牙周菌斑生物膜的体外模型建立

目的观察血链球菌、牙龈卟啉单胞菌和具核梭杆菌在人工牙根面上随培养时间变化形成单菌种、双菌种和三菌种生物膜的能力,期望在体外建立龈下菌斑生物膜模型。方法培养血链球菌、牙龈卟啉单胞茵和具核梭杆菌试验菌株,制备胶原包被羟磷灰石的人工牙根面,在人工牙根面上培养形成试验菌株的单菌种、双菌种和多菌种生物膜,并用扫描电镜观察三种试验菌株分别在培养24、48和72h后形成单菌种、双菌种和三菌种生物膜的情况。结果单独培养24h的血链球菌在人工牙根面上形成的完整生物膜,具备三维立体结构;培养48和72h后,细菌密度逐渐增大,生物膜更加成熟。单独培养48h的牙龈卟啉单胞菌形成的完整生物膜,具备三维立体结构;培养72h后,细菌密度有所降低。牙龈卟啉单胞菌和具核梭杆菌培养24h形成的完整的双菌种生物膜,具备三维立体结构;培养48和72h后已看不出完整的生物膜结构,两菌的数量大幅度降低。血链球菌、牙龈卟啉单胞菌和具核梭杆菌在培养24h时形成的较完整的三菌种生物膜,初步具备三维立体结构;三菌中,血链球菌、具核梭杆菌所占比列远大于牙龈卟啉单胞菌;培养48h后,三菌种生物膜更加成熟,牙龈卟啉单胞菌所占比例有所增加,此时的生物膜已具备三维立体结构;培养72h后,三菌种仍保持较完整的生物膜结构,但数量均有所下降。结论血链球菌、牙龈卟啉单胞菌和具核梭杆菌在培养24h时间点已基本形成了完整的单菌种、双菌种和三菌种生物膜结构,故在今后建立龈下菌斑生物膜模型时可选取24h这一时间点。     

血链球菌细菌素对唾液生物膜中牙龈卟啉单胞菌和具核梭杆菌拮抗作用的实验研究

目的:提取血链球菌标准菌株(ATCC10556)细菌素,研究其对唾液生物膜中牙龈卟啉单胞菌和具核梭杆菌的拮抗作用.方法:通过超声破碎,高速离心,盐析等方法提取血链球菌细菌素;采用二倍稀释法,测定血链球菌细菌素对浮游状态下牙龈卟啉单胞菌和具核梭杆菌的最小抑制浓度(MIC);在体外建立牙龈卟啉单胞菌和具核梭杆菌唾液生物膜模...     

中草药提取物联合应用锌离子对口臭相关致病菌的体外抑菌效果

目的 评价中草药提取物与锌离子对口臭相关致病菌的体外抑菌效果;探讨其联合应用是否具有协同作用.方法 选用厚朴酚、小檗碱、黄芩素、鞣酸、银杏双黄酮、苦参碱、绿原酸、丹皮酚和甘草酸等9种中草药提取物与锌离子,采用倍比梯度稀释法,分别测定对口臭相关致病菌牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)、具核梭杆菌(Fusobacterium nucleatum,Fn)、牙周有益菌血链球菌(Streptococcussanguis,Ss)的最小杀菌浓度值(Minimum Bactericidal Concentration,MBC);采用杯碟法分析筛选出的中草药成分与锌离子联合应用对Pg和Fn的抑制作用.实验数据使用SPSS19.0软件进行单因素方差分析.结果 厚朴酚、小檗碱、绿原酸、银杏双黄酮、锌离子对Pg和Fn抑制效果明显,且对Ss的MBC高于其它两种口臭相关致病菌;厚朴酚与锌离子联合应用组作用于Pg或Fn所形成的抑菌环直径均大于各单独应用组的抑菌环直径(P＜0.05);小檗碱联合应用锌离子对Fn形成的抑菌环大小与两种成分单独应用时的抑菌环直径之间也存在显著差异(P＜0.05).结论 厚朴酚、小檗碱、绿原酸、银杏双黄酮、锌离子是较为理想的抑菌成分.厚朴酚、小檗碱联合应用锌离子对口臭相关致病菌的抑制具有协同作用.     

鞣花酸对牙龈卟啉单胞菌和具核梭杆菌的体外抑菌效果

目的 观察鞣花酸对牙龈卟啉单胞菌和具核梭杆菌的生长、黏附的影响。方法 将鞣花酸单体溶于二甲基亚砜（DMSO）,用二倍梯度稀释法测定最低抑菌浓度和最低杀菌浓度。0.5 g/L氯己定作为阳性对照组,无药液组为阴性对照组,采用紫外分光光度计测定细菌黏附能力及生长曲线;通过生物膜结晶紫染色法测定生物膜抑制浓度和生物膜清除浓度。结果 石榴皮生物活性成分鞣花酸对牙龈卟啉单胞菌和具核梭杆菌最低抑菌浓度均为1 g/L,最低杀菌浓度均为2 g/L。在1 g/L时鞣花酸对牙龈卟啉单胞菌和具核梭杆菌的细菌粘附能力的抑制率为80.68%和83.53%(P<0.05)。鞣花酸对牙龈卟啉单胞菌和具核梭杆菌的生长影响为将牙龈卟啉单胞菌对数生长期由8 h推迟至12 h,将具核梭杆菌的对数生长期由4 h推迟至12 h。鞣花酸对牙龈卟啉单胞菌和具核梭杆菌的生物膜抑制浓度及生物膜清除浓度均为4 g/L和8 g/L。结论 鞣花酸对牙周主要致病菌牙龈卟啉单胞菌和具核梭杆菌都有显著的抑制作用,有望成为一种牙周病防治的辅助治疗药物。     

牙周病与恶性肿瘤相关性的研究进展

牙周病是一种常见的慢性感染性疾病,是菌斑生物膜、环境、遗传和表观基因组学等多种风险因素相互作用的结果。近年来,越来越多的研究发现牙周病与恶性肿瘤密切关联。文章就牙周病与口腔癌、胃癌、结直肠癌、胰腺癌、肺癌、乳腺癌、前列腺癌相关的流行病学研究进展,以及牙周病与恶性肿瘤相关致病机制做一综述,为相关疾病的早期防治和临床研究提供新思路。     

戈登链球菌对牙龈卟啉单胞菌生物膜形成的影响

目的 观察戈登链球菌（S.gordonii）对牙龈卟啉单胞菌（P. gingivalis）生物膜超微结构及生物膜中牙龈卟啉单胞菌数量的影响。方法 分别形成P. gingivalis单菌种生物膜和P. gingivalis－S.gordonii混合生物膜,利用扫描电镜观察其超微结构,定量PCR法对生物膜中P. gingivalis数量进行定量分析。采用SPSS 13.0软件包对数据进行统计学分析。结果 P. gingivalis生长72 h,与单菌种生物膜相比,P. gingivalis-S.gordonii混合生物膜形成量明显增多;而且混合生物膜的超微结构更规则、有序、多孔隙。在生物膜形成24、48、72 h后,混合生物膜中P. gingivalis的数量分别是单菌种生物膜中P. gingivalis数量的5.4、3.8和4.4倍。结论 早期定植菌S.gordonii的存在,使P. gingivalis更容易定植、形成生物膜。     

具核梭杆菌黏附和侵入上皮细胞的初步研究

目的:本研究以P.gingivalisATCC33277为阳性对照,用KB细胞体外模拟牙龈上皮细胞,初步研究F.nucleatum对上皮细胞的黏附和侵入能力。方法:将实验菌株P.gingivalisATCC33277、F.nucleatumATCC10953以及F.nucleatumWCD05-1分别配制成2×107CFU/mL的菌悬液,加入培养有KB细胞的24孔细胞培养板中。在37℃,50 mL二氧化碳的细胞培养箱中孵育2 h,以P.gingivalisATCC33277为阳性对照,观察F.nucleatum对KB细胞的黏附和侵入。结果:两种F.nucleatum均能黏附和侵入KB细胞。与P.gingivalisATCC33277相比较,F.nucleatumATCC10953的黏附和侵入能力稍弱,而F.nucleatumWCD05-1的黏附和侵入能力更强。结论:F.nucleatum能黏附和侵入上皮细胞,至少在短时间内能保持一定的黏附和侵入量,在细胞内保持一定的活力。F.nucleatum的不同菌株之间黏附和侵入能力存在差异,临床株的毒力明显强于标准株。     

聚合酶链式反应在3种牙周病原菌检测中的应用

应用聚合酶链式反应检测临床标本中的Pg、Aa与Fn具有很高的敏感性和特异性。聚合酶链式反应及其扩增产物测序结果以及AP-PCR指纹图分析提示不同标本分离的Pg与Aa菌株所携带的毒力基因有差异。分析不同Pg、Aa与Fn等牙周致病菌基因型与其致病力之间的关系,对于阐明牙周炎的发病机制、改善诊断方法、采取更有效的治疗措施等方面具有重要意义。     

聚合酶链式反应在3种牙周病原菌检测中的应用

应用聚合酶链式反应检测临床标本中的Pg、Aa与Fn具有很高的敏感性和特异性。聚合酶链式反应及其扩增产测结果以及AP－PCR指纹图分析提示不同标本分离的Pg与Aa菌株所携带的毒力基因有差异。分析了不同Pg、Aa与Fn等牙周致病菌基因型与其致病力之间的。对于阐明牙周炎的发病机制、改善诊断方法、采取更有效的治疗措施等方面具有重要意义。     

牙龈卟啉单胞菌和具核梭杆菌多克隆抗体的制备及应用效果评价

目的    制备高效价高纯度的牙龈卟啉单胞菌（P. gingivalis）和具核梭杆菌（F. nucleatum）多克隆抗体，并评价其应用效果。方法    将新鲜培养的P. gingivalis和F. nucleatum菌液灭活后与佐剂乳化混匀，作为抗原皮下多点注射免疫雄性新西兰大白兔，定期免疫并在抗体效价达到预期后采用颈动脉取血获得抗血清。采用间接ELISA法测定多克隆抗体交叉反应。应用硫酸铵沉淀法纯化多克隆抗体，并通过Western-blot方法鉴定抗体的纯度。荧光显微镜观察多克隆抗体应用于免疫荧光实验的效果。结果    免疫6周后，血清抗体效价达到1∶320 000 ~ 1∶640 000，相互之间未出现交叉反应，且纯度较高。免疫荧光实验中可观察到明显的荧光，并可见P. gingivalis的球杆状形态和F. nucleatum的短杆状形态。结论    成功制备了P. gingivalis和F. nucleatum多克隆抗体，其特异性良好，为后续的免疫学相关实验奠定了基础。     

Fusobacterium nucleatum impairs serum binding to Porphyromonas gingivalis biofilm

Mouse immune sera obtained by immunization with Fusobacterium nucleatum and then Porphyromonas gingivalis demonstrated an impaired binding capacity to P. gingivalis-biofilm and lower avidity to P. gingivalis when compared with sera obtained from mice immunized with P. gingivalis alone.     

Stimulation of Fusobacterium nucleatum biofilm formation by Porphyromonas gingivalis

BACKGROUND/AIMS: Bacterial infection is a major cause of periapical periodontitis. Eradication of these microorganisms from apical lesions is essential to the success of endodontic treatment. The aim of this study was to clarify the molecular interaction between Fusobacterium nucleatum, Porphyromonas gingivalis and other microorganisms associated with periapical periodontitis. METHODS: Microorganisms isolated from periapical lesions were inoculated into type-I collagen-coated polystyrene microtiter plates and maintained at 37 degrees C under anaerobic conditions for 2 days, after which, the quantity of organized biofilm on the plates was evaluated by crystal violet staining. Growth enhancement via soluble factor was evaluated by separated coculture using a 0.4-mum membrane filter. RESULTS: F. nucleatum exhibited strong adherence to type-I collagen-coated polystyrene microplates. Biofilm formation by F. nucleatum was significantly enhanced by P. gingivalis. It was complemented by compartmentalized coculture with P. gingivalis. Enhancement of biofilm formation by P. gingivalis was only slightly reduced by inactivation of its autoinducer-2-producing gene luxS. CONCLUSION: The results suggest that P. gingivalis enhances biofilm formation by F. nucleatum by releasing diffusible signaling molecules other than autoinducer-2.     

Inhibitory effect of cranberry polyphenol on biofilm formation and cysteine proteases of Porphyromonas gingivalis

BACKGROUND AND OBJECTIVE: The purpose of this study was to investigate the effects of cranberry polyphenol fraction on biofilm formation and activities of Arg-gingipain and Lys-gingipain in Porphyromonas gingivalis. MATERIAL AND METHODS: The polyphenol fraction was prepared by using a glass column packed with Amberlite XAD 7HP and 70% aqueous ethanol as an elution solvent. RESULTS: Synergistic biofilm formation by P. gingivalis and Fusobacterium nucleatum was significantly inhibited by the polyphenol fraction at a concentration of 250 microg/mL compared with untreated controls (p < 0.01). Arg-gingipain and Lys-gingipain activities in P. gingivalis ATCC 33277 and FDC 381 were inhibited significantly at a polyphenol fraction concentration of > or = 1 microg/mL (p < 0.05). CONCLUSION: These findings indicate that the polyphenol fraction inhibits biofilm formation and the Arg-gingipain and Lys-gingipain activities of P. gingivalis.     

Serum immunoglobulin G antibodies to Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum and Streptococcus sanguis during experimental gingivitis in young adults

Twenty-eight young, healthy adults completed an experimental gingivitis study in which blood and clinical recordings were obtained at baseline; after a 4-week period of thorough oral hygiene; after a subsequent 3-week period of plaque accumulation; and after another 2 weeks of thorough oral hygiene. Serum immunoglobulin G antibodies against whole cells of Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum and Streptococcus sanguis were determined using enzyme-linked immunosorbent assay. Mean serum immunoglobulin G antibody levels to P. intermedia, F. nucleatum and S. sanguis remained essentially constant during the experiment, whereas the immunoglobulin G antibodies to P. gingivalis declined during the initial period of oral hygiene and the subsequent period of plaque accumulation to an average of 84.5% of the baseline value. This reduction could be attributed to the people who developed marked gingival inflammation during the period of plaque accumulation, indicating that the systemic host response may be associated with local tissue responses to variations in oral hygiene. These people were, however, also characterized by higher initial serum immunoglobulin G responses to P. gingivalis than people who developed less pronounced gingival inflammation during the experiment. The variability and individuality noted in the host response to potential pathogens have important implications for attempts to use such measures for establishing a diagnosis or prognosis for the individual patient.     

Kinetics of coaggregation of Porphyromonas gingivalis with Fusobacterium nucleatum using an automated microtiter plate assay

Coaggregation between Porphyromonas gingivalis and Fusobacterium nucleatum strains was previously studied using either a semi-quantitative macroscopic assay or radioactive tracer assays. A new automated microtiter plate assay is introduced, in which the plate reader (Vmax) was adapted to allow quantitative evaluation of the kinetics of coaggregation. F nucleatum PK 1594 coaggregated with P. gingivalis HG 405 with a maximal coaggregation rate of 1.05 mOD/min, which occurred at a P. gingivalis to F. nucleatum cell ratio of 1 to 2. F. nucleatum PK 1594 failed to do so with P. gingivalis strains A 7436 or ATCC 33277. Galactose inhibition of this coaggregation could be quantitatively measured over a wide range of concentrations to demonstrate its dose-dependent manner. P. gingivalis HG 405 failed to coaggregate with F. nucleatum strains ATCC 25586 and ATCC 49256. The assay used in the present study is a sensitive and efficient quantitative automated tool to study coaggregation and may replace tedious radioactive tracer assays.     

CD45RA and CD45RO positive CD4 cells in human peripheral blood and periodontal disease tissue before and after stimulation with periodontopathic bacteria

Flow cytometric analysis was used to examine naive and primed or memory CD4 cells extracted from periodontal lesions compared with cells from peripheral blood of healthy subjects before and after stimulation with the periodontopathic bacteria, Porphyromonas gingivalis and Fusobacterium nucleatum. In peripheral blood, approximately 60% and 40% of CD4 cells were CD45RO+ and CD45RA+ respectively at day 0. Phytohaemagglutinin (PHA) induced CD45RO expression on almost 100% of CD4 cells. However, P. gingivalis and F. nucleatum stimulation did not cause any significant change in percentage of CD45RO+ CD4 cells except for a loss of antigen at day 6 together with re-expression at day 7, which also occurred on cells cultured in medium only. CD45RA expression on PHA and bacterial-stimulated peripheral blood CD4 cells remained fairly stable for the 10-d culture period. Greater than 90% CD4 cells extracted from healthy or marginal gingivitis (H/MG) and adult periodontitis (AP) lesions were CD45RO+ and this was maintained on AP cells throughout the 6-d culture period, except for a small decrease in the percentage of positive cells induced by P. gingivalis at day 3. Approximately 9% CD4 cells from H/MG tissue were CD45RA+, but about 22% AP cells expressed this antigen, and this increased again in P. gingivalis- and F. nucleatum-stimulated cultures after 3 d. Therefore, in peripheral blood P. gingivalis and F. nucleatum do not act as nonspecific T-cell mitogens and, in AP cells, these bacteria induce changes in phenotype, supporting previous data that although they may be polyclonal B-cell activators, they activate antigen specific T-cells.     

Effect of an essential oil-containing antiseptic mouthrinse on induction of platelet aggregation by oral bacteria in vitro

BACKGROUND: With an increasing body of data suggesting an association between periodontitis and cardiovascular disease, studies have been conducted to elucidate potential mechanisms by which oral bacteria might exert systemic effects. 2 oral bacteria, Streptococcus sanguis and Porphyromonas gingivalis, have been shown to induce platelet aggregation in vitro. This study was conducted to determine the effect of treatment with an essential oil mouthrinse (Listerine Antiseptic) on the platelet-aggregating activity of these organisms. METHOD: Bacteria were grown under standard culture conditions. S. sanguis ATCC strain 10556 was exposed for 3 min to the essential oil mouthrinse at either full strength or a 1:1 dilution, while P. gingivalis FDC strain 381 was exposed to the essential oil mouthrinse at a 1:10 dilution. Positive control cells were treated with Hanks balanced salt solution (HBSS). Aggregation was measured using a recording platelet aggregometer. The assay of each organism in its respective mouthrinse dilution(s) or HBSS was repeated 5 times. RESULTS: In all cases, the HBSS-treated organisms induced platelet aggregation, with mean(+/-S.E.) lag times of 12.30 (+/-1.36) min and 11.36 (+/-0.58) min for P. gingivalis and S. sanguis, respectively. In contrast, treatment with the essential oil mouthrinse completely inhibited the platelet aggregating activity of P. gingivalis and of S. sanguis exposed to the 1:1 mouthrinse dilution in all assays; the aggregating activity of S. sanguis treated with full-strength mouthrinse was completely inhibited in 4 of 5 assays, and inhibited by 75% in the 5th, for a mean inhibition of 95 +/- 1.5%. CONCLUSION: This study provides additional evidence that the essential oil mouthrinse can interfere with bacterial cell surface-associated activities which may have clinical relevance.     

Synergy between Tannerella forsythia and Fusobacterium nucleatum in biofilm formation

During dental plaque formation, the interaction of different organisms is important in the development of complex communities. Fusobacterium nucleatum is considered a 'bridge-organism' that facilitates colonization of other bacteria by coaggregation-mediated mechanisms and possibly by making the environment conducive for oxygen intolerant anaerobes. These studies were carried out to determine whether coaggregation between F. nucleatum and Tannerella forsythia is important in the formation of mixed species biofilms. Further, the role of BspA protein, a surface adhesin of T. forsythia, in coaggregation and biofilm formation was investigated. The results showed the development of synergistic mixed biofilms of F. nucleatum and T. forsythia when these bacteria were cocultured. The BspA protein was not involved in biofilm formation. Though BspA plays a role in coaggregation with F. nucleatum, presumably other adhesins are also involved. The synergistic biofilm formation between the two species was dependent on cell-cell contact and soluble components of the bacteria were not required. This study demonstrates that there is a positive synergy between F. nucleatum and T. forsythia in the development of mixed biofilms and that the cell-cell interaction is essential for this phenomenon.