods.     

Assessment of periradicular microbiota by DNA-DNA hybridization

Abstract – In the present study the "checkerboard" DNA-DNA hybridization technique was used to identify bacteria in periapical endodontic lesions of asymptomatic teeth. Thirty-four patients with root-filled teeth and apical periodontitis were divided into two groups, each containing 17 patients. In Group 1, a marginal incision was performed during surgery to expose the lesion, and in Group 2, a submarginal incision was applied. The gingiva and mucosa were swabbed with an 0.2% chlorhexidine gluconate solution prior to surgery. Bacterial DNA was identified in all samples from the two groups using 40 different whole genomic probes. The mean number (±SD) of species detected was 33.7±3.3 in Group 1 and 21.3±6.3 in Group 2 ( P <0.001). The majority of the probe-detected bacteria were present in more lesions from Group1 than from Group 2. The differences were most notable for Campylobacter gracilis, Porphyromonas endodontalis, Propionibacterium acnes, Capnocytophaga gingivalis, Fusobacterium nucleatum ssp . nucleatum, Fusobacterium nucleatum ssp . polymorphum, Prevotella intermedia, Treponema denticola, Streptococcus constellatus and Actinomyces naeslundii I. Bacterial species such as Actinobacillus actinomycetemcomitans and Bacteroides forsythus were detected in more than 60% of the lesions from both groups. Also, P. endodontalis was abundant in periapical tissue. The data supported the idea that following a marginal incision, bacteria from the periodontal pocket might reach the underlying tissues by surgeon-released bacteremia. The study provided solid evidence that bacteria invade the periapical tissue of asymptomatic teeth with apical periodontitis. The detection of much more bacteria with the "checkerboard" DNA-DNA hybridization method than has previously been recovered by anaerobic culture indicated that the endodontic (and periodontal) microfloras should be redefined using molecular methods.     

Geographical differences in bacteria detected in endodontic infections using polymerase chain reaction

The polymerase chain reaction (PCR) is an innovative nucleic acid-based assay that has the highest sensitivity of any microbiological technique for the detection of bacteria. The purpose of this study was to use PCR to detect the presence of specific species of bacteria in samples collected from two geographical locations. Microbial samples from abscesses of endodontic origin were collected from patients in Portland, Oregon, and Rio de Janeiro, Brazil. PCRs with species-specific oligonucleotide primers for the 16S ribosomal RNA gene were used for detection of the bacteria after DNA extraction from each clinical sample. Statistical analysis revealed that there was a significant difference in detection of the bacteria between the two geographical locations for Prevotella intermedia, Prevotella nigrescens, Prevotella tannerae, Fusobacterium nucleatum, and Porphyromonas gingivalis, but not for Porphyromonas endodontalis, Fusobacterium necrophorum, and Enterococcus faecalis. These results suggest that differences in bacteria detected or cultured in studies can be associated with geographical location.     

顽固性根尖周感染根管内常见致病微生物的研究

目的:研究根管内常见致病微生物在顽固性根尖周感染根尖组织中的检出情况。方法:收集26例顽固性根尖周感染病人的根尖周病变组织,抽提组织DNA成分,用二步聚合酶链反应(polymerase chainreaction,PCR)技术检测根尖周病变组织内的可疑致病菌的存在情况,包括粪肠球菌(E.faecalis,Ef)、牙龈卟啉单胞菌(P.gingivalis,Pg)、牙髓卟啉单胞菌(P.endodontalis,Pe),中间普氏菌(P.intermedia,Pi)、具核梭杆菌(F.nucleatum,Fn)、伴放线放线杆菌(A.actinomycetemcomitans,Aa)、福赛特纳菌(T.forsythensis,Tf)和齿垢密螺旋体(T.denticola,Td)。结果:在26例根尖周组织样本中有14例中出现目标微生物感染,其中Ef感染7例(27%),Pg感染5例(19%),Pe感染5例(19%),Pi感染3例(12%),Fn感染3例(12%),Aa感染1例(4%),Tf感染4例(15%)、Td感染2例(8%)。结论:顽固性根尖周感染的根尖周病变组织中有微生物存在。     

Survey for collagenase gene prtC in Porphyromonas gingivalis and Porphyromonas endodontalis isolated from endodontic infections

Collagenase is a potential virulence factor shown to be expressed by Porphyromonas gingivalis associated with periodontal disease. The purpose of this study was to use the polymerase chain reaction (PCR) to detect the presence of the collagenase gene (prtC) in 21 strains of Porphyromonas species isolated from endodontic infections. Type strains for P. gingivalis (ATCC 33277), P. endodontalis (ATCC 35406), Prevotella intermedia (ATCC 25611), and Prevotella nigrescens (ATCC 33563) were used as controls. When PCR primers specific for the 16S ribosomal RNA gene of P. gingivalis or P. endodontalis were used, 16 of the strains were identified as P. gingivalis, and five strains were identified as P. endodontalis. The presence of the prtC gene for collagenase was detected using PCR. Amplicons were analyzed by agarose gel electrophoresis, with an 815 bp amplicon representing the presence of the collagenase gene. Type strain ATCC 33277 and all 16 clinical isolates of P. gingivalis produced the collagenase gene amplicon. Neither type strain ATCC 35406 nor the five strains from clinical isolates of P. endodontalis produced the collagenase gene amplicon. These results indicate that P. gingivalis from endodontic infections possesses the prtC gene. P. endodontalis does not seem to exhibit prtC. The virulence of P. gingivalis may be related to its production of collagenase.     

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.     

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.     

Susceptibility of some oral microorganisms to chlorhexidine and paramonochlorophenol

Since the use of antimicrobial agents is required in endodontic therapies, this study aimed at determining the minimum inhibitory concentrations (MICs) of chlorhexidine digluconate and paramonochlorophenol (PMC) against microorganisms commonly found in endodontic infections. Both agents were tested by agar dilution tests against Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Candida albicans, Prevotella intermedia, Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella denticola and Prevotella melaninogenica. The MIC of chlorhexidine ranged from 2.67 to 80.00 microg/ml, and the MIC of PMC from 46.67 to 213.33 microg/ml. The highest MIC value of PMC was detected for E. faecalis whereas E. coli was the most susceptible microorganism to this agent. The highest MIC values of chlorhexidine were observed for P. aeruginosa whereas E. coli and P. denticola were the most susceptible microorganisms to this agent. Since the MIC values observed are much lower than the concentrations currently used in the endodontic therapy, it is suggested that both agents are effective in reducing the microbiota in the root canal.     

Microbiological examination of infected dental root canals

OBJECTIVES: The aim of this study was to investigate the root canal microbiota of primary and secondary root-infected canals and the association of constituent species with specific endodontic signs and symptoms. METHODS: Microbial samples were taken from 60 root canals, 41 with necrotic pulp tissues (primary infection) and 19 with failed endodontic treatment (secondary infection). Strict anaerobic techniques were used for serial dilution, plating, incubation and identification. RESULTS: A total of 224 cultivable isolates were recovered belonging to 56 different bacterial species. Individual root canals yielded a maximum of 10 bacterial species. Of the bacterial isolates, 70% were either strict anaerobes or microphilic. The anaerobes most frequently isolated were: Peptostreptococcus micros (35%), Fusobacterium necrophorum (23.3%), Fusobacterium nucleatum (11.7%), Prevotella intermedia/nigrescens (16.7%), Porphyromonas gingivalis (6.7%) and Porphyromonas endodontalis (5%). The root canal microflora of untreated teeth with apical periodontitis was found to be mixed, comprising gram-negative and gram-positive and mostly anaerobic microorganisms and usually containing more than 3 species per canal. On the other hand, facultative anaerobic and gram-positive bacteria predominated in canals with failed endodontic treatment, which harbored 1-2 species per canal. Suggested relationships were found between anaerobes, especially gram-negatives, and the presence or history of pain, tenderness to percussion and swelling (P<0.05). In particular, associations were found between: a) pain (n=29) and P. micros (P<0.01), P. intermedia/nigrescens and Eubacterium spp. (both P<0.05); b) history of pain (n=31) and P. micros (P<0.01) Porphyromonas and Fusobacterium spp. (P<0.05); c) tenderness to percussion (n=29) and Porphyromonas spp. (P<0.01), Peptostreptococcus and Fusobacterium spp. (P<0.001); d) swelling (n=20) and Peptostreptococcus spp. (P<0.01), Porphyromonas and Enterococcus spp. (P<0.05); e) wet canals (n=33) and Porphyromonas and Fusobacterium spp. (P<0.05); f) purulent exudate (n=20) and Porphyromonas, Peptostreptococcus and Fusobacterium spp. (P<0.05); previous endodontic treatment and Enterococcus faecalis, Streptococcus spp., P. micros, F. necrophorum (P<0.05). CONCLUSIONS: Our findings indicate potential complex interactions of species resulting in characteristic clinical pictures which cannot be achieved by individual species alone. They also indicate that the microbiota of primary infected canals with apical periodontitis differs in number and in species from the secondary infected canals by using the culture technique.     

Positive and negative bacterial associations involving Dialister pneumosintes in primary endodontic infections

Dialister pneumosintes is an anaerobic Gram-negative rod that has been recently implicated as a candidate endodontic pathogen. In this study, samples taken from abscessed teeth and infected root canals associated with asymptomatic or symptomatic periradicular lesions were examined for the occurrence of bacterial associations involving D. pneumosintes. DNA was extracted from the samples, and the presence of D. pneumosintes and 16 other bacterial species was determined by means of species-specific nested polymerase chain reaction. Positive and negative associations involving D. pneumosintes were investigated by computing the odds ratio of D. pneumosintes being found in a sample from endodontic infection in co-infection with one of the other target species. The association between the pairs containing D. pneumosintes and the occurrence of pain also was evaluated. D. pneumosintes was always detected in mixed infections with at least two of the other target species. D. pneumosintes was positively associated with Treponema denticola, Porphyromonas endodontalis, Fusobacterium nucleatum, Peptostreptococcus micros, Campylobacter rectus, Prevotella intermedia, T. pectinovorum, and T. vincentii. Negative associations were observed with Bacteroides forsythus, P. gingivalis, and Actinomyces israelii. No pair containing D. pneumosintes was found to be significantly associated with symptomatic cases (p > 0.01). The findings of this study lend considerable support to the notion of D. pneumosintes being an important endodontic pathogen, usually in a mixed infection. Positive associations of this species with other highly prevalent species, such as T. denticola and P. endodontalis, suggest that bacterial synergism can occur and thereby play an important role in the pathogenesis of different forms of periradicular lesions.     

Selected endodontic pathogens in the apical third of infected root canals: a molecular investigation

Bacteria located at the apical portion of the root canals are conceivably in a strategic position to induce damage to the periradicular tissues and resulting inflammatory diseases. This study sought to investigate the prevalence of 11 selected putative endodontic pathogens in the apical third of infected root canals associated with periradicular lesions. The apical root portion of 23 extracted teeth with carious pulpal exposures and attached periradicular lesions was sectioned, and the root canals were sampled for microbiological investigation. DNA was extracted from the samples and analyzed for the presence of 11 bacterial species using a nested polymerase chain reaction assay. The results showed that Pseuramibacter alactolyticus occurred in 10 cases (44%), Treponema denticola in 6 (26%), Fusobacterium nucleatum in 6 (26%), Porphyromonas endodontalis in 4 (17%), Filifactor alocis in 2 (9%), Dialister pneumosintes in 1 (4%), Porphyromonas gingivalis in 1 (4%), and Tannerella forsythensis in 1 (4%). No sample yielded Prevotella intermedia, Prevotella nigrescens, or Campylobacter rectus. Of the samples examined, 17 were positive for at least 1 of the target species. Occurrence of these bacterial species in the apical third of infected root canals suggests that they can be involved in causation of periradicular lesions.     

Evaluation of the microbiota of primary endodontic infections using checkerboard DNA-DNA hybridization

BACKGROUND/AIMS: The aim of this study was to evaluate the composition of the microbiota of primary endodontic infections in 111 selected cases of single-rooted teeth with necrotic pulp. METHODS: Samples were collected from the root canals using #15 Hedstr?en-type files and two sterile paper points, which were introduced 1 mm short of the apical foramen. The presence, levels, and proportions of 40 different bacterial species in each sample were determined using DNA probes and checkerboard DNA-DNA hybridization techniques. RESULTS: The mean number of species per sample was 22. Enterococcus faecalis (89.3%), Campylobacter gracilis (89.3%), Leptotrichia buccalis (89.3%), Neisseria mucosa (87.5%), Prevotella melaninogenica (86.6%), Fusobacterium nucleatum ssp. vincentii (85.7%), Eubacterium saburreum (75.9%), Streptococcus anginosus (75%), and Veillonella parvula (74.1%) were the most prevalent species. The species found in highest mean counts (over 10(5)) were F. nucleatum ssp. vincentii (13.14 x 10(5)), E. saburreum (5.67 x 10(5)), E. faecalis (5.38 x 10(5)), N. mucosa (4.19 x 10(5)), V. parvula (3.63 x 10(5)), C. gracilis (3.46 x 10(5)), Treponema socranskii (3.34 x 10(5)), Porphyromonas endodontalis (2.96 x 10(5)), Porphyromonas gingivalis (2.85 x 10(5)), Micromonas micros (2.81 x 10(5)), Prevotella nigrescens (2.68 x 10(5)) and Fusobacterium nucleatum ssp. nucleatum (2.64 x 10(5)). Most of these species were also found in high proportions. CONCLUSIONS: Our results suggest that several bacterial species considered to be oral pathogens seem to be implicated in the etiology of primary endodontic infections.     

Detection of Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia, and Prevotella nigrescens in chronic endodontic infection

Black-pigmented anaerobic rods such as Prevotella spp. and Porphyromonas spp. are involved in the etiology and perpetuation of endodontic infections. The aim of this study was to evaluate the prevalence of these species in chronic endodontic infections by using culture and polymerase chain reaction (PCR) techniques. Samples of 100 patients with root canals displaying chronic endodontic infections were obtained by sterilized paper points. Bacterial identification was performed by using culture and PCR techniques. By culture, in 33% of the samples, P. intermedia-P. nigrescens (75.8%), P. gingivalis (27.3%), and P. endodontalis (9.1%) were identified, and by PCR 60% of the samples harbored P. nigrescens (43.3%), P. gingivalis (43.3%), P. intermedia (31.7%), and P. endodontalis (23.3%). The presence of these black-pigmented anaerobic rods alone or in association in chronic endodontic infections seems to be frequent. PCR is a very sensitive technique for detecting DNA from bacterial cells. Culturing is only able to reveal living bacteria and is less sensitive for the identification of low numbers of bacterial cells.     

PCR detection of 5 putative periodontal pathogens in dental plaque samples from children 2 to 12 years of age

BACKGROUND, AIMS: The purpose of this study was to detect the presence of Prevotella intermedia, P. nigrescens, Bacteroides forsythus, Treponema denticola, and Campylobacter rectus in plaque samples from 119 children, collected from their toothbrushes using a polymerase chain reaction (PCR). METHOD: The subjects were 24, 83, and 12 children with healthy gingiva, gingivitis, and periodontitis, respectively, ranging in age from 2-12 years old. Plaque samples were collected from all erupted teeth sites using a sterile toothbrush. The mean concentration of DNA recovered from the plaque samples was approximately 640 microg/ml, which was deemed sufficient for performing a PCR-based survey. RESULTS: The prevalence by PCR in healthy, gingivitis, and periodontitis subjects was 0.0%, 6.0% and 25.0% for P. intermedia, 45.8%, 79.5% and 50.0% for P. nigrescens, 33.3%, 63.9% and 58.3% for B. forsythus, 0.0%, 18.1% and 16.7% for T. denticola, and 100% in duplicate for C. rectus, respectively. CONCLUSION: Our survey indicated that P. intermedia and T. denticola were more associated with periodontal diseases, B. forsythus and P. nigrescens had a moderate prevalence in all clinical groups, while C. rectus were the most commonly detected species in the oral cavities of children suggesting establishment in their early years.     

Quantitative detection of periodontal pathogens using real-time polymerase chain reaction with TaqMan probes

Quantitative analysis, with identification of periodontopathic bacteria, is important for the diagnosis, therapeutic evaluation and risk assessment of periodontal disease. We developed a highly sensitive and specific method using real-time polymerase chain reaction (PCR) to detect and quantify six periodontal bacteria: Porphyromonas gingivalis, Tannerella forsythia, Actinobacillus actinomycetemcomitans, Treponema denticola, Prevotella intermedia, and Prevotella nigrescens. Species-specific TaqMan probe/primer sets were designed according to 16S ribosomal RNA gene sequences. Plaque and tongue debris specimens were collected from 10 patients with advanced periodontitis and 10 periodontal healthy individuals and analyzed. All species, except for P. nigrescens, were detected in samples from diseased sites in significantly greater numbers than in those from healthy sites, whereas greater numbers of P. nigrescens were found in the controls. These results suggest that the present real-time PCR method with the designed probe/primer sets enabled sensitive detection of the six periodontal bacteria, and may also assist future microbial studies of periodontal diseases.