应用16S rDNA结合膜芯片检测3种产黑色素牙周可疑致病菌

目的:探讨采用16SrDNA结合膜芯片检测牙龈卟啉菌Pg、中间普氏菌Pi和变黑普氏菌Pn的价值。方法:利用Genebank中细菌16SrDNA保守区序列,设计一对通用引物,通过已知Pg、Pi和Pn的16SrDNA序列,设计合成对应的特异性寡核苷酸探针。先用通用引物PCR扩增所有标准菌株的DNA,PCR产物和已点样有Pg、Pi和Pn的3种探针的膜芯片杂交,进行分析。结果:膜芯片上的Pg、Pi和Pn3种探针只能与相对应的Pg、Pi和Pn的PCR产物反应,而与其他标准菌株的PCR产物无反应。结论:用16SrDNA结合膜芯片,可准确检测Pg、Pi和Pn,具有很高的特异性,有望成为一种有效的临床检测方法。     

感染根管致病菌及其控制方法的研究进展

细菌与牙髓以及根尖周组织炎症的发生发展关系密切,根管治疗术通过去除根管系统内的病原菌,促进根尖周组织愈合。其中通过根管预备清除根管系统内残余牙髓组织、微生物碎屑以及细菌毒素是根管治疗成功的关键,本文就近年来感染根管致病菌及其临床控制措施的研究进展作一综述。     

基因芯片技术分析慢性根尖周炎患牙产黑色素菌

目的：利用基因芯片技术分析慢性根尖周炎患牙根管中牙髓卟啉单胞菌（Porphyromonas endodontalis,Pg）、牙龈卟啉单胞菌（Porphyromonas gingivalis,聊和中间普氏菌（Irevotella intermedia,Pi）三种产黑色素菌（blackpigmented bacteria,BPB）。方法：收集76例慢性根尖周炎患牙根管细菌学样本,经DNA抽提,PCR荧光标记后,与点样有忍、段和只三种菌特异寡核苷酸探针的基因芯片进行杂交,用激光共聚焦扫描仪分析杂交结果,最后进行统计分析。结果：慢性根尖周炎患牙中Pe、Pg和Pi三种菌的检出率分别为48．68％,32．89％,42．11％。Pe和Pg两菌同时被检出具有统计学意义（P〈0．05）。Pe和Pg与瘘管显著相关（p〈0．01）,Pe和Pg同时感染与瘘管、脓肿显著相关如〈0．01）。结论：产黑色素菌与慢性根尖周炎关系密切,Pe、Pg和瘘管、脓肿显著相关。用基因芯片技术分析慢性根尖周炎患牙中细菌具有快速、灵敏、高效的特点,而且可以通过增加基因芯片上探针数目扩展被检出菌的种类。     

感染根管中产黑色素杆菌检测率的影响因素

产黑色素杆菌与感染根管密切相关,但各项研究得出的检测率存在一定差异.本文通过对国内外诸多文献中的试验数据进行比较,分析了影响感染根管中产黑色素杆菌检测率的主要因素,其中包括样本量、样本来源地区、取样部位、患者的临床症状和检测方法,希望将来能通过控制影响因素更全面地评价该细菌在根尖周感染中所占的地位.     

感染根管内产黑色素类杆菌的定植

目的:了解感染根管内产黑色素类杆菌(BPB)的定植情况.方法:采用16S rDNA PCR技术检测5种BPB在牙髓炎、慢性根尖周炎患牙根管内的定植情况.统计学分析5种BPB在感染根管内检出率的差异及菌种间相互关系.结果:BPB在感染根管内总检出率为60%,其中牙髓卟啉单胞菌(Pe)、变黑普氏菌(Pn)、牙龈卟啉单胞菌(Pg)、产黑普氏菌(Pm)、中间普氏菌(Pi)检出率分别为38%、32%、30%、18%、14%.Pe、Pn检出率明显高于Pm、Pi(P<0.05).Pe在慢性根尖周炎组检出率(50%)明显高于牙髓炎组检出率(20%)(P<0.05).Pn、Pg、Pi和Pm在慢性根尖周炎组检出率(43.33%、36.67%、16.67%、16.67%)与牙髓炎组检出率(20%、20%、10%、15%)无明显差异(P>0.05).Pg/Pe、Pg/Pn、Pe/Pn之间存在正相关.结论:BPB是感染根管内的优势菌群,是牙髓炎和慢性根尖周炎共有的致病菌.Pg/Pe、Pg/Pn、Pe/Pn常定植于同一根管.     

4种根管消毒药物用于乳磨牙管感染的近期疗效比较

本组分别应用碘伏、5 g/L甲硝唑液、20 mL/L戊二醛水溶液、甲醛甲酚液作为根管消毒剂,对它们的疗效进行临床观察,并做一比较.     

4种根管消毒药物用于乳磨牙根管感染的近期疗效比较

本组分别应用碘伏、5ｇ/Ｌ甲硝唑液、2 0ｍＬ/Ｌ戊二醛水溶液、甲醛甲酚液作为根管消毒剂 ,对它们的疗效进行临床观察 ,并做一比较。1　材料和方法1 .1　病例选择和分组各种原因引起的乳磨牙根管感染 2 4 0例 ,共 32 0个患牙 ,其中男 1 1 9例 ,女 1 2 1例 ,年龄 3～ 8岁。随机分成Ａ、Ｂ、Ｃ、Ｄ 4组 ,每组 80个牙。1 .2　材料和治疗方法Ａ组 :用碘伏 (云南康宁药业公司生产 )原液配蒸馏水稀释 1 0倍。Ｂ组 :5ｇ/Ｌ甲硝唑液 (昆明南疆制药厂 )。Ｃ组 :2 0ｍＬ/Ｌ戊二醛水溶液 (云南康宁药业公司生产 )。Ｄ组 :甲醛甲酚液 (上海第二医科大学…     

常用根管消毒药物对犬牙根管内优势致病菌消毒作用研究

目的:对比研究碘仿糊剂、FC、Vitapex和氢氧化钙糊剂4种根管消毒药物对犬牙根管内根尖周病优势致病菌的抗菌活性。方法:健康成年杂种犬4只,随机选取单根管前牙,建立犬牙根尖周病模型。取根管预备前后根管内细菌进行培养和计数,将碘仿糊剂、FC、Vitapex、氢氧化钙糊剂分别封入4组根管内,对照组封无菌纸捻,7 d后再取样进行细菌培养和计数,对细菌计数结果进行统计学分析。结果:①根管预备后的各种细菌数量均较根管预备前明显减少,差异有统计学意义(P<0.05);②封药后根管内细菌数量大部分为0,但4种消毒药物抗菌活性比较无显著统计学意义(P>0.05)。结论:完善的根管预备能有效去除大部分根管内优势致病菌,4种消毒药物对犬牙根管内的优势致病菌均有抗菌作用。     

感染根管暴发疼痛者根管内厌氧菌的培养研究

对26例感染根管暴发疼痛者根管内厌氧菌进行了培养研究。21例治疗前疼痛者根管内分离、鉴定出厌氧菌46株,分布于5个属8个种的范围内,主要为拟杆菌属、丙酸杆菌属、梭杆菌属、消化链球菌屈和真杆菌周。厌氧菌检出率为90.5％,从5例治疗过程中暴发疼痛者根管培养未发现厌氧菌的局部繁殖与其有关。揭示感染根管内的细菌学与临床关系的复杂性。     

感染根管一次性根管治疗的疗效观察

目的评价感染根管一次性根管治疗的临床效果和可靠性。方法选择临床诊断为牙髓坏死、慢性根尖周炎、急性根尖周炎的单根管患牙138颗为研究对象。76颗患牙在一次治疗内完成根管预备和根管充填,设为一次组;62颗患牙经2次治疗后完成根管充填（第1次治疗后氢氧化钙封药1周）,设为二次组。观察2组在根管治疗术前、术后的疼痛状况,评价术后6月、1年和2年的治愈率,比较2组的临床疗效。结果根管治疗后,一次组和二次组术后疼痛率的差异无统计学意义。术后6月、1年和2年,一次组的治愈率为68.4%、92.1%、98.7%,二次组分别为64.5%、91.9%、96.8%,2组治愈率的差异也无统计学意义（P>0.05）。结论与2次治疗后完成根管充填相比,一次性根管治疗的术后疼痛和2年治愈率差异不大,且疼痛期较短,具有一定的临床可行性。     

Detection of Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia, and Prevotella nigrescens in chronic endodontic infection

Black-pigmented anaerobic rods such as Prevotella spp. and Porphyromonas spp. are involved in the etiology and perpetuation of endodontic infections. The aim of this study was to evaluate the prevalence of these species in chronic endodontic infections by using culture and polymerase chain reaction (PCR) techniques. Samples of 100 patients with root canals displaying chronic endodontic infections were obtained by sterilized paper points. Bacterial identification was performed by using culture and PCR techniques. By culture, in 33% of the samples, P. intermedia-P. nigrescens (75.8%), P. gingivalis (27.3%), and P. endodontalis (9.1%) were identified, and by PCR 60% of the samples harbored P. nigrescens (43.3%), P. gingivalis (43.3%), P. intermedia (31.7%), and P. endodontalis (23.3%). The presence of these black-pigmented anaerobic rods alone or in association in chronic endodontic infections seems to be frequent. PCR is a very sensitive technique for detecting DNA from bacterial cells. Culturing is only able to reveal living bacteria and is less sensitive for the identification of low numbers of bacterial cells.     

Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia and Prevotella nigrescens in endodontic lesions detected by culture and by PCR

he aim of this study was to investigate the presence of four black-pigmented bacteria, Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia and Prevotella nigrescens, in endodontic infections by culture and polymerase chain reaction (PCR) analyses. Microbial samples were obtained from 50 teeth with untreated necrotic pulps (primary infection) and from 50 teeth with failing endodontic treatment (secondary infection). Microbiological strict anaerobic techniques were used for serial dilution, plating, incubation, and identification. For PCR detection, the samples were analyzed using species-specific primers of 16S rDNA and the downstream intergenic spacer region. Culture and PCR detected the test species in 13/100 and 50/100 of the study teeth, respectively. The organisms were cultured from 11/50 (22%) of primarily infected root canal samples and from 2/50 (4%) of secondary root canal samples. PCR detection identified the target species in 32/50 (64%) and 18/50 (36%) of primary and secondary infections, respectively. P. gingivalis was rarely isolated by culture methods (1%), but was the most frequently identified test species by PCR (38%). Similarly, P. endodontalis was not recovered by culture from any tooth studied, but was detected by PCR in 25% of the sampled teeth. PCR-based identification also showed higher detection rates of P. intermedia (33%) and P. nigrescens (22%) than culture (13%). In conclusion, P. gingivalis, P. endodontalis, P. intermedia, and P. nigrescens were identified more frequently in teeth with necrotic pulp than in teeth with failing endodontic treatment. Also, a higher frequency of black-pigmented species was detected by PCR than by culture.     

Possible potentiation of toxins from Prevotella intermedia, Prevotella nigrescens, and Porphyromonas gingivalis by cotinine

BACKGROUND: Smoking is a recognized risk factor for the initiation and progression of periodontitis. However, the mechanism by which smoking induces its negative effects on the periodontium is not clear. This study aimed to test the hypothesis that synergy may occur between cotinine and bacterial products isolated from 3 putative periodontopathogens. METHODS: A chick embryo toxin assay was used to investigate bacterial toxins (cell-free extracellular toxins and cell-free cell lysates) from 5 species with and without cotinine. A total of 9 putative periodontopathogens (3 species) and 2 non-oral controls (2 species) were studied. The periodontal species were: Prevotella intermedia (n = 4), Prevotella nigrescens (n = 4), and Porphyromonas gingivalis (n = 1). The control species tested were: Staphylococcus aureus (n = 1) and Escherichia coli (n = 1). RESULTS: The toxicity kill was significantly greater than expected by simple addition alone (P <0.05, Fisher's exact test) between cotinine (800 ng/ml) and 1) the cell-free extracellular toxins of P. nigrescens MH1 and 2) the cell-free cell lysates of P. intermedia MH2. Synergy occurred with cotinine plus the cell-free extracellular toxins in all but 3 periodontal isolates, and the cell-free cell lysates in all but 2 periodontal isolates. Cotinine significantly (P <0.05, Fisher's exact test) enhanced the effects of cell-free extracellular toxins and cell lysates from one control species (E. coli), but not the other (S. aureus). CONCLUSIONS: These findings indicate that synergy in an in vitro assay can occur between cotinine and toxins from putative periodontopathogens. This may be one important mechanism by which smoking increases the severity of periodontitis.     

Dipeptide utilization by the periodontal pathogens Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Fusobacterium nucleatum

Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Fusobacterium nucleatum, which can frequently be isolated from periodontal pockets, preferentially utilize proteins and peptides as growth substrates. In this study, we determined the size of peptide that is preferentially utilized as a source of energy and material for cell growth by P. gingivalis, P. intermedia, P. nigrescens and F. nucleatum using various sizes of poly amino acids consisting of two to approximately 100 molecules of aspartate or glutamate. Resting cells of P. gingivalis, P. intermedia and P. nigrescens utilized aspartylaspartate, while cells of P. gingivalis and F. nucleatum utilized glutamylglutamate. The addition of aspartylaspartate to the culture medium increased the growth of P. gingivalis, P. intermedia and P. nigrescens, while the addition of glutamylglutamate promoted the growth of P. gingivalis and F. nucleatum. These results clearly indicate that dipeptides such as aspartylaspartate and glutamylglutamate can be utilized as growth substrates for P. gingivalis, P. intermedia, P. nigrescens and F. nucleatum.     

Optimized oligonucleotides for the differentiation of Prevotella intermedia and Prevotella nigrescens

The gram-negative, anaerobic bacterium Prevotella intermedia plays an important role in the progression of periodontitis, whereas the etiological role of the closely related but phenotypically indistinguishable species Prevotella nigrescens is controversial. To differentiate between these species properly, 16S rDNA/RNA directed, computer-optimized oligonucleotides were designed and tested with 26 P. intermedia , 26 P. nigrescens and a number of closely and more distantly related strains. The oligonucleotides were used as primers in a polymerase chain reaction and could be demonstrated to be species specific with a detection limit of 50 bacterial cells, which could also be detected when diluted 1:1⁵ with different plaque bacteria. In addition, the described oligonucleotides were digoxigenin-labeled at the 3' end and used as DNA probes in a dot blot hybridization assay. This assay, although slightly less sensitive than the polymerase chain reaction-based method, gave species-specific reactions and also allowed, (semi-)quantification of bacterial cells in clinical specimens.     

Presence and antibiotic resistance of Porphyromonas gingivalis,Prevotella intermedia,and Prevotella nigrescens in children

BACKGROUND/AIMS: Only limited information exists about the prevalence in children of pathogens associated with periodontitis. The aim of the present study was to determine by culture whether 8-11-year-old children carry Porphyromonas gingivalis, Prevotella intermedia, and/or P. nigrescens in samples from the gingiva and/or the buccal mucosa taken before, and after caries treatment and oral hygiene instruction. A second aim was to assess the proportion of subjects who had gram-negative anaerobes carrying the tet(Q) and erm(F) genes, suggesting antibiotic resistance to tetracycline or erythromycin. METHOD: A total of 150 children provided gingival and buccal swab bacterial samples that were cultured for P. gingivalis, P. intermedia, and P. nigrescens. The species was verified using DNA-DNA hybridization with species-specific probes made from the variable region of the 16S rRNA sequences. Antibiotic-resistant genes, tet(Q) and erm(F), were identified using specific DNA-DNA hybridization with specific DNA probes. RESULTS: A total of 116 isolates of black-pigmented bacteria were cultured from 47 (31%) of 150 children. Five isolates were identified as P. gingivalis, 29 as P. intermedia, 33 as P. nigrescens, and 49 as other species. In general, the bacteria were not culturable at more than one time period. We found that 55% of these 47 children harbored black pigmented bacteria that carried either one or both of the two antibiotic-resistant genes studied (tet(Q), and erm(F)). CONCLUSION: The present study demonstrated that children not exposed to regular dental treatment carry bacteria outside the gingival sulcus that have been associated with periodontitis, and that standard treatment procedures may not clear the presence of the putative pathogens. In addition, antibiotic-resistant genes are common in identifiable gram-negative anaerobes, including putative pathogens.     

应用16SrDNA序列同源性分析鉴定放射线照射后的口腔链球菌

目的 对受放射线影响，糖发酵特征发生改变的口腔链球菌进行鉴定。方法 生理、生化试验；16S rDNA序列同源性分析：提取待鉴定细菌的基因组DNA，用多聚酶链式反应(PCR)扩增16S rDNA，对其测序后输入GenBank中与已知序列进行比较。结果 应用16S rDNA序列同源性分析方法可对因受放射线影响，糖发酵特征发生改变而无法通过生化试验进行鉴定的菌株做出准确鉴定。结论 在无法通过生化试验方法对细菌作出准确鉴定时，16S rDNA序列同源性分析是一种可靠的鉴定方法。