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

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

Molecular and cultural analysis of the microflora associated with endodontic infections

Cultural studies have indicated that a subset of the oral microflora is responsible for endodontic infections. Approximately 50% of oral bacteria are unculturable, so it is likely that currently unknown bacteria are present in such infections. In this study, cultural and molecular analyses were performed on the microflora in aspirate samples collected from 5 infected root canals. 16S rDNA sequences from 261 isolates and 624 clones were identified by comparison with database sequences. Sixty-five taxa were identified, of which 26 were found by the molecular method alone. A mean of 20.2 taxa was found in each sample. A new species of Dialister was the only organism present in all 5 samples. Twenty-seven novel taxa were detected, 18 of which belonged to the phylum Firmicutes and 8 to Bacteroidetes. Culture-independent, molecular analysis has revealed a more diverse microflora associated with endodontic infections than that revealed by cultural methods alone.     

Effect of endodontic procedures on enterococci, enteric bacteria and yeasts in primary endodontic infections

AIM: To detect enterococci, enteric bacteria and yeast species from the canals of teeth with primary endodontic infections before and after canal preparation and to test the antibiotic susceptibility of enterococcal strains isolated from infected root canals. METHODOLOGY: Twenty-five single-rooted teeth with pulp necrosis, intact pulp chambers and periradicular lesions were selected for study. Samples were collected from canals before and after instrumentation. Amongst isolated microorganisms from infected root canals only enterococci, enteric bacteria and yeasts were identified by biochemical tests. The in vitro antimicrobial susceptibility of isolated enterococci strains was evaluated by the Etest system. RESULTS: Microorganisms were isolated from 92% of the samples following intracoronal access, 22% were enterococci, enteric bacteria or yeast species. After biomechanical preparation, these species were no longer detected. After 7 days without intracanal dressing, 100% of the canals contained microorganisms, 52% of which were target species. However, after using paramonochlorophenol [PRP (2.0 g), Rinosoro and polyethylene glycol (400 equal parts up to 100 mL)] as an intracanal dressing for 7 days, enteric bacteria and yeasts were not detected; only enterococci were still present. All strains of enterococci were susceptible to ampicillin, but exhibited variable susceptibility to rifampin and ciprofloxacin. CONCLUSIONS: Enterococci, enteric bacteria and yeasts were present in primary endodontic infections. Enterococci, particularly Enterococcus faecalis and E. faecium were resistant to removal by root canal preparation followed by intracanal dressing.     

Prevalence and clonal analysis of Porphyromonas gingivalis in primary endodontic infections

This study investigated the prevalence of Porphyromonas gingivalis in 62 teeth with primary endodontic infections by using a species-specific 16S rRNA gene-based nested polymerase chain reaction assay. P. gingivalis isolates recovered from 2 infected root canals were also analyzed for clonal diversity by using arbitrarily primed PCR. Overall, P. gingivalis was found in 48% of the samples. This species was specifically detected in 36% of canals of teeth with chronic apical periodontitis, in 46% of the cases of acute apical periodontitis, and in 67% of acute apical abscesses. P. gingivalis was significantly more frequent in abscess aspirates than in canals of teeth with chronic apical periodontitis (P < .05). Typing of colonies retrieved from 2 infected canals revealed 2 clones per individual. These findings confirmed that P. gingivalis can be an important endodontic pathogen, mostly associated with acute abscesses, and demonstrated that different clonal types of this species can colonize the root canal in the same individual.     

Molecular analysis of bacteria in asymptomatic and symptomatic endodontic infections

The purpose of the present study was to use terminal restriction fragment length polymorphism analysis and the 16S rRNA gene clone library to investigate the diversity of the microbiota associated with asymptomatic and symptomatic endodontic infections and to compare the bacterial community structure in these two clinical conditions. Samples were taken from asymptomatic endodontic infections associated with chronic periradicular lesions and from symptomatic infections clinically diagnosed as acute abscesses. 16S rRNA genes from DNA isolated from clinical samples were used to construct clone libraries or were subjected to terminal restriction fragment length polymorphism analysis. Sequence analysis of 186 clones revealed 42 taxa; 23 (55%) were uncultivated phylotypes, of which seven were unique to endodontic infections. Clone sequencing and terminal restriction fragment length polymorphism analysis revealed that the most commonly detected taxa were Fusobacterium nucleatum (including terminal restriction fragment types 1 and 2), Peptostreptococcus micros/Peptostreptococcus sp. oral clone AJ062/BS044/FG014, Prevotella species, Dialister species, Mogibacterium species, Lachnospiraceae oral clone 55A-34, Filifactor alocis, Megasphaera sp. oral clone CS025/BS073, and Veillonella sp. oral clone BP1-85/Veillonella dispar/V. parvula. Bacteroides-like sp. oral clone X083/Bacteroidales oral clone MCE7_20 and Dialister sp. oral clone BS016/MCE7_134 were detected only in asymptomatic teeth. On the other hand, F. nucleatum terminal restriction fragment type 2, Prevotella intermedia, Dialister pneumosintes, and some phylotypes were exclusively detected in symptomatic samples. Bacterial profiles of symptomatic endodontic infections generated by terminal restriction fragment length polymorphism analysis were clearly different from those of asymptomatic infections. Overall, the average number of terminal restriction fragments in symptomatic samples was significantly larger than in asymptomatic samples. Molecular analysis of the microbiota associated with symptomatic or asymptomatic endodontic infections indicates that the endodontic bacterial diversity is greater than previously described by culture methods and that the structure of the microbiota differ significantly between asymptomatic and symptomatic infections.     

感染根管中6种厌氧菌与症状或体征的相关性研究

目的：应用16S rRNA—PCR技术测定感染根管内6种细菌的检出率,分析根管内细菌种类与临床症状及体征的关系。方法：采集48例感染根管样本,按照临床症状或体征分为自发痛、叩痛、窦道3组,提取样本细菌基因组DNA,用PCR扩增细菌16S rRNA基因片段的方法检测细菌种类,计算检出率。结果：共检测48例样本,其中35例检测到待检细菌,检出率达72.9%。检出率最高的是牙髓卟啉单胞菌（35.4%）,其次是牙龈卟啉单胞菌（31.2%）和粪肠球菌（29.1%）,咽峡炎链球菌为（18.7%）,有2例能检出古菌,轻链球菌未检出。统计学分析显示,牙龈卟啉单胞菌与自发痛、粪肠球菌与窦道分别存在相关性（P〈0.05）。结论：根管感染是由多种细菌造成的;5种细菌是感染根管的优势菌;感染根管内牙龈卟啉单胞菌和粪肠球菌检出与临床相应症状或体征有相关性。     

Characterization of Dialister species in infected root canals

Members of the Dialister genus are asaccharolytic obligately anaerobic gram-negative coccobacilli that are culture-difficult or remain uncultivated. Their participation in endodontic infections has been only consistently demonstrated after advent of molecular biology approaches. This study was undertaken to characterize Dialister species in samples from primary endodontic infections using a devised 16S rRNA gene-based group-specific heminested PCR assay followed by sequencing of PCR products. Genomic DNA was isolated directly from clinical samples and used as template for PCR. Amplicons from positive specimens were sequenced and phylogenetically analyzed to determine species identity. Ten of 21 clinical samples yielded sequences with the highest percent similarities to oral Dialister species/phylotypes. Seven sequences were from Dialister invisus, and the other three sequences belonged to Dialister pneumosintes, Dialister oral clone BS095 and Dialister sp. clone IS013B24. Findings demonstrated that different Dialister species can take part in the microbiota associated with apical periodontitis lesions.     

根管感染中产甲烷古细菌基于 mcrA 序列的系统发育分析

目的：分析根管感染中产甲烷古细菌的多样性,建立基于功能基因--甲基辅酶 M 还原酶（methyl coenzyme M reduc-tase,MCR）基因α亚基（mcrA）的系统发育树。方法：利用一对特异性引物选择性扩增感染根管中产甲烷古细菌的 mcrA 片段,在此基础上建立 mcrA 克隆文库。通过蓝白斑筛选的方法,筛选出阳性重组克隆子,进一步通过基因测序获得重组克隆子中的插入片段 mcrA 序列。利用 Clustalx 和 Mega 4软件包分析 mcrA 序列,对其进行同源性比较和系统发育学分析。结果：4例根管样本的其中1例检出了产甲烷古细菌;构建了基于 mcrA 序列的系统发育树。随机选出的8个 mcrA 克隆片段高度同源,均为类口腔甲烷短杆菌序列型。结论：本例根管感染中产甲烷古细菌的多样性局限于类口腔甲烷短杆菌序列型。     

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.     

Antibiotic resistance gene transfer between Streptococcus gordonii and Enterococcus faecalis in root canals of teeth ex vivo

Multiple bacterial species coexisting in infected root canals might interact, but evidence for interspecies gene transfer is lacking. This study tested the hypothesis that horizontal exchange of antibiotic resistance can occur between different bacterial species in root canals. Transfer of the conjugative plasmid pAM81 carrying erythromycin resistance between 2 endodontic infection-associated species, Streptococcus gordonii and Enterococcus faecalis, was investigated in an ex vivo tooth model. Equal numbers of each species (one with pAM81 and the other plasmid-free) were combined in prepared root canals of sterilized teeth and incubated at 37 degrees C. At 24 and 72 hours, bidirectional interspecies antibiotic resistance gene transfer was evident in microorganisms recovered from teeth; average transfer frequencies from S. gordonii to E. faecalis were 10(-3) transconjugants per donor and from E. faecalis to S. gordonii were 10(-6) and 10(-7) transconjugants per donor at 24 and 72 hours, respectively. Microbial accumulations were observed on root canal walls with scanning electron microscopy. Horizontal genetic exchange in endodontic infections might facilitate adoption of an optimal genetic profile for survival.     

Photodynamic therapy for endodontic disinfection

The aims of this study were to investigate the effects of photodynamic therapy (PDT) on endodontic pathogens in planktonic phase as well as on Enterococcus faecalis biofilms in experimentally infected root canals of extracted teeth. Strains of microorganisms were sensitized with methylene blue (25 microg/ml) for 5 minutes followed by exposure to red light of 665 nm with an energy fluence of 30 J/cm2. Methylene blue fully eliminated all bacterial species with the exception of E. faecalis (53% killing). The same concentration of methylene blue in combination with red light (222 J/cm2) was able to eliminate 97% of E. faecalis biofilm bacteria in root canals using an optical fiber with multiple cylindrical diffusers that uniformly distributed light at 360 degrees. We conclude that PDT may be developed as an adjunctive procedure to kill residual bacteria in the root canal system after standard endodontic treatment.     

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.     

Effectiveness of three endodontic irrigants at various tubular depths in human dentin

Bacteria from infected root canals can invade dentinal tubules, thus dentin disinfection is an important aspect of endodontic therapy. This study compares three endodontic irrigants for efficiency in killing bacteria established within human dentinal tubules. Root canals in extracted teeth were prepared and sterilized. Broth cultures of Enterococcus faecalis were allowed to grow within the canals to penetrate dentinal tubules. The infected canals were exposed individually to each of the irrigants for 1 min. Irrigants were 0.525% sodium hypochlorite, Tubulicid (0.2% EDTA), and 0.12% chlorhexidine (Peridex). Sterile water was the control. Viable bacteria were analyzed by drilling incrementally into dentin from the cementum toward the canal. Smaller diameter drills were used for each depth. Shavings were cultured at three depths, for each of three root levels: coronal, midroot, and apical. Although considerable variation occurred between roots, sodium hypochlorite seemed to be superior. Tubulicid and Peridex were better than water. More bacteria remained viable at greater distances from the pulp. These observations apparently apply to all levels in the canal.     

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.     

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.     

根管治疗期间急性发作与慢性根尖周炎患牙根管细菌组成比较

目的:比较根管治疗期间炎症急性发作及慢性根尖周炎患牙根管内8种厌氧菌检出情况,分析急性发作时根管内定植细菌种类及其相关性。方法:分别提取26例根管预备后约诊期间炎症急性发作患牙根管样本和23例慢性根尖周炎患牙根管样本,提取样本细菌DNA,利用细菌16S rRNA引物通过PCR扩增方法鉴定细菌。结果:慢性根尖周炎样本根管细菌检出率达100%(23/23),根管治疗期间急性发作样本细菌检出率为92.31%(24/26);产黑普氏菌、齿垢密螺旋体和直肠弯曲杆菌在急性发作样本中的检出率较慢性根尖周炎样本显著增高(P<0.05)。急性发作样本中牙龈卟啉菌与福赛氏类杆菌的检出显著相关(OR>2,P<0.05)。结论:根管内感染是根管治疗期间炎症急性发作的重要原因,厌氧菌在急性发作的发生中起重要作用。     

New Bacterial Composition in Primary and Persistent/Secondary Endodontic Infections with Respect to Clinical and Radiographic Findings

Introduction

The aim of the present study was to analyze the microbiota of primary and secondary/persistent endodontic infections of patients undergoing endodontic treatment with respect to clinical and radiographic findings.

Methods

Samples from the root canals of 21 German patients were taken using 3 sequential sterile paper points. In the case of a root canal filling, gutta-percha was removed with sterile files, and samples were taken using sterile paper points. The samples were plated, and microorganisms were then isolated and identified morphologically by biochemical analysis and sequencing the 16S rRNA genes of isolated microorganisms.

Results

In 12 of 21 root canals, 33 different species could be isolated. Six (50%) of the cases with isolated microorganisms were primary, and 6 (50%) cases were endodontic infections associated with root-filled teeth. Twelve of the isolated species were facultative anaerobic and 21 obligate anaerobic. Monomicrobial infections were found for Enterococcus faecalis and Actinomyces viscosus. E. faecalis was most frequently isolated in secondary endodontic infections (33%). Moraxella osloensis was isolated from a secondary endodontic infection that had an insufficient root canal filling accompanied by a mild sensation of pain. A new bacterial composition compromising Atopobium rimae, Anaerococcus prevotii, Pseudoramibacter alactolyticus, Dialister invisus, and Fusobacterium nucleatum was recovered from teeth with chronic apical abscesses.

Conclusions

New bacterial combinations were found and correlated to clinical and radiographic findings, particularly to chronic apical abscesses. M. osloensis was detected in root canals for the second time and only in German patients.     

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

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

从微生物学角度思考感染根管的治疗

根管治疗术已被公认为治疗感染根管的有效方法,日渐受到临床医师的重视和采用。但随着此技术的推广应用,特别是用于根管形态较复杂的磨牙,发生了一些问题,如过度根管预备引起的管壁侧穿、根尖偏移、约诊间痛、牙根纵裂以及根充后的再感染等。如何解释这些问题,采用什么方法防治这些问题,使我们感到有必要从现代微生物学的观点探讨细菌在感染根管中生存的状态,并对治疗感染根管的方法进行思考和评估。     

Loop-mediated isothermal amplification method for the rapid detection of Enterococcus faecalis in infected root canals

INTRODUCTION: Enterococcus faecalis is a major pathogen in the etiology of apical periodontitis after root canal treatment. A loop-mediated isothermal amplification method, which amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand-displacement activity, was developed for the rapid detection of E. faecalis in clinical specimens from root canals. METHODS: Primers for detecting E. faecalis from the azoA gene were designed. The specificity of this assay was evaluated using various oral bacteria and the sensitivity was evaluated using serially diluted E. faecalis chromosomal DNA. In addition, loop-mediated isothermal amplification assays were applied to the rapid detection of E. faecalis from endodontic samples. RESULTS: The loop-mediated isothermal amplification products amplified with the primer set were specific for E. faecalis. To confirm the specificity of the amplicon, the amplified products were digested with the restriction endonuclease Sau3AI. The lower detection limit of the E. faecalis primer set without the loop primer set was 10 microg/tube for a 50-min loop-mediated isothermal amplification reaction. Using loop primers increased the detection sensitivity by several orders of magnitude. Furthermore, E. faecalis was detected with the loop-mediated isothermal amplification assay in four root canals from 18 individuals and the detection results were consistent with those of conventional polymerase chain reactions. CONCLUSION: These results indicate that the loop-mediated isothermal amplification assay is very useful for rapid detection of E. faecalis and diagnosis of endodontic infection.