Trypsin-like activity levels of Treponema denticola and Porphyromonas gingivalis in adults with periodontitis

Abstract Treponema denticola (Td) and Porphyromonas gingivalis (Pg) are associated with human moderate and severe adult periodontal diseases. This study Quantifies these two anaerobes and their trypsin-like (TL) activities in subgingival plaque collected from both clinically healthy and periodontally diseased sites of human periodontitis patients. Antigen levels of the microorganisms were determined by monoclonal antibodies and TL activities were measured by the fluorescent substrate Z-gly-gly-arg-AFC in a disc format. Significant positive correlations were observed between the antigen levels and the TL activities when the data were subjected to statistical analyses both on a site-specific and on a patient basis. Anaerobe synergism was found between Td and Pg in a continental US population, and positive correlations were found between anaerobe levels (individually and total) and clinical indicators of adult periodontitis.     

Virulence of a polymicrobic complex,Treponema denticola and Porphyromonas gingivalis,in a murine model

The effect of a polymierobic infection employing Treponema denticola and Porphyromonas gingivalis in the murine lesion model was used to determine the synergistic virulence of these two periodontopathic bacteria. At high doses of P. gingivalis W50, addition of T. denticola in the infection mixture had no effect on the formation and size of the spreading lesion caused by this microorganism. However, at low P. gingivalis challenge doses, T. denticola significantly enhanced the virulence of P. gingivalis compared with monoinfection of this microorganism. A potential role of the trypsin-like protease enzyme activity of P. gingivalis in this synergistic virulence was tested using P. gingivalis mutants deficient (i.e., BEI) or devoid (i.e., NG4B19) of this protease activity. These findings demonstrated that T. denticola—P. gingivalis complexes exhibit enhanced virulence in this model and that even using a polymicrobic challenge infection, the trypsin-like protease activity was important to P. gingivalis virulence expression.     

Bacterial synergy of Treponema denticola and Porphyromonas gingivalis in a multinational population

Treponema denticola and Porphyromonas gingivalis have been associated with human adult severe periodontitis. In this study, we quantified these putative pathogens in subgingival plaque samples collected from 74 Fijians, 74 Colombians and 73 U.S. Americans stationed at the Multinational Force and Observers encampment in the Sinai Desert, Egypt. A contingency table of T. denticola and P. gingivalis frequency revealed a highly significant synergistic relationship. We discovered that the occurrence of T. denticola apparently requires the presence of P. gingivalis. This represents the first observation of a synergistic relationship between these putative oral pathogens associated with adult severe periodontal disease.     

齿垢密螺旋体在慢性牙周炎龈下菌斑中的分布

目的:采用聚合酶链反应(PCR)技术,检测慢性牙周炎患者患病部位和相对健康部位龈下菌斑中齿垢密螺旋体(Treponema denticola,Td),了解齿垢密螺旋体在不同部位龈下菌斑中的分布情况,并探讨其检出率与慢性牙周炎的关系。方法:选择58例慢性牙周炎患者患病部位和相对健康部位龈下菌斑,对齿垢密螺旋体tdpA基因片段进行扩增和克隆测序。结果:34例(58.6%)患病部位PCR为阳性,相对健康部位中5例(8.62%)PCR为阳性,患病部位齿垢密螺旋体检出率高于相对健康部位(P<0.001)。对tdpA基因克隆后测序,结果与B last比对与Gen Bank中已登陆的tdpA基因同源性为94%。结论:在慢性牙周炎患者患病部位龈下菌斑中,齿垢密螺旋体检出率高于相对健康部位,与慢性牙周炎关系密切,利用PCR方法检测Td,简便、特异性高。     

Demonstration of a bimodal coaggregation reaction between Porphyromonas gingivalis and Treponema denticola

This study demonstrates a strong coaggregation reaction between 2 suspected periodontopathogens: Porphyromonas gingivalis and Treponema denticola. Other black-pigmented oral bacterial species tested did not coaggregate with T. denticola. This specific interbacterial aggregation was bimodal, since heating of both cell types was required to completely eliminate the reaction. The coaggregation reaction occurred between pH 4 and 9. Under some conditions, arginine and D-galactosamine were effective in preventing the coaggregation. The heat-sensitive receptor on P. gingivalis was found to be loosely bound and could be released by a light ultrasonic treatment of the cells. It is suggested that the bacterial interaction described may participate in the establishment of a potentially pathogenic subgingival plaque.     

Distribution of Porphyromonas gingivalis and Treponema denticola in human subgingival plaque at different periodontal pocket depths examined by immunohistochemical methods

Localization of Porphyromonas gingivalis and Treponema denticola in different areas of subgingival plaque from advanced adult periodontitis patients was studied immunohistochemically using sensitive immunogold-silver staining and immunoelectron microscopy. Fourteen periodontally diseased teeth were extracted without damaging the subgingival plaque, fixed, and embedded. The subgingival plaque samples were sectioned according to four different pocket depths (0–2 mm, 2–4 mm, 4–6 mm and ≤6 mm). Serial thin sections were stained using specific antisera to P. gingivalis or T. denticola and then with secondary antibody labelled with colloidal-gold. Cells of both P. gingivalis and T. denticola were predominantly found in subgingival plaque located at depths of more than 4 mm in periodontal pockets. T. denticola cells were found in the surface layers of subgingival plaque, and P. gingivalis were predominant beneath them. However, in the deeper subgingival plaque, the coexistence of P. gingivalis and T. denticola was observed. The present findings suggest that P. gingivalis and T. denticola play important roles in the pathogenicity of periodontal disease and provide the useful information for elucidating the pattern of colonization of microorganisms in the periodontal pocket.     

Immunization of Macaca fascicularis against experimental periodontitis using a vaccine containing cysteine proteases purified from Porphyromonas gingivalis

INTRODUCTION: Periodontitis is a common infectious disease to which Porphyromonas gingivalis has been closely linked, in which the attachment tissues of the teeth and their alveolar bone housing are destroyed. We conducted a study to determine if immunization using a purified antigen could alter the onset and progression of the disease. METHODS: Using the ligature-induced model of periodontitis in Macaca fascicularis, we immunized five animals with cysteine protease purified from P. gingivalis and used an additional five animals as controls. Alveolar bone loss was measured by digital subtraction radiography. RESULTS: Immunization induced high titers of specific immunoglobuin G serum antibodies that were opsonic. Total bacterial load, levels of P. gingivalis in subgingival plaque and levels of prostaglandin E(2) in gingival crevicular fluid were significantly reduced. Onset and progression of alveolar bone loss was inhibited by approximately 50%. No manifestations of toxicity were observed. CONCLUSIONS: Immunization using a purified protein antigen from P. gingivalis inhibits alveolar bone destruction in a ligature-induced periodontitis model in M. fascicularis.     

牙龈卟啉单胞菌PG1055基因在临床菌株中的表达

目的应用基因芯片技术检测PG1055基因在不同人群的牙龈卟啉单胞菌(P.gingivalis)中分布,探讨这些基因与牙周临床指数之间的关系。方法取龈下菌斑进行细菌分离培养,以临床采集样本提取的DNA为探针,以抑制消减杂交技术获得P.gingivalisW83的特异基因片段PG1055为目标序列,采用Cy5荧光标记目标序列。应用基因芯片技术检测PG1055基因在牙周病患者及健康人群的牙龈卟啉单胞菌中的分布。结果PG1055基因在牙周病患者及健康人群中的检出率有统计学差异,并且与牙周临床指数相关。结论PG1055基因与P.gingivalis的致病性有关。     

Antibody reactive with Porphyromonas gingivalis hemagglutinin in chronic and generalized aggressive periodontitis

BACKGROUND: Porphyromonas gingivalis, a black-pigmented, gram-negative anaerobe, is found in periodontitis lesions and its presence in subgingival plaque significantly increases the risk for periodontitis. We have previously shown that patients with aggressive forms of periodontitis that are seropositive for P. gingivalis have less attachment loss than those that are seronegative. This suggests that antibody reactive with antigens of P. gingivalis may be protective and decrease disease severity and extent. Recent studies in the murine abscess model and in the host antibody response in chronic periodontitis patients suggest that antibody reactive with P. gingivalis hemagglutinin may be an important protective antibody response. OBJECTIVES: In this study, we tested the hypothesis that there was a significant relationship between antibody reactive with P. gingivalis hemagglutinin and measures of periodontal attachment loss. METHODS: We determined the immunoglobulin G (IgG) antibody concentration reactive with recombinant P. gingivalis hemagglutinin in 117 chronic periodontitis and 90 generalized aggressive periodontitis patients. We also determined the IgG subclass distribution for antibody reactive with P. gingivalis hemagglutinin. RESULTS AND CONCLUSIONS: We found IgG reactive with P. gingivalis hemagglutinin in both chronic periodontitis and generalized aggressive periodontitis patients. Most of this IgG antibody was of the IgG1 and IgG3 subclasses. Antibody reactive with P. gingivalis hemagglutinin, however, did not have a significant relationship with measures of periodontal attachment loss.     

慢性牙周炎栖牙密螺旋体和牙周袋内硫化物水平的相关性研究

目的 研究慢性牙周炎病人牙周袋内栖牙密螺旋体和牙周袋内硫化物水平的关系。方法 临床上选取17例诊断为慢性牙周炎的病人,采用金刚探针/牙周诊断仪检测牙周袋内硫化物水平,记录牙周袋探诊深度、临床附着丧失以及探诊出血相关牙周指标。同时,采用16S rRNA PCR检测相同位点的栖牙密螺旋体。结果 慢性牙周炎病人栖牙密螺旋体检出率为88.2%,硫化物阳性位点和硫化物阴性位点中栖牙密螺旋体的检出率分别为68.5%和43.2%。硫化物阳性位点与阴性位点中牙周附着丧失（clinical attachment loss, CAL）平均值分别为（2.84±2.33）mm和（1.83±1.60）mm,两者差异有统计学意义（P<0.01）。硫化物阳性位点与阴性位点牙周探诊深度（probing depth, PD）平均值分别为（4.20±1.57）mm和（3.83±1.30）mm,两者差异无统计学意义(P＞0.05)。硫化物阳性位点中牙周探诊出血（bleeding on probing, BOP）阳性检出为率92.5%,大于硫化物阴性位点（75.8%）,两者差异有统计学意义（P<0.01）。结论 慢性牙周炎病人牙周袋内的硫化物水平能反映牙周栖牙密螺旋体分布情况,与牙周附着丧失存在相关性。     

Progression of chronic periodontitis can be predicted by the levels of Porphyromonas gingivalis and Treponema denticola in subgingival plaque

Introduction:  Chronic periodontitis is an inflammatory disease of the supporting tissues of the teeth associated with bacteria. Diagnosis is achieved retrospectively by clinical observation of attachment loss. Predicting disease progression would allow for targeted preventive therapy. The aim of this study was to monitor disease progression in patients on a maintenance program and determine the levels of specific bacteria in subgingival plaque samples and then examine the ability of the clinical parameters of disease and levels of specific bacteria in the plaque samples to predict disease progression.
Methods:  During a 12-month longitudinal study of 41 subjects, 25 sites in 21 subjects experienced disease progression indicated by at least 2 mm of clinical attachment loss. Real-time polymerase chain reaction was used to determine the levels of Porphyromonas gingivalis , Treponema denticola , Tannerella forsythia , Fusobacterium nucleatum , and Prevotella intermedia in subgingival plaque samples.
Results:  No clinical parameters were able to predict periodontal disease progression. In sites undergoing imminent periodontal disease progression within the next 3 months, significant partial correlations were found between P. gingivalis and T. forsythia ( r  = 0.55, P  < 0.001) and T. denticola and T. forsythia ( r  = 0.43, P  = 0.04). The odds of a site undergoing imminent periodontal disease progression increased with increasing levels of P. gingivalis and T. denticola .
Conclusion:  Monitoring the proportions of P. gingivalis and T. denticola in subgingival plaque has the potential to help identify sites at significant risk for progression of periodontitis, which would assist in the targeted treatment of disease.     

K-antigens in Porphyromonas gingivalis are associated with virulence

We investigated antigens in spreading and non-spreading Porphyromonas gingivalis strains. On the basis of differences in virulence in the mouse model, 8 strains were selected for antiserum production in rabbits. Hyperimmune sera were tested by double immunoprecipitation and immunoelectrophoresis. Besides a common antigen, differences in antigenic composition were observed in the thermolabile antigens between all strains tested. Two different heat-stable antigens were found after heating at 120°C. One such antigen was detected after sonication of the pellet fraction of autoclaved P. gingivalis cells. This antigen cross-reacted with 6 of the 8 immune sera. This somatic antigen was almost neutrally charged and sensitive to sodium periodate treatment, suggestive of lipopolysaccharide. A second heat-stable antigen was detected in the supernatant of autoclaved strains of W 83, W50, HG 184 and A7A1-28. These non-somatic antigens were strain-specific, i.e., no cross-reactivity was found with heterologous hyperimmune sera. An exception was strain W50, which had a non-somatic heat-stable antigen which was recognized by W83 antiserum. These antigens were resistant to DNAse, RNAse and proteinase-K treatment but were degraded by sodium periodate. In immunoelectrophoresis, these antigens appeared to be negatively charged. These properties are characteristics of a K-antigen, which likely represent a thermostable carbohydrate capsule. The presence of K-antigen correlates very well with the serum resistance, the low chemiluminescence, the resistance to phagocytosis and the need for opsonization with specific antibodies for complement-mediated killing of virulent P. gingivalis strains. We propose to designate these antigens in P. gingivalis W83 and W50 as K1, in HG 184 as K2 and in A7A1-28 as K3.     

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目的研究能够快速建立稳定、可靠、重复性好的大鼠实验性牙周炎模型的方法,摸索建模的最佳时间并对比研究结扎与非结扎区域的牙槽骨破坏情况。方法本研究于2010年7月至2011年1月在中国医科大学基础医学院微生物学实验室及口腔医学院中心实验室完成。将18只SD大鼠随机分为对照组和4周、8周实验组。采用牙颈部结扎合并口腔接种牙周致病菌的方法,建立大鼠实验性牙周炎模型。分别于4周、8周时进行牙周检查,并利用放射影像和体视显微镜检查对比大鼠的牙周骨组织破坏情况。结果4周和8周实验组大鼠结扎区都出现牙龈红肿,探诊出血,均可探及较深的牙周袋。对照组和4周、8周实验组大鼠上颌三颗磨牙平均骨丧失量分别为（357．63±284．96）μm、（929．56±366．43）μm和（941．80±354．87）μm。与对照组相比,4周和8周实验组大鼠上颌磨牙的骨丧失量均显著增加,差异有统计学意义（P〈0．05）,但4周与8周实验组之间差异无统计学意义（P〉0．05）。结论采用牙颈部结扎合并口腔接种牙周致病菌的方法,4周左右即可形成稳定可靠的大鼠牙周炎模型。大鼠的牙周骨破坏在4周之后速度减缓。     

Confounding and interaction effect of Treponema denticola salivary carriage in chronic periodontitis

Oral Diseases (2010) 16 , 278–285 Aim: To evaluate the salivary carriage of Treponema denticola and its association with demographic variables in the etiopathogenesis of chronic periodontitis. Subjects and methods: Ninety‐seven chronic periodontitis (CP) patients and a control group of 51 healthy subjects (HC) were selected. Periodontal status was assessed by criteria based on probing depth, attachment loss, extent, and severity of periodontal breakdown. A polymerase chain reaction method was used to determine the occurrence of T. denticola in saliva samples. Risk indicators for CP were assessed individually and adjusted for confounding and/or interaction using a logistic regression model. Results: Although univariate analysis revealed a positive association of age ≥30 years, smoking, and salivary carriage of T. denticola with CP, after logistic regression analysis, the association between age ≥30 years/smoking and CP persisted, whereas salivary carriage of T. denticola failed to achieve statistical significance. An interaction effect was significantly detected between these three variables. Conclusion: Although salivary carriage of T. denticola may be a risk indicator for CP, its pathogenicity should not be exclusively endorsed to its detection in saliva, but it might be associated with the synergistic biological interaction of the bacterium with some demographic characteristics of the susceptible host.     

牙龈卟啉单胞菌PG0717基因与慢性牙周炎的相关性研究

目的检测慢性牙周炎患者和牙周健康者龈下菌斑中牙龈卟啉单胞菌（P.gingivalis）PG0717基因,探讨PG0717基因与牙周临床指数之间的关系。方法选取慢性牙周炎（CP）患者90例和牙周健康者90例,共采集龈下菌斑标本540个;记录临床牙周指数（牙周探诊深度、临床附着丧失和探诊出血）;设计特异性引物检测P.gingivalis阳性龈下菌斑标本的PG0717基因。结果在P.gingivalis阳性龈下菌斑中,CP组PG0717基因检出率显著高于对照组,分别为56.22%和41.27%（掊2=4.50,P＜0.05）;随着牙周探诊深度、临床附着丧失加重和探诊出血趋势的增加,CP组该基因检出率呈现增高趋势。结论PG0717基因与P.gingivalis的致病性有关。     

Serum IgA and IgG antibodies to Treponema vincentii and Treponema denticola in adult periodontitis, juvenile periodontitis and periodontally healthy subjects

13 patients with untreated adult periodontitis (AP) were compared to 8 subjects free of periodontal disease (H) with respect to plaque index (PlI), gingival index (GI), probing depth (PD) and differential counts of subgingival bacterial morphotypes from a pooled sample of 6 surfaces with the greatest probing depth. Serum antibody levels to T. vincentii and T. denticola strains were also determined in these subjects as well as in the sera from 5 subjects with localized juvenile periodontitis (LJP). Subjects with AP had significantly elevated proportions of spirochetes and motile rods and lower proportions of coccoid cells than H subjects. They also exhibited significantly higher PlI and GI scores and greater probing depths. Antibody levels were normalized against a standard serum and expressed as ELISA units (EU). IgA and IgG antibody levels to all tested spirochete strains were significantly elevated in AP subjects as compared to subjects in group H or subjects with LJP. No significant differences in antibody titers were detectable between the H and LJP groups with respect to any of the tested strains. No significant correlation could be demonstrated between serum antibody titers to any of the oral spirochete strains tested and the proportions of oral spirochetes determined microscopically.     

牙龈卟啉单胞菌与动脉粥样硬化发病的关系

慢性牙周炎是心血管疾病特别是冠心病的危险因素之一,然而其相关的生物学基础目前尚不清楚.笔者下面就与慢性牙周炎关系最为密切的牙龈卟啉单胞菌与动脉粥样硬化的相关性和牙龈卟啉单胞菌致动脉粥样硬化的可能机制以及Toll样受体在牙龈卟啉单胞菌与动脉粥样硬化相关性中的作用作一综述.