再治疗根管内粪肠球菌检出及其与临床表征关系的分析研究

目的检测临床再治疗根管内的粪肠球菌,分析粪肠球菌检出率与临床症状及体征之间的关系。方法临床收集需根管再治疗的患牙108颗,记录症状和体征,根管内采集细菌样本,提取细菌基因组DNA,用聚合酶链反应定性检测粪肠球菌。结果根管内粪肠球菌的检出率为47.2%。在有症状病例、有体征病例、既有症状又有体征病例中,根管内粪肠球菌的检出率分别为52.6%、57.9%、62.5%,其中,有体征病例与无体征病例粪肠球菌的检验率差异有统计学意义（P＜0.05）,既有症状又有体征病例与无体征病例组粪肠球菌的检出率差异有统计学意义（P＜0.05）;在有症状的病例组中,有咬合痛的病例粪肠球菌的检出率为66.7%,与无咬合痛病例组相比差异有统计学意义（P＜0.05）。结论再治疗根管内粪肠球菌的存在与临床症状或体征密切相关。     

饥饿期粪肠球菌生物膜毒力因子明胶酶E表达研究

目的:研究粪肠球菌生物膜形成相关毒力因子明胶酶E(gelE)在饥饿期及药物作用后的表达情况。方法:体外建立对数期、稳定期、饥饿期粪肠球菌生物膜模型,分别以1%、2.5%、5.25%次氯酸钠溶液作用于各时期粪肠球菌生物膜后,用Real-time PCR对gelE的基因表达相对量进行检测。结果:gelE基因表达相对量,饥饿期高于稳定期及对数期(P<0.05);用药后,3个期gelE基因表达相对量,5.25%次氯酸钠溶液组低于2.5%次氯酸钠溶液组和1%次氯酸钠溶液组(P<0.05)。结论:饥饿期粪肠球菌生物膜形成相关毒力因子gelE较对数期及稳定期表达增强。次氯酸钠浓度依赖性可抑制粪肠球菌毒力因子gelE的表达。     

再治疗根管内粪肠球菌的分离鉴定及其相关特性研究

目的:研究粪肠球菌在再治疗根管内的检出率及其相关特性(生长情况、生物膜形成能力及耐药性)。方法:收集临床病例样本54例,使用粪肠球菌选择性培养基和胆盐七叶苷琼脂进行分离培养。分离株通过16SrRNA测序法鉴定。将鉴定正确的粪肠球菌临床分菌株和作为对照的粪肠球菌标准株(EfATCC29212)用BHI培养基进行培养,分别对比其生长曲线、生物膜形成能力及耐药性。结果:收集临床病例样本54例,分离鉴定为粪肠球菌的有18例,检出率为33.33%。经统计分析:粪肠球菌在再治疗根管内的检出率在病人性别、年龄、患牙尖周稀疏大小及距离上次根管治疗时间长短等方面无统计学差异(P>0.05);但与上次根管治疗根充是否到位有关,欠填根管中粪肠球菌的检出率明显高于恰填根管(P<0.05),但不同欠填长度组间粪肠球菌的检出率无统计学差异(P>0.05)。此外,粪肠球菌临床分离株与对照菌株在生长曲线、生物膜形成能力及耐药性等方面均无统计学差异(P>0.05)。结论:粪肠球菌在再治疗根管内的检出率为33.33%,其检出率与上次根管治疗根充情况有关。同时,粪肠球菌临床分离株与标准株的生长情况、生物膜形成能力及耐药性基本一致。     

粪肠球菌与复发性根尖周炎的相关性及其机制

粪肠球菌可在恶劣的环境中持续生长和繁殖,而在生物膜中具有的极强的生存和致病能力,使其成为根尖周炎复发的重要的致病因素。粪肠球菌的毒力因子,可引起健康组织损伤,增强细菌的黏附能力。粪肠球菌的检出率,在原发性感染根管内为7.5%,但在治疗失败的感染根管中可达到70%以上,即粪肠球菌在原发性根尖周炎患牙的根管内并非主要致病菌。粪肠球菌在根管冠1/3段的感染较重,在根中1/3和根尖1/3段的感染较轻。粪肠球菌对常规的根管消毒和抗菌药物有极强的耐药性,很难用传统的方法将其于根管内彻底清除,因此研发可以有效地抑制或杀灭粪肠球菌药物十分必要。     

根管治疗失败病例根管内微生物的分子生物学检测

目的:通过对根管治疗失败病例根管内微生物进行分子生物学检测,研究失败病例根管内各种细菌的检出率及优势菌;分析临床症状和体征与特殊微生物的关系.方法:选择40 例根管治疗失败病例共40 颗患牙,根据症状分为疼痛组、窦道组和无症状组,去除根管内充填物,根管内细菌取样,PCR检测鉴定.结果: 根管治疗失败病例根管主要呈混合感染,共检出6 种细菌,粪肠球菌是最常检出的细菌,产黑普氏菌与疼痛、衣氏放线菌与瘘管有一定的相关性.结论:根管治疗失败的主要原因是根管内的微生物感染持续存在,根管治疗失败病例根管内微生物组成有其特殊性.     

慢性根尖周炎感染根管内粪肠球菌和白色念珠菌的检测

目的检测未经治疗和根管再治疗的慢性根尖周炎感染根管内粪肠球菌和白色念珠菌,探讨粪肠球菌和白色念珠菌与慢性根尖周炎发病的关系。方法选取未经根管治疗和需根管再治疗的慢性根尖周炎病例各30例,采集根管内细菌样本,提取DNA,分别设计粪肠球菌和白色念珠菌的引物进行PCR检测。结果所有根管内均可检测到细菌。粪肠球菌在慢性根尖周炎根管再治疗病例中检出率为53.3%(16/30),慢性根尖周炎未经根管治疗病例的根管为10%(3/30)。白色念珠菌仅检出2例,均在慢性根尖周炎根管再治疗病例中检出(6.67%),而未经根管治疗病例的检出率为0。结论粪肠球菌和白色念珠菌在原发的慢性根尖周炎感染根管中检出率较低,在再治疗根管中检出率增高,可能与根管治疗时再感染有关。     

粪肠球菌及其在牙本质小管内的检测和鉴定

粪肠球菌的抗饥饿能力较强，可在非常苛刻的环境下生存。在根管内的不同部位，粪肠球菌的定植密度不同。粪肠球菌具有的多种毒力因子，可在肠球菌菌种之间转移扩散，耐受宿主的非特异性免疫应答，增强其致病性和生存能力。本文就粪肠球菌的特点，牙本质小管内粪肠球菌的检测与鉴定等研究进展作一综述。     

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。     

根管内粪肠球菌感染的研究进展

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

再治疗根管粪肠球菌生物膜形成与临床表现相关性分析

目的检测粪肠球菌形成生物膜的能力,探讨其生物膜形成能力与临床表现之间的关系。方法采用96孔板法形成生物膜,结合结晶紫染色,检测临床样本中分离的53株粪肠球菌形成生物膜的能力,分析其生物膜形成能力与患牙临床表现之间的关系。结果53株粪肠球菌中,40株（75.47%）具有生物膜形成能力;在患牙的多种临床表现中,瘘道与再治疗根管粪肠球菌生物膜形成具有相关性,结果具有统计学意义（P＜0.05）。结论再治疗根管中,无瘘道的患牙分离出来的粪肠球菌生物膜形成能力强于有瘘道的患牙,临床治疗中应予以注意。     

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.     

Isolation and identification of Enterococcus faecalis from necrotic root canals using multiplex PCR

This study was designed to survey the incidence of Enterococcus faecalis infection in symptomatic and asymptomatic root canals of necrotic teeth using PCR and to isolate the bacterium for further screening. Sixty patients categorized according to their clinical symptoms were used for sampling by insertion of paper points into the root canals and absorbing all the fluids present within them. The samples were incubated in 1.0 ml 2xYT (containing 16 g bacto tryptone, 10 g yeast extract and 5.0 g NaCl per liter) for 24 h at 37 degrees C without aeration prior to multiplex PCR analysis. To assist the isolation of E. faecalis, sub-samples were further grown in the same medium supplemented with 6.5% NaCl and back-inoculated into bile esculin. Using multiple cultivation-dependent and PCR analyses, 6 cases (10%) of E. faecalis were identified. Four isolates were obtained from asymptomatic cases of chronic apical periodontitis, and the other two were associated with phoenix abscess and acute apical abscess, respectively. No E. faecalis infection was found in 5 patients with acute apical periodontitis or in 9 with chronic suppurative periodontitis. Our results indicate that there is no significant difference in the incidence of E. faecalis between symptomatic and asymptomatic necrotic dental root canals (P > 0.05).     

Virulence, phenotype and genotype characteristics of endodontic Enterococcus spp

BACKGROUND/AIMS: Enterococci have been implicated in persistent root canal infections but their role in the infection process remains unclear. This study investigated the virulence, phenotype and genotype of 33 endodontic enterococcal isolates. METHODS: Phenotypic tests were conducted for antibiotic resistance, clumping response to pheromone, and production of gelatinase, hemolysin and bacteriocin. Genotype analysis involved polymerase chain reaction amplification of virulence determinants encoding aggregation substances asa and asa373, cytolysin activator cylA, gelatinase gelE, gelatinase-negative phenotype ef1841/fsrC, adherence factors esp and ace, and endocarditis antigen efaA. Physical DNA characterization involved pulsed-field gel electrophoresis of genomic DNA, and plasmid analysis. RESULTS: Potential virulence traits expressed included production of gelatinase by Enterococcus faecalis (n=23), and response to pheromones in E. faecalis culture filtrate (n=16). Fourteen strains produced bacteriocin. Five strains were resistant to tetracycline and one to gentamicin, whereas all were susceptible to ampicillin, benzylpenicillin, chloramphenicol, erythromycin, fusidic acid, kanamycin, rifampin, streptomycin and vancomycin. Polymerase chain reaction products encoding efaA, ace, and asa were detected in all isolates; esp was detected in 20 isolates, cylA in six isolates, but asa373 was never detected. The gelatinase gene (gelE) was detected in all isolates of E. faecalis (n=31) but not in Enterococcus faecium (n=2); a 23.9 kb deletion sequence corresponding to the gelatinase-negative phenotype was detected in six of the eight E. faecalis isolates that did not produce gelatinase. Pulsed-field gel electrophoresis and plasmid analyses revealed genetic polymorphism with clonal types evident. Plasmid DNA was detected in 25 strains, with up to four plasmids per strain and a similar (5.1 kb) plasmid occurring in 16 isolates. CONCLUSIONS: Phenotypic and genotypic evidence of potential virulence factors were identified in endodontic Enterococcus spp., specifically production of gelatinase and response to pheromones.     

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.     

Comparative analysis of virulence determinants and mass spectral profiles of Finnish and Lithuanian endodontic Enterococcus faecalis isolates

INTRODUCTION: Putative virulence factors of Enterococcus faecalis have been proposed by several workers and, by analogy, these have been linked to strains of endodontic origin. However, their distribution within the cell population is unknown. In the present study, isolates were taken from the dental root canals of two defined human populations, Lithuanian and Finnish, and examined for a range of virulence properties. In addition, surface-associated molecules and intracellular proteins were compared using matrix-assisted laser desorption-ionization/mass spectrometry (MALDI-TOF-MS) and ProteinChip capture/MS (SELDI-TOF-MS), respectively. METHODS: Twenty-three Lithuanian and 35 Finnish dental root canal isolates were included. The esp, gelE, ace and efaA genes were detected by polymerase chain reaction, and cytolysin and gelatinase phenotypes were determined by hydrolysis of horse blood agar and gelatine agar, respectively. Protein extracts and surface-associated molecules of whole cells were analysed by SELDI-TOF-MS and MALDI-TOF-MS, respectively. RESULTS: Presence of esp (n = 15), cytolysin (n = 9), ace (n = 55) and efaA (n = 58) was not statistically different in the two samples, whereas gelE and gelatinase production was detected more frequently in the Finnish material (chi-squared, P < 0.01). Analysis of protein profiles by SELDI-TOF-MS showed clustering of cytolysin-producing strains, whereas MALDI-TOF-MS generated profiles that clustered according to the samples' origin and, furthermore, to atypical quinupristin-dalfopristin susceptibility. CONCLUSION: A high prevalence of virulence factors was demonstrated in both population types. SELDI-TOF-MS and MALDI-TOF-MS proved useful in distinguishing between different E. faecalis phenotypes and they may be useful technologies for elucidating the eco-distribution of E. faecalis in humans.     

Calcium hydroxide inactivates lipoteichoic acid from Enterococcus faecalis

Calcium hydroxide is a widely used endodontic medicament for eliminating viable bacteria and inactivating virulence factors. Enterococcus faecalis, a pathogenic gram-positive bacterium, has been associated with refractory apical periodontitis. Because lipoteichoic acid (LTA) is a major virulence factor of gram-positive bacteria, we examined whether calcium hydroxide could detoxify LTA from E. faecalis. An enzyme-linked immunosorbent assay showed that calcium hydroxide-killed E. faecalis was less potent than heat-killed bacteria in stimulating the release of tumor necrosis factor-alpha by a murine macrophage line, RAW 264.7 (P < 0.05). Pretreatment of LTA with calcium hydroxide remarkably abrogated the ability of LTA to induce the release of tumor necrosis factor-alpha (P < 0.05). Furthermore, calcium hydroxide-treated LTA was not able to stimulate Toll-like receptor 2, which recognizes functionally intact LTA. These results suggest that calcium hydroxide could detoxify LTA, resulting in attenuation of the inflammatory responses to E. faecalis and its LTA.     

Failure of Regenerative Endodontic Procedures: Case Analysis and Subsequent Treatment Options

IntroductionRegenerative endodontic procedures (REPs) are considered effective treatments for immature necrotic permanent teeth, with favorable outcomes. However, failed cases require subsequent treatment. This study aimed to review and analyze failed cases after REPs and suggest a treatment algorithm to aid clinical decision-making.MethodsA total of 111 REP cases were selected that were conducted between 2015 and 2020. Clinical outcomes were assessed based on clinical and radiographic evaluations. The criteria for failure included persistence of clinical signs or symptoms and/or periapical radiolucency showing persistent apical periodontitis. Cases requiring any treatment intervention, including extraction, were also considered failures.ResultsSixteen cases were included as failures. The etiology of pulpal disease was stratified into dental trauma (56%), dens evaginatus (25%), and dental caries (12.5%), with the remaining one case having an undocumented cause. The primary reasons for treatment failure were persistent infection (81.3%) and root resorption (18.7%). The identification time of failure varied, with 6 cases (37.5%) detected in less than 6 months and 10 cases (62.5%) later than 6 months after REPs. Sixteen failed cases received 5 different interventions: second REPs, apexification, conventional root canal treatment, surgical approach, and extraction.ConclusionsInterventions for failed REPs are challenging. Consideration of the treatability of the tooth, accessibility to the canal, and the presence of an apical seat might be key factors in clinical decision-making to obtain a successful outcome.     

微小小单胞菌与慢性根尖周炎临床症状的相关性研究

目的：研究慢性根尖周炎感染根管中微小小单胞菌( Parvimonas micra,Pm)的检出情况以及与慢性根尖周炎临床症状、体征的关系。方法采集104名慢性根尖周炎患者的120颗患牙根管内标本,其中包括初次治疗组(A组)和再次治疗组(B组)各60颗患牙。利用16S rDNA聚合酶链反应技术检测标本中的微小小单胞菌16S rDNA的表达;用比值比( OR值)的方法,分析Pm检出率与慢性根尖周炎临床症状及体征的关系。结果慢性根尖周炎初次治疗组检出Pm 24例,再次治疗组检出Pm 3例。2组结果通过比值比分析发现,随检出Pm几率增加,慢性根尖周炎根管内出现恶臭(A组OR=3．35,B组OR=14．29)或根尖出现大于5mm的根尖病变(A组OR=2.75, B组OR=6.70)的几率随之增加,95%可信区间下限>1,结果有显著性差异(P<0．05)。结论无论在初次治疗或再次治疗的慢性根尖周炎感染根管中均有Pm 检出。 Pm 可能在该疾病的发生发展过程中发挥一定作用。