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。     

慢性根尖周炎感染根管内牙龈卟啉单胞菌和福赛斯拟杆菌的定植

目的：研究慢性根尖周炎患牙感染根管内牙龈卟啉单胞菌和福赛斯拟杆菌的定植情况,探讨两者间的定植关系.方法：采集31例慢性根尖周炎患者的38颗患牙根管内标本,加热裂解法获得细菌DNA,菌种特异引物16SrDNA PCR法检测标本的牙龈卟啉单胞菌和福赛斯拟杆菌,四格表确切概率检验福赛斯拟杆菌在有、无牙龈卟啉单胞菌定植根管内的检出率,比数比（odds ratio,OR）单因素分析两者间的定植关系.结果：牙龈卟啉单胞菌和福赛斯拟杆菌的检出率分别为39.5%、26.3%,其中前者单独检出7颗患牙,后者2颗患牙,两者同时检出8颗患牙.福赛斯拟杆菌在有、无牙龈卟啉单胞菌定植根管内的检出率分别为53.33%、8.70%（P=0.0036）,相关分析两菌间在慢性根尖周炎根管中的OR＞2（OR=12）,呈正相关关系（P＜0.05）.结论：牙龈卟啉单胞菌和福赛斯拟杆菌为慢性根尖周炎感染根管的定植菌,两者呈正相关定植.     

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。     

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.     

慢性根尖周炎根管中8种厌氧菌检出分析

目的应用16SrRNA-PCR技术检测慢性根尖周炎患牙根管内8种厌氧菌的定植情况,分析根管细菌与患牙临床症状的关系。方法采集23例慢性根尖周炎患牙根管样本,提取样本细菌DNA,用细菌16SrRNA引物通过PCR扩增细菌基因片段检测细菌种类。结果23例样本均检出有细菌存在,待检细菌检出率达73.91%(17/23)。其中检出率最高的是中间普氏菌(39.13%),其次是牙龈卟啉菌(30.43%)和福赛斯类杆菌(21.74%),变黑普氏菌、齿垢密螺旋体和啮蚀艾肯氏菌均为13.04%,伴放线放线杆菌有1例检出,直肠弯曲杆菌未检出。中间普氏菌在有自发痛症状组检出率高于无自发痛症状组(P<0.05),其他细菌检出率组间比较差异无显著性。结论慢性根尖周炎患牙根管内以厌氧菌感染为主;根管内中间普氏菌感染与患牙自发痛症状相关。     

根管治疗一次法和两次法清除感染根管内细菌能力的比较研究

目的 比较根管治疗一次法和两次法清除慢性根尖周炎患牙根管内细菌的能力。方法 慢性根尖周炎患者100例共100个牙,随机分为一次法组和两次法组, 分别在根管预备前、预备后(封药后)取样做细菌培养,对比两组根管预备前后菌落数的变化。取根管预备后(封药后)的样本,提取细菌DNA, PCR扩增,测序,确定细菌的种属进行比较。结果 比较一次法组根备后和二次法组封药后菌量,差异无统计学意义（P>0.05）。两组根管内的口腔链球菌、嗜酸乳杆菌、粪肠球菌、内氏放线菌、厌氧消化链球菌、中间普雷沃菌的检出差异无统计学意义（P>0.05）,但两次法组的具核梭杆菌和牙龈卟啉单胞菌检出率高于一次法组,差异有统计学意义（P<0.05）。结论 根管治疗两次法并不能明显降低感染根管内细菌的数量和种类,与一次法的疗效不存在明显差异。     

根管充填失败后根管内微生物的分子生物学分析

目的：基于16S rRNA,构建治疗失败根管内微生物的16S rRNA基因文库,并通过分子生物学方法分析其微生物的多样性。方法：选取经完善根管治疗2年后失败的患牙20例,根管内取样提取细菌DNA;然后用PCR扩增细菌16S rRNA基因,并构建其基因文库;最后通过测序比对构建系统发育树以确定微生物种类。结果：PCR检测显示,所有样本中有细菌出现;20个样本中共发现15种不同的系统发育型细菌（phlylotype）,分属4个细菌门（phylum）：厚壁菌门（Firmicutes）、变异菌门（Proteobacteria）、拟杆菌门（Bacte-roidetes）、放线菌门（Actinobacteria）;每个样本中检出1～6种不同的细菌系统发育型,平均每个样本中可检测到2．7种;在检出的15种细菌中,以粪肠球菌（Enterococcus faecali）的出现频次最高（60％,12／20）,微小消化链球菌（Peptostreptoccus micros）次之（40％,8／20）,而另外13种细菌的出现频次较低,其中有4种系统发育型细菌（Rummeliibacillus、Exiguobacterium aurantiacum、Pseudomonas veronii、phocaeicola abscessus ）只出现了1次。结论：根管治疗失败主要是由多种细菌感染所致,其中以粪肠球菌的检出率最高。     

口腔微生物与免疫学

次氯酸钠对粪肠球菌杀菌效果的体外研究；牙龈卟啉单胞菌红细胞凝集素A黏附和入侵人牙龈上皮细胞的研究；柞蚕溶菌酶基因在酵母中的表达及对变形链球菌的作用；慢性牙周炎龈下菌斑中五种牙周可疑致病微生物的分布；根管治疗失败病例根管内微生物检测及药敏试验.     

根管治疗对牙周源性牙周牙髓联合病变细菌谱的影响

目的:分析牙周源性牙周牙髓联合病变根管治疗前、后牙周微生物的差异,探讨根管治疗对牙周牙髓联合病变预后的影响.方法:收集2018～2020年新疆维吾尔自治区人民医院接诊的牙周源性牙周牙髓联合病变并发逆行性牙髓炎的患牙31例,采集根管治疗前、后牙周袋和根管治疗前根管内标本,提取DNA采用荧光定量PCR对福赛斯坦纳菌、牙龈卟啉单胞菌、具核梭杆菌、中间普氏菌、齿垢密螺旋体、消化性链球菌、粪肠球菌、牙髓卟啉单胞菌数量进行检测.结果:根管治疗后牙周袋内细菌数量较根管治疗前明显降低,差异有统计学意义(P<0.05);其中牙髓卟啉单胞菌、中间普氏菌、牙龈卟啉单胞菌差异最明显.结论:牙周源性牙周牙髓联合病变以牙周可疑致病菌感染为主,完善的根管治疗术对牙周病引起的牙周牙髓联合病变的预后具有促进作用.     

两种根管消毒药物体外抑菌作用的比较研究

目的：探讨氢氧化钙制剂ApexCal、2%洗必泰（CHX）及两者配伍制剂对感染根管内常见菌的体外抑制作用。方法：采用琼脂扩散实验测定ApexCal、2%CHX及ApexCal与2%CHX1∶1混合对粪肠球菌、粘性放线菌、牙髓卟啉单胞菌、牙龈卟啉单胞菌和中间普氏菌形成的抑菌环直径并进行统计学比较。结果：2%洗必泰的抑菌作用显著高于ApexCal,ApexCal/2%CHX的抑菌作用显著低于2%CHX,但对粪肠球菌的抑菌作用明显优于Apex-Cal。结论：2%CHX和ApexCal/2%CHX可以作为有效的根管消毒剂,具有临床实用价值。     

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.     

Microbial analysis of canals of root-filled teeth with periapical lesions using polymerase chain reaction

The objective of the present study was to investigate the presence of nine bacterial species in root-filled teeth associated with periapical lesions using a polymerase chain reaction analysis and to correlate these species with clinical features of the cases. DNA was extracted from 45 canal samples of root-filled teeth with periapical lesions. A PCR assay using species-specific primers of 16S rDNA and the downstream intergenic spacer region was used for microbial detection. Enterococcus faecalis was the most prevalent species, detected in 77.8% of the study teeth, followed by Peptostreptococcus micros, detected in 51.1%. Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia, and Prevotella nigrescens were detected in 35.6%, 22.2%, 11.1%, and 11.1% of the sampled teeth, respectively. Moreover, PCR detected Filifactor alocis in 26.7%, Treponema denticola in 24.4%, and Tannerella forsythia in 4.4% of the samples. T. denticola and P. micros were statistically associated with tenderness to percussion (p < 0.05). P. nigrescens was associated with the presence of spontaneous pain and abscess (p < 0.05). P. endodontalis and P. nigrescens were associated with purulent exudates (p < 0.05). Synergistic relationship was also observed between some species. The results of this study indicated that E. faecalis was the most frequently identified test species by PCR in teeth with failing 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.     

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。     

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。     

Polymerase chain reaction identification of microorganisms in previously root-filled teeth in a South Korean population

A large body of evidence indicates that microorganisms are the primary causative agents of endodontic treatment failures. This study intended to assess the occurrence of nine putative endodontic pathogens in root-filled teeth associated with periradicular lesions in a South Korean population using a culture-independent molecular approach. Fourteen root-filled teeth with persistent periradicular diseases were selected for retreatment. After removal of the root canal filling, the canals were sampled, and a polymerase chain reaction assay using taxon-specific oligonucleotide primers was used for microbial detection. Bacteria were present in all cases, as revealed by amplification using ubiquitous 16S rDNA primers. The most frequently detected taxon was Enterococcus faecalis (64%), followed by Streptococcus spp. (21%) and Tannerella forsythensis (14%). The results of this study using a highly sensitive identification method are concurrent with those from other geographical locations using diverse identification methods in that E. faecalis is the main species found in cases of root-filled teeth associated with periradicular lesions.     

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.     

Detection of Porphyromonas endodontalis in infected root canals by 16S rRNA gene-directed polymerase chain reaction

Porphyromonas endodontalis has been isolated from the endodontic infections mainly in symptomatic teeth. This study evaluated the occurrence of P. endodontalis in both symptomatic and asymptomatic endodontic infections using 16S rRNA gene-directed polymerase chain reaction. P. endodontalis was detected in 39.5% of the cases (17 of 43 teeth). It was present in 4 of the 6 cases with acute periradicular abscess (66.7%) and in 13 of the 37 other cases (35.1%). The presence of P. endodontalis was associated with an asymptomatic periradicular lesion in 6 cases (25%) and in 10 teeth with tenderness to percussion (52.6%). P. endodontalis was also found in one asymptomatic case without evidence of periradicular pathosis. Our results indicated that, although P. endodontalis is commonly detected in symptomatic cases, it can be present in asymptomatic root canal infections. Further studies should determine if this bacterial species is really an important endodontopathogen.     

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.