PCR检测慢性根尖周炎临床标本中牙髓卟啉单胞菌

目的:检测慢性根尖周炎临床标本中牙髓卟啉单胞菌(Porphyromonas endodontalis, Pe),以反映Pe在慢性根尖周炎中的存在情况. 方法:采用聚合酶链反应(PCR)检测100例慢性根尖周炎临床标本中Pe 16S rDNA,电泳,照相后计算其检出率. 结果:PCR检测慢性根尖周炎临床标本中Pe的检出率为50%.结论:Pe较高的检出率提示Pe可能与根管感染密切相关.     

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.     

牙髓源性牙周牙髓联合病变牙周袋内牙龈卟啉单胞菌定量PCR检测

目的 比较牙髓源性牙周牙髓联合病变与重度慢性牙周炎患牙牙周袋内牙龈卟啉单胞菌的数量差异,为临床诊断牙髓源性牙周牙髓联合病变提供科学依据.方法 采集牙髓源性牙周牙髓联合病变、重度慢性牙周炎牙周袋内的龈下菌斑及正常人群龈沟液标本,应用实时定量PCR法检测牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)数量.结果 3组标本中Pg检出率差异无统计学意义;Pg数量差异有统计学意义.牙髓源性牙周牙髓联合病变中Pg数量显著性低于重度慢性牙周炎患牙(P<0.01),与正常人群Pg数量差异无统计学意义.结论 牙髓源性牙周牙髓联合病变中Pg数量低于重度慢性牙周炎患牙,与正常人龈沟液内的水平一致,Pg数量有可能作为鉴别牙髓源性与牙周源性牙周牙髓联合病变的指标.     

3种根管冲洗液对根管内可疑致病菌的抑制作用分析

目的对慢性根尖周炎患牙根管治疗过程中采用3种不同根管冲洗液进行根管预备,分析治疗前后根管内4种可疑致病菌的检出情况,了解不同冲洗液对这些可疑致病菌的抑制作用,为根管预备时冲洗液的选择和应用提供微生物学依据。方法选取60颗慢性根尖周炎患牙(单根管),根据根管预备时采用不同冲洗液随机分为3组(每组20颗):Na Cl O组(根管冲洗液为2.5%次氯酸钠,sodium hypochlorite); H2O2组(根管冲洗液为3%双氧水,hydrogen peroxide); PVP-I组(根管冲洗液为1%聚维酮碘,Povidone iodine,怡速欣)。分析根管治疗前后临床指数(clinical periapical index,CPI);同时采集根管预备前后患牙根管内感染物,提取细菌基因组DNA,合成针对粪肠球菌(Enterococcus faecalis,E.f)、具核梭杆菌(Fusobacterium nucleatum,F.n)、牙龈卟啉单胞菌(Porphyromonas gingivalis,P. g)和牙髓卟啉单胞菌(Porphyromonas endodontalis,P. e)的16S r DNA基因的特异性引物,运用实时荧光定量PCR(qRT-PCR)技术,比较分析E.f、F.n、P.g及P.e的检出率和检出量。结果 3组治疗前、后E.f、F.n、P.g和P.e的检出率相同,但可疑致病菌的检出量均有下降。其中3组间比较E.f、F.n和P.g检出量的下降率经统计学检验有差异(P<0.05),而P.e检出数量下降无显著差异(P>0.05); 2组间比较发现Na Cl O组和PVP-I组间E.f、F.n和P.g检出量的下降率统计学检验无差异(P>0.05); Na Cl O组和PVP-I组分别与H2O2组比较,E.f、F.n和P.g检出量的下降率大,差异有统计学意义(P<0.05)。PVP-I组P.e检出量的下降率大于H2O2组,差异有统计学意义(P<0.05)。结论使用3种不同根管冲洗液进行根管预备,能明显地减少感染根管内的E.f、F.n、P.g和P.e的量;与3%双氧水比较,用2.5%NaClO和1%PVP-I根管冲洗液对E.f、P.g和F.n抑制作用更明显;用1%PVP-I对P.e的抑制作用更明显。     

牙龈卟啉单胞菌对牙髓成纤维细胞胞内感染的研究

目的研究牙龈卟啉单胞菌活菌细胞内感染牙髓成纤维细胞的体外模型。方法将牙龈卟啉单胞菌和牙髓细胞以100∶1比例共同孵育0.5、1.0、2.0 h,倒置显微镜观察牙髓细胞形态。加入双抗和甲硝唑杀死胞外细菌,牙髓细胞用蒸馏水裂解后厌氧培养细胞裂解液,观察活细菌是否进入牙髓细胞胞内。将牙龈卟啉单胞菌和牙髓细胞以多重感染比（MOI）100、50共同孵育1.0 h,采用MTT法检测感染牙龈卟啉单胞菌后牙髓细胞存活率。结果倒置显微镜下显示,被胞内感染的牙髓细胞培养2.0 h未见胞膜破裂,形态完整。采用双抗能彻底杀灭胞外培养基中的细菌而对胞内细菌无影响,共同孵育1.0 h和2.0 h活细菌能进入牙髓细胞。MTT法显示细菌感染后牙髓细胞仍有一定存活率,其存活率分别是MOI 100组74.43%、MOI 50组99.07%。结论成功建立了牙龈卟啉单胞菌对牙髓成纤维细胞胞内感染的模型。     

黄连对牙髓卟啉单胞菌抑菌作用的体外研究

目的研究中药黄连对牙髓卟啉单胞菌的抑菌作用。方法采用杯碟法进行抑菌实验,以氢氧化钙为对照,测定各浓度黄连水煎剂对牙髓卟啉单胞菌的抑菌环直径。结果各浓度黄连水煎剂对牙髓卟啉单胞菌均具有抑菌作用,随浓度增加,抑菌环直径增加,呈现正相关。抑菌作用强于25%氢氧化钙。结论黄连水煎剂对牙髓卟啉单胞菌有较强的抑菌作用,用于根管消毒具有较好的应用前景。     

牙龈卟啉单胞菌与牙周基础治疗关系的实验研究

目的应用TaqMan实时荧光定量聚合酶链反应法检测慢性牙周炎患者牙周洁刮治术（SRP）治疗前后龈下菌斑中牙龈卟啉单胞菌（P. gingivalis）的变化,分析P. gingivalis与SRP疗效之间的关系,探讨应用实时荧光定量聚合酶链反应监测和评价SRP的可能性。方法选择20例中重度慢性牙周炎患者为研究对象,检查SRP治疗前后的临床指标,包括探诊深度（PD）、临床附着丧失（CAL）和探诊出血（BOP）;采集SRP治疗前后的龈下菌斑共142个样本,应用TaqMan实时荧光定量聚合酶链反应检测样本中的P. gingivalis。构建含有P. gingivalis扩增片段的重组质粒,建立定量标准。结果慢性牙周炎患者SRP治疗后PD、CAL和BOP均明显下降（P<0.001）;治疗前P. gingivalis检出率为80.28%,治疗后下降为54.23%;治疗前P. gingivalis数量与PD相关,Kendall相关系数为0.70（P<0.001）,治疗后牙周袋内细菌数量明显减少（P<0.001）。治疗前牙周袋内P. gingivalis的定植数量与PD、CAL和BOP的改善无相关性（P>0.05）,但治疗后治疗有效位点P. gingivalis数量减少程度明显大于治疗无效位点（P<0.05）,细菌减少量与PD改善弱相关（r=0.25,P=0.04）。结论SRP治疗可以明显改善临床症状,降低P. gingivalis检出率和绝对数量;治疗前P. gingivalis定植水平对临床指标的改善没有影响,治疗后P. gingivalis数量下降程度可以反映治疗效果。TaqMan实时荧光定量聚合酶链反应可以用于牙周炎治疗效果的监测和评价。     

感染根管中牙龈卟啉单胞菌fimA基因多态性研究

目的研究根尖周炎患牙感染根管内牙龈卟啉单胞菌(P.gingivalis)不同fimA基因型的分布情况。方法收集200例感染根管内样本,经DNA抽提后使用16S r DNA PCR方法检测P.gingivalis。在P.gingivalis检出阳性样本中,根据各fimA基因型的特异引物,采用PCR检测不同fimA基因型菌株的分布,并通过Pearsonχ2检验对不同fimA基因型与临床症状进行相关性分析。结果感染根管中P.gingivalis的检出率为48%,其中79例样本只检测到1种fimA基因型P.gingivalis菌株(82.3%);12例样本检测到2种或2种以上fimA基因型P.gingivalis(12.5%)。各fimA基因型P.gingivalis的检出情况:Ⅰ型29.2%、Ⅰb型8.3%、Ⅱ型37.5%、Ⅲ型14.6%、Ⅳ型19.8%。各型fimA基因与临床症状之间无统计学相关性(P<0.05)。结论感染根管内P.gingivalis的fimA基因存在基因多态性,其中以fimAⅡ型P.gingivalis菌株检出率最高,其次为Ⅰ型和Ⅳ型。     

感染根管机械预备联合不同化学方法预备后细菌残余量的实时定量检测

目的:探讨快速、敏感定量检测感染根管病原菌的方法;并在RNA水平评估不同根管化学预备方法清除根管细菌的效果.方法:选择慢性根尖周炎单根管患牙24颗,随机分为实验组与对照组.常规根管机械预备后,对照组用3%H2O2、1%NaClO交替冲洗根管;实验组加用EDTA凝胶根管化学预备,联合3%H2O2、1%NaClO交替冲洗.预备前后根管采样.提取总RNA并反转录为cDNA,进行实时PCR定量分析,采用SAS6.12软件包对数据进行方差分析.结果:机械预备联合化学预备后,根管内细菌数量显著降低(P＜0.01);其中实验组清除病原菌的效果显著优于对照组(P＜0.05).结论:实时PCR定量检测技术可以快速、敏感的检测根管内病原菌,机械预备联合化学预备可有效降低根管内细菌的数量.     

牙髓卟啉单胞菌的毒力因子及其致病性

牙髓及根尖周病是最为常见的口腔疾患,主要病因是牙髓的细菌感染及感染菌在根管系统中的持续存在.牙髓卟啉单胞菌是感染根管的优势菌,在牙髓、根尖周组织病的发生、发展中起着重要的作用,并与牙髓坏死、根尖部疼痛肿胀、叩痛等症状和窦道形成密切相关,近年来成为研究热点.本文从牙髓卟啉单胞菌的毒力因子、致病机制、临床意义等方面综述.     

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.     

Invasion of vascular cells in vitro by Porphyromonas endodontalis

AIM: The objective of this study was to determine whether laboratory strains and clinical isolates of microorganisms associated with root canal infections can invade primary cultures of cardiovascular cells. METHODOLOGY: Quantitative levels of bacterial invasion of human coronary artery endothelial cells (HCAEC) and coronary artery smooth muscle cells (CASMC) were measured using a standard antibiotic protection assay. Transmission electron microscopy was used to confirm and visualize internalization within the vascular cells. RESULTS: Of the laboratory and clinical strains tested, only P. endodontalis ATCC 35406 was invasive in an antibiotic protection assay using HCAEC and CASMC. Invasion of P. endodontalis ATCC 35406 was confirmed by transmission electron microscopy. DISCUSSION: Certain microorganisms associated with endodontic infections are invasive. If bacterial invasion of the vasculature contributes to the pathogenesis of cardiovascular disease, then microorganisms in the pulp chamber represent potential pathogens.     

Molecular detection and identification of Synergistes phylotypes in primary endodontic infections

Aim:  Uncultivated phylotypes from the Synergistes group have recently emerged as suspected endodontic pathogens. This study aimed to investigate the presence and identity of these bacteria in primary endodontic infections using a 16S rRNA gene-based group-specific heminested polymerase chain reaction (PCR) protocol.
Materials and methods:  Genomic DNA was isolated directly from clinical samples and used as templates for PCR. Amplicons from positive specimens were sequenced and phylogenetically analyzed to determine species identity.
Results:  Overall, about one-third of the samples harbored Synergistes bacteria. The following phylotypes were disclosed: oral clones W028, BA121/P4G_18 P1, W090, BH017 and E3_33.
Conclusions:  These results demonstrated that as-yet uncultivated Synergistes phylotypes are present in the endodontic microbiota and a role in causation of apical periodontitis is suggested though remains unproven.     

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.     

Bacterial reduction and persistence after endodontic treatment procedures

Bacteria that persist after endodontic disinfection procedures may lead to treatment failure. Over 50% of the bacteria found in endodontic infections are as-yet-uncultivated so investigations of bacteria that endure treatment procedures should include techniques that side-step cultivation. This culture-independent study evaluated the bacterial reduction promoted by intracanal disinfection procedures and identified the taxa persisting after treatment. Samples taken from the infected canals of teeth with apical periodontitis before treatment (S1), after instrumentation using NaOCl as irrigant (S2) and after interappointment medication with a calcium hydroxide paste (S3) were subjected to 16S rRNA gene clone library and real-time polymerase chain reaction analyses. The S2 and S3 samples from five of the 15 canals showed negative results. In the other cases, instrumentation and instrumentation/medication promoted a significant reduction (99.67% and 99.85%, respectively) in the number of bacteria when compared to S1 samples. Forty-three distinct bacterial taxa were identified, of which 24 (56%) were as-yet-uncultivated phylotypes. Nineteen of these 43 taxa (including eight as-yet-uncultivated phylotypes) were disclosed in post-treatment samples, with streptococci being the most prevalent taxa. Findings demonstrated that culture-independent methods provide a detailed insight into the effects of intracanal disinfection protocols, helping to define more effective strategies to deal with endodontic bacteria, including as-yet-uncultivated phylotypes.     

不同氢氧化钙制剂对牙髓卟啉单胞菌的杀菌效果

目的:比较Endocal、Calxyl、国产氢氧化钙糊剂、Vitapex 4种氢氧化钙制剂对牙髓根尖周主要致病菌牙髓卟啉单胞菌(Porphyromonas endodontalis,P.e)活性的影响。方法:(1)通过动态比浊法比较在直接接触条件下不同氢氧化钙制剂不同作用时间对牙髓卟啉单胞菌的抑制作用。(2)选取新鲜拔除的人单根管牙85颗,经根管预备、高压消毒灭菌后,置入P.e菌悬液,建立牙髓卟啉单胞菌感染模型。将模型采用抽签法随机分为5组,即Endocal组、Calxyl组、国产氢氧化钙糊剂组、Vitapex组、无菌去离子水对照组。分别于封药前和封药7d后,用无菌纸捻在根管中取样,细菌培养计数菌落,并完成细菌24h回复实验。采用SPSS11.0软件包对数据进行统计学处理。结果:Endocal、Calxyl、国产氢氧化钙糊剂、Vitapex均能有效减少牙髓卟啉单胞菌的数量(P<0.05),细菌清除率均达95%以上。动态比浊法检测结果显示,Endocal、国产氢氧化钙糊剂抑菌作用优于其他组,有显著性差异(P<0.05);细菌培养法结果显示,Endocal组细菌清除率显著大于Vitapex组(P<0.05)。结论:Endocal、Calxyl、国产氢氧化钙糊剂、Vitapex 4种药物对牙髓卟啉单胞菌均有较好的抑制作用,其中,Endocal的抗菌作用优于Vitapex。     

慢性牙周炎患者龈下菌斑中不同基因型牙龈卟啉单胞菌的检测

摘要 目的:建立临床标本中牙龈卟啉单胞菌(P.g)的PCR检测方法,探讨慢性牙周炎患者不同牙位的龈下菌斑中P.g基因型的差异。方法:采用培养法分离鉴定慢性牙周炎患者不同牙位龈下菌斑中P.g,同时采用PCR检测 P.g16SrDNA、prtC和fimA基因。部分扩增产物测定了核苷酸序列。结果:在66例患者的127个龈下菌斑标本中, P.g16SrDNA、prtC和fimA多重引物扩增的阳性率为9814%;PCR阳性率显著高于培养法P.g的检出率(P< 0101)。3010%的患者(18/60)同时感染了不同基因型的P.g菌株。P.g16SrDNA、prtC和fimA扩增片段的核苷酸序列同源性在98162%~100%之间。结论:本文所建立的P.g的PCR检测方法具有较高的敏感性和特异性,适用于P.g的快速临床诊断。同一患者可被不同感染来源的多株P.g同时感染。