Oral treponemes in primary root canal infections as detected by nested PCR

Aim  To investigate the prevalences of four Treponema species in primary root canal infections using a nested PCR assay.     

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.     

Clonality of Fusobacterium nucleatum in root canal infections

Fusobacterium nucleatum is a gram-negative non-spore-forming, non-motile, obligate anaerobic rod that is normally isolated from the oral cavity. Several studies have reported a significant heterogeneity within the F. nucleatum species. The aim of the present study was to analyze the clonal diversity of F. nucleatum strains isolated from intracanal infections and to evaluate the presence of Enterobacterial Repetitive Intergenic Consensus (ERIC)-like sequences in the genome of F. nucleatum . Samples were collected from 13 single-root teeth from adult patients, all having carious lesions, necrotic pulps and radiographic evidence of periradicular bone loss. F. nucleatum was isolated from two different patients (subjects 5 and 7) by culture. Amplification of 19 colonies from subject 5 and 15 colonies from subject 7 using ERIC primers resulted in four clonal types, two per subject. An intense amplicon of approximately 700 bp was generated by ERIC-PCR for all F. nucleatum isolates and F. nucleatum ssp. polymorphum ATCC 10953. The amplification reaction using primer 1254 confirmed the results obtained with the ERIC primer. Our findings indicate that DNA fingerprints provided by ERIC- and Arbitrarily Primed (AP)-PCR may constitute a powerful tool for investigating F. nucleatum clonal diversity.     

Analysis of symptomatic and asymptomatic primary root canal infections in adult Norwegian patients

Introduction

This molecular study analyzed the microbiota of primary root canal infections from adult Norwegian patients.

Methods

Samples were taken from the necrotic root canals of teeth with symptomatic (n = 13) or asymptomatic (n = 21) apical periodontitis and chronic apical abscesses (n = 9). DNA was extracted from samples, and bacterial identifications were performed by a closed-ended reverse-capture checkerboard approach targeting 50 candidate endodontic pathogens.

Results

Bacterial DNA was detected in all cases. In teeth with asymptomatic apical periodontitis, the most frequent taxa were Dialister invisus (71%), Fusobacterium nucleatum (62%), and Porphyromonas endodontalis (62%). In chronic apical abscesses, the most prevalent taxa were P. endodontalis (100%), D. invisus (89%), Parvimonas micra (78%), and Solobacterium moorei (78%). In teeth with symptomatic apical periodontitis, the most prevalent taxa were D. invisus, P. endodontalis, S. moorei, Propionibacterium acnes, and Streptococcus species (all in 69%). None of the targeted taxa were significantly associated with either sinus tract or pain (P > .05), except for Selenomonas sputigena, which was more frequently found in painful cases (P = .04). No taxa were found in significantly higher levels in any conditions (P > .05). Cluster analyses revealed bacterial groupings that differed between cases with and without pain.

Conclusions

Although basically the same species were highly prevalent in the different conditions examined and none of the most prevalent taxa were positively associated with symptoms, results revealed that species formed different partnerships and associations in samples from teeth with or without pain. Therefore, it is possible that more virulent multispecies communities can form as a result of overall bacterial combinations and give rise to acute inflammation.     

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%.     

Quantification and characterization of Synergistes in endodontic infections

INTRODUCTION: Bacterial species belonging to the poorly characterized division Synergistes have recently been reported in endodontic infections, and therefore may be part of the etiology of periradicular diseases. The objective of this study was to characterize and quantify the predominant Synergistes phylotypes in infected root canals. METHODS: We analyzed 32 necrotic teeth, each from a different patient, with radiographic evidence of apical periodontitis and with primary endodontic infections. RESULTS: Using real-time quantitative polymerase chain reaction based on Synergistes-specific primers, seven of the 32 cases were found to be positive. Comparative sequence analysis showed that each of the seven samples was infected by one numerically dominant phylotype. Diversity among phylotypes was such that they could be grouped into three major evolutionary branches within the Synergistes division. The size of the total Synergistes population ranged from 4.5 x 10(4) to 1.5 x 10(6) 16S rRNA gene copies, and the median proportion accounted for 0.79% of the total bacterial community. For comparison, we also quantified such recognized endodontic pathogens as Prevotella intermedia, Porphyromonas gingivalis, and Treponema. The first two species were found in five and nine cases, respectively, with a median proportion below 0.01%, while Treponema was found in 18 cases with a median proportion of 1.48%. CONCLUSION: Thus, the prevalence and quantity of Synergistes was clearly within the range of the other analyzed pathogens, suggesting their clinical relevance in endodontic infections. Furthermore, the diversity of Synergistes found at the diseased sites designates infected root canals as an important human ecosystem providing several unique micro-niches for this novel group of bacteria.     

PCR-based identification of Treponema maltophilum,T amylovorum,T medium,and T lecithinolyticum in primary root canal infections

Objective: Molecular genetic methods have significantly contributed to the knowledge about the microbiota associated with infected root canals. Albeit spirochetes have been commonly observed in primary root canal infections, only recently they have been identified. The purpose of the present study was to investigate the occurrence of four treponemes—Treponema maltophilum, Treponema lecithinolyticum, Treponema amylovorum, and Treponema medium—in cases of primary endodontic infections associated with different forms of periradicular diseases through a 16S rDNA-based nested PCR assay. Design: Samples were taken from thirty-one infected root canals associated with either asymptomatic or symptomatic apical periodontitis. 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 the 16S rDNA of each target Treponema species. Results: All cases were positive for the universal bacterial primers, indicating that samples contained bacterial DNA. Of the four target species, T. maltophilum was the most prevalent, being detected in 39% of the cases (33% of the asymptomatic cases and 50% of the symptomatic cases). T. lecithinolyticum was the next more prevalent among the species tested, being found in 26% of the samples (33% of asymptomatic cases and 10% of the symptomatic cases). T. amylovorum was found in 7% of the cases (5% of the asymptomatic cases and 10% of the symptomatic cases), while T. medium was in 13% of the cases (14% of the asymptomatic cases and 10% of the symptomatic cases). None of the species tested was significantly associated with clinical symptoms. Conclusions: This was possibly the hitherto first study to report the occurrence of T. lecithinolyticum, T. amylovorum, and T. medium in infections of endodontic origin. Overall, findings suggested that these oral treponemes, particularly T. maltophilum and T. lecithinolyticum, can be involved in the pathogenesis of periradicular diseases.     

牙髓卟啉单胞菌在原发性感染和再感染根管内的定植

目的 采用16s rDNA PCR和实时荧光定量聚合酶链反应（RTFQ-PCR）分析和比较牙髓卟啉单胞菌（P. endodontalis）endodontalis）在原发性感染和再感染根管中的定植情况。方法 将120例慢性根尖周炎患者的120颗单根管患牙按原发性感染和再感染分为2组,每组60颗。采用16s rDNA PCR分析P. endodontalis在两种感染根管内的检出率,对于P. endodontalis endodontalis检出阳性者用RTFQ-PCR比较P. endodontalis在两种感染根管内的DNA相对表达量。结果 P. endodontalis在原发性感染根管内的检出率明显高于再感染根管的检出率（P=0.001）。RTFQ-PCR检测结果表明P. endodontalis在原发性感染根管内的DNA表达量和再感染根管内DNA表达量差异无统计学意义（P=0.303）。结论 P. endodontalis在原发性感染和再感染根管内均有定植,但与原发性感染根管关系更为密切。     

慢性牙周炎龈下菌斑中五种牙周可疑致病微生物的分布

目的研究五种牙周可疑致病微生物在慢性牙周炎患者龈下菌斑的分布。方法选择27例慢性牙周炎患者,每位患者选取牙周袋最深的两个位点作为观察位点,采集龈下微生物样本,采用多重聚合酶链反应和反杂交的方法对伴放线菌嗜血菌、牙龈卟啉单胞菌、福赛斯坦纳菌、中间普雷沃菌和齿垢密螺旋体五种微生物进行半定量检测。结果在所检测的54个位点中,牙龈卟啉单胞菌、中间普雷沃菌、福赛斯坦纳菌和齿垢密螺旋体均有较高的检出率,分别为98.15%、92.59%、100%和98.15%;伴放线菌嗜血菌检出率较低,为20.37%。牙龈卟啉单胞菌和福赛斯坦纳菌的检出量明显高于其他三种微生物,其差异有统计学意义（P<0.05）。结论慢性牙周炎患者多存在牙龈卟啉单胞菌、福赛斯坦纳菌、中间普雷沃菌和齿垢密螺旋体的同时感染,且牙龈卟啉单胞菌和福赛斯坦纳菌的感染量较高。     

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%.     

Occurrence of two newly named oral treponemes - Treponema parvum and Treponema putidum - in primary endodontic infections

Background/aims:  Recent evidence from molecular genetic studies has revealed that oral Treponema species are involved in infections of endodontic origin. This study assessed the occurrence of two newly named oral treponemes −  Treponema parvum and Treponema putidum –  in primary endodontic infections using a culture-independent identification technique.
Methods:  Genomic DNA was isolated directly from clinical samples, and a 16S rRNA gene-based nested polymerase chain reaction (PCR) assay was used to determine the presence of T. parvum and T. putidum . Species-specific primer pairs were developed by aligning closely related 16S rRNA gene sequences. The specificity for each primer pair was validated by running PCR against a panel of oral bacteria and by sequence analysis of PCR products from positive clinical samples.
Results:  T. parvum was detected in 52% of the root canals associated with chronic apical periodontitis, in 20% of the cases diagnosed as acute apical periodontitis, and in no abscessed case. In general, T. parvum was detected in 26% of the samples from primary endodontic infections. T. putidum was found in only one case of acute apical periodontitis (2% of the total number of cases investigated).
Conclusions:  The devised nested PCR protocol was able to identify both T. parvum and T. putidum directly in clinical samples and demonstrated that these two treponemes can take part in endodontic infections.     

Apical periodontitis development and bacterial response to endodontic treatment. Experimental root canal infections in monkeys with selected bacterial strains

In six monkeys, 160 root canals were inoculated with a combination of four bacterial strains belonging to species Streptococcus milleri, Peptostreptococcus anaerobius, Prevotella oralis, and Fusobacterium nucleatum. In two other monkeys, 24 root canals were inoculated with a five-strain combination consisting of these strains and a strain of Enterococcus faecalis. All strains were previously isolated from an infected monkey root canal. After 8-12 months, survival of the strains was recorded bacteriologically, and the reaction in the periapical region was radiographed. From 180 of 184 root canals, one or more of the bacterial strains were reisolated. The two facultative strains were more frequently reisolated than the anaerobic strains. Apical periodontitis was registered in the periapical region of more than 96% of root canals with reisolated bacteria but in none of those without reisolated bacteria. Endodontic treatment was carried out in two sessions with an interval of 14 d without interappointment dressings, and the effect was evaluated bacteriologically before and after each treatment. The chemo-mechanical treatment reduced significantly the number of strains and bacterial cells. The facultative bacteria were more resistant to the treatment than the anaerobic bacteria. The five-strain combination had a higher survival rate than the four-strain combination.     

Occurrence of two newly named oral treponemes –Treponema parvum and Treponema putidum– in primary endodontic infections

Background/aims: Recent evidence from molecular genetic studies has revealed that oral Treponema species are involved in infections of endodontic origin. This study assessed the occurrence of two newly named oral treponemes ? Treponema parvum and Treponema putidum – in primary endodontic infections using a culture‐independent identification technique. Methods: Genomic DNA was isolated directly from clinical samples, and a 16S rRNA gene‐based nested polymerase chain reaction (PCR) assay was used to determine the presence of T. parvum and T. putidum. Species‐specific primer pairs were developed by aligning closely related 16S rRNA gene sequences. The specificity for each primer pair was validated by running PCR against a panel of oral bacteria and by sequence analysis of PCR products from positive clinical samples. Results: T. parvum was detected in 52% of the root canals associated with chronic apical periodontitis, in 20% of the cases diagnosed as acute apical periodontitis, and in no abscessed case. In general, T. parvum was detected in 26% of the samples from primary endodontic infections. T. putidum was found in only one case of acute apical periodontitis (2% of the total number of cases investigated). Conclusions: The devised nested PCR protocol was able to identify both T. parvum and T. putidum directly in clinical samples and demonstrated that these two treponemes can take part in endodontic infections.     

5种牙周龈下可疑致病微生物与慢性牙周炎局部不同牙周状态间的关系

目的 观察5种龈下微生物检出水平与慢性牙周炎局部牙周状态的关系。方法 选择20例慢性牙周炎患者的80个位点及10例牙周健康者的20个位点为观察位点,采集龈下微生物样本,记录牙周探诊深度（PD）,根据所测位点的PD进行分组。PD≤4 mm为A组,4 mm＜PD≤6 mm为B组,PD>6 mm为C组,健康对照组为H组。通过聚合酶链反应（PCR）和DNA探针反杂交技术半定量检测各组伴放线菌嗜血菌、牙龈卟啉单胞菌、福赛斯坦纳菌、齿垢密螺旋体和中间普氏菌的检出率和检出水平。结果 B、C组牙龈卟啉单胞菌、福赛斯坦纳菌、齿垢密螺旋体和中间普氏菌的检出率和检出水平均高于H组,A组牙龈卟啉单胞菌的检出率和检出水平也高于H组,C组福赛斯坦纳菌和齿垢密螺旋体检出水平高于B组,以上差异均有统计学意义（P<0.05）;伴放线菌嗜血菌在各组间的检出率及检出水平都无明显差异。结论 随着牙周袋的加深,牙龈卟啉单胞菌、福赛斯坦纳菌、齿垢密螺旋体和中间普氏菌体的阳性检出率和检出水平都有随之增加的趋势;牙龈卟啉单胞菌与慢性牙周炎的早期炎症关系较为密切,而福赛斯坦纳菌和齿垢密螺旋体与中重度慢性牙周炎炎症位点的严重程度有关。     

Spirochaetes in oral infections

Abstract Oral Spirochaetes, which are small-, medium- or large-sized, include species of the genus Treponema, many of which have not yet been cultured. They are found in root canal infections, pericoronitis, gingivitis and periodontitis, constituting up to 10% of the flora in endodontic abscesses, 30% in acute necrotizing ulcerative gingivitis, and 36% in advanced marginal periodontitis. The strong proteolytic activity of these organisms probably make them causes of infection rather than consequences. Being able to penetrate tissue, they bring their enzymes, metabolic products, and endotoxins, in direct contact with target cells. This may perturb essential functions of host cells and immunoglobulins. Enzyme activities may also help fulfil the complex growth requirements of Spirochaetes in vivo. Reaction between infected periodontal tissue and monoclonal antibodies to Treponema pallidum has suggested that uncharacterized pathogen-related oral spirochaetes have surface structures and functions analogue to this well recognized pathogen. This warrants a more intensified search for the role of spirochaetes in oral infections.     

根管治疗难治病例临床分析

目的 :探讨根管治疗难治病例出现的临床因素。方法 :以根管扩大至根管充填所增加的次数为指标 ,抽取作者 1988年 - 1998年间根管治疗所出现的 316例难治病例 ,分析造成难治病例出现的临床因素。结果 :根管治疗难治病例以感染根管出现最多占 6 8.99% ;拔髓根管占 31.0 1% ;难治病例与牙齿病变的关系 :根尖暗影占 45 .2 5 % ;根分叉暗影占 18.99% ;牙隐裂占 2 4.6 8% ;药物性尖周炎占 4.43% ;机械损伤占 5 .70 % ;不明原因占 0 .95 %。结论 :根管治疗难治病例的发生主要与牙体、根尖病变的程度有关 ,医生扩管操作不当也是造成难治病例的一个因素     

Periodontal pathogens in atheromatous plaques. A controlled clinical and laboratory trial

OBJECTIVE: A possible relationship between periodontitis and cardiovascular disease has been suggested. The aims of this controlled clinical study were: (i) to ascertain the presence of periodontal bacteria DNA [Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis (formerly Bacteroides forsythus)] in carotid atheromatous plaques and (ii) to assess the concomitant presence of the same periodontal bacteria DNA, if any, in periodontal pockets and in carotid atheroma in the same patient. METHODS: A total of 52 patients scheduled for carotid endarderectomy were enrolled in this study. The test group consisted of 26 dentate patients; the control group included 26 edentulous patients. A complete periodontal examination, including radiographic orthopanoramic and subgingival plaque sample, was performed in the test population. Oral and X-ray examinations were performed in the control group. Atheromatous plaques were harvested during surgical procedure for each dentate and edentulous patient and then sent to the microbiological laboratory. Subgingival plaque samples and carotid specimens were examined using the polymerase chain reaction (PCR) technique by means of specific primers for periodontal bacteria. Amplification of extracted DNA was tested using human beta-globin specific-primers. RESULTS: Out of 52 endarterectomy samples, 12 (seven dentate, five edentulous patients) were excluded as negative to DNA amplification. In subgingival plaque samples of 19 test patients, T. forsythensis (79%), F. nucleatum (63%), P. intermedia (53%), P. gingivalis (37%) and A. actinomycetemcomitans (5%) were found. No periodontal bacteria DNA was detected by PCR in any of the carotid samples in either patient group. CONCLUSION: The presence of periodontal bacteria DNA in atheromatous plaques could not be confirmed by this study and thus no correlation could be established between species associated with periodontal disease and putative bacteria contributing to atheromatous plaques.     

Polyamine analysis of infected root canal contents related to clinical symptoms

Abstract The contents of infected root canals were collected by mechanical and chemical debridement. Intracanal polyamines were identified and quantified by HPLC in all canals examined, however, no histamine was detected in any canal. The relationship between the amount of each polyamine and clinical signs of the teeth was analyzed statistically. The teeth without clinical signs tended to have limited varieties and smaller amounts of polyamines. Using %2, the amounts were significantly greater in teeth with spontaneous pain, swelling and putrescent odor (p<0.01), with exudate (p<0.025) and with percussion pain (p<0.1) than in teeth without. Amounts of putrescine (p<0.05) and total polyamines (p<0.01) were greater in teeth with spontaneous pain and percussion pain than in teeth without clinical sign, and those with root canal exudate also had greater amounts of total polyamines than those without (p<0.01). The amounts of cadaverine, however, from the teeth with gingival fistulae, was greater than from those without (p<0.05). No significant relationship was established between the amount of each polyamine and the presence of putrescent odor or gingival swelling. Intracanal polyamines, especially putrescine may leak out through apical foramen and may be implicated in pain production by eliciting acute inflammatory response in the periapical tissues.