长春市某小学7～12岁儿童福赛斯坦氏菌和中间普氏菌分布状态调查

目的 应用聚合酶链反应(PCR)法对儿童口腔内福赛斯坦氏菌(Tannerella forsythus,Tf)和中间普氏菌(Prevotella intermedia,Pi)进行检测,探讨检出结果与牙周临床指标之间的关系.方法 选取长春市自强小学151名7至12岁儿童为研究对象,选择右上颌中切牙和第一磨牙为被检牙位,取龈上菌斑、记录探诊出血(bleeding on probing,BOP)、探诊深度(probing depth,PD)、牙龈指数(gingival index,GI),应用PCR法对儿童龈上菌斑中的Tf和Pi进行检测.结果 儿童龈上菌斑中Tf、Pi的检出率分别为40.3%(1 18/293)、46.4%(136/293);6颊面Tf、Pi的检出率分别为55.2%(80/145)、53.8%(78/145)均显著高于(1)唇面的Tf和Pi的检出率[分别为25.7%(38/148)、39.2%(58/148)],差异有统计学意义(P＜0.01);Pi检出率随年龄增长呈逐渐减低趋势,Tf检出率在7～8岁组最高,其次为11～12岁和9～10岁组;BOP阳性部位的Tf、Pi检出率[分别为43%(35/81)、49%(40/81)]均高于BOP阴性部位二者的检出率[分别为37%(72/194)、45%(87/194)].在BOP阳性部位,随PD加深Tf检出率逐渐增高,特别是在PD 4 mm以上时Tf检出率明显增高(P＜0.01),Tf检出率与BOP阳性、PD增加呈正相关.结论 7～12岁儿童龈上菌斑中高频度分布着Tf和Pi;上颌前牙区与磨牙区菌丛构成不同,Tf、Pi在磨牙区检出率更高,Tf的检出率与PD呈正相关,Tf检出率在7～8岁组最高,Pi检出率随年龄增长呈减低趋势,儿童时期进行牙周病早期预防是非常必要的.

Abstract：

Objective To detect the presence of Tannerella forsythus(Tf)and Prevotella intermedia (Pi) using polymerase chain reaction(PCR) in the oral plaque samples from children and investigate the relationship between bacteria and clinical parameters. Methods A total of 151 children aged 7 to 12 years were selected from Changchun primary school. The supragingival plaque sample was collected from the mesiobuccal and labial surfaces of the right maxillary central incisor ( FDI 1 ) and the right maxillary first molar ( FDI 6 ). Extracted DNA from plaque samples was used for PCR analysis. Intraoral examination,probing depth (PD) and bleeding on probing (BOP) were performed and recorded. Results The detection rate for Tf was 40. 3% ( 118/293 ) and Pi was 46. 4% ( 136/293 ) in supragingival plaque. The detection rates for Tf and Pi in molars were much higher than those in incisors( P ＜0. 01 ). The detection rate of Tf and Pi was positively related to BOP + and PD. The detection rate for Pi decreased gradually with age, and the detection rate for Tf was highest in the group aged 7 to 8 and the detection rates for Tf and Pi were higher in the gingiva with BOP + than that with BOP - ( P ＞ 0. 05 ). The detection rates for Tf increased remarkably with BOP + and especially when PD was greater than 4 mm. Conclusions Detection rates of putative periodontal pathogens from healthy children of 7 to 12 years of age were high. The detection rates for Tf and Pi in molars were much higher than those in incisors, and the presence of Tf and Pi in supragingival plaque was related to periodontal parameters.     

Mixed infection of Porphyromonas gingivalis and Bacteroides forsythus in a murine abscess model: involvement of gingipains in a synergistic effect

Several microorganisms including Porphyromonas gingivalis and Bacteroides forsythus have been implicated to be etiologically important agents of periodontal disease. In this study, we determined the ability of combinations of periodontopathogenic microorganisms to cause tissue destruction in a murine abscess model. Although all bacterial combinations used in this study produced larger abscesses than did monoinfection of each bacterium, the combination of P. gingivalis and B.forsythus showed a synergistic effect on abscess formation. Since these two bacteria have been frequently found together in lesions of periodontitis, these results suggest the significance of their co-infection in the progression of periodontitis. P. gingivalis produces extracellular and cell-associated cysteine proteinases (gingipains) which appear to be involved in its virulence. The rgpA rgpB double and kgp mutants induced significantly smaller abscesses than the wild type. Moreover, the rgpA rgpB kgp triple (gingipain-null) mutant hardly showed lesion formation at all with the experimental conditions used in this study, indicating that these genes encoding gingipains are important for virulence of P. gingivalis. Mixed infection of these P. gingivalis mutants with B. forsythus showed an additive effect on abscess formation, indicating that the gingipains of P. gingivalis may play an important role in the pathological synergism between P. gingivalis and B. forsythus.     

Endpoint quantitative PCR assays for Bacteroides forsythus,Porphyromonas gingivalis,and Actinobacillus actinomycetemcomitans

BACKGROUND: Conventional polymerase chain reaction (PCR) assays for periodontal pathogens are so sensitive that they detect infections of no clinical significance. Quantitative PCR (qPCR) may provide a solution to this problem. However, most qPCR systems require expensive real-time thermal cyclers. OBJECTIVE: Our goal was to develop qPCR assays which would allow endpoint quantification. MATERIALS AND METHODS: 16S rRNA primers for Bacteroides forsythus, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans were adapted to the Amplifluor qPCR system, which incorporates fluorescein into the PCR product so that endpoint fluorescence is proportional to the original amount of template. DNA dilutions representing known numbers of cells were used as standard curves. Pooled subgingival plaques from the four deepest pockets of 21 severe adult periodontitis patients were assayed. Buccal molar supragingival plaque from 35 dental students provided healthy controls. Endpoint fluorescence was measured with a microplate reader. RESULTS: Optimized standard curves were linear in log-log or semilog fits over a range of 100-10(6) cells. Countable B. forsythus was present in all patients, with counts (as logs) from 2.4 to 7.3 (mean = 5.0), and 11 controls with counts from 2.1 to 4.5 (mean = 3.0). P. gingivalis was present in 11 patients and no controls, with counts from 2.2 to 4.7 (mean = 3.2). A. actinomycetemcomitans was present in two patients, with counts of 1.5 and 3.5. CONCLUSIONS: Amplifluor qPCR assays discriminated between plaque samples differing by one log or more, allowing major infections to be distinguished from minor ones. This approach allows high-throughput qPCR of plaque samples, using equipment available to many laboratories.     

PCR detection of 5 putative periodontal pathogens in dental plaque samples from children 2 to 12 years of age

BACKGROUND, AIMS: The purpose of this study was to detect the presence of Prevotella intermedia, P. nigrescens, Bacteroides forsythus, Treponema denticola, and Campylobacter rectus in plaque samples from 119 children, collected from their toothbrushes using a polymerase chain reaction (PCR). METHOD: The subjects were 24, 83, and 12 children with healthy gingiva, gingivitis, and periodontitis, respectively, ranging in age from 2-12 years old. Plaque samples were collected from all erupted teeth sites using a sterile toothbrush. The mean concentration of DNA recovered from the plaque samples was approximately 640 microg/ml, which was deemed sufficient for performing a PCR-based survey. RESULTS: The prevalence by PCR in healthy, gingivitis, and periodontitis subjects was 0.0%, 6.0% and 25.0% for P. intermedia, 45.8%, 79.5% and 50.0% for P. nigrescens, 33.3%, 63.9% and 58.3% for B. forsythus, 0.0%, 18.1% and 16.7% for T. denticola, and 100% in duplicate for C. rectus, respectively. CONCLUSION: Our survey indicated that P. intermedia and T. denticola were more associated with periodontal diseases, B. forsythus and P. nigrescens had a moderate prevalence in all clinical groups, while C. rectus were the most commonly detected species in the oral cavities of children suggesting establishment in their early years.     

Prevalence of Bacteroides forsythus, Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans in subgingival microflora of Japanese patients with adult and rapidly progressive periodontitis

BACKGROUND, AIMS: This study investigated the prevalence of Bacteroides forsythus, Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans among various periodontitis patients and healthy individuals in Japan, and correlated it with clinical parameters. METHOD: Subgingival plaque samples were collected from 21 patients with adult periodontitis (AP), 8 with rapidly progressive periodontitis (RPP) and 15 healthy individuals. RESULTS: The frequency detected in culture was as follows: B. forsythus was found in 47.6% of AP sites and in 37.5% of RPP sites. P. gingivalis was identified in 64.3% of AP and 59.4% of RPP sites. A. actinomycetemcomitans was detected in 4.8% of AP and 3.1% of RPP sites. The 3 species were detected in only 2 of the healthy individuals. The proportion of B. forsythus in the total microflora in culture was 0.07% in the healthy group, 4.1% in AP and 2.4% in RPP. The proportions of P. gingivalis were 0% in the healthy group, 18.8% in AP and 16.2% in RPP. The proportion of A. actinomycetemcomitans was very low in all 3 groups. A DNA probe detected B. forsythus in 78.6% of AP and 65.6% of RPP sites, as well as P.gingivalis in 58.3% of AP and 59.4% of RPP sites. A. actinomycetemcomitans was detected in only 1.2% of AP sites. The 3 species were undetectable in the healthy group. CONCLUSIONS: The prevalence and the proportion of B. forsythus and P. gingivalis were significantly correlated with clinical parameters, suggesting that B. forsythus and P. gingivalis are closely related to AP and RPP in the Japanese population.     

Bacteroides forsythus prtH genotype in periodontitis patients: occurrence and association with periodontal disease

Bacteroides forsythus has been described as a periodontopathogen and its presence in the subgingival plaque can lead to periodontal disease. Recently, a cysteine protease designated as prtH was isolated and characterized from B. forsythus ATCC 43037. The purpose of this study was to determine the prevalence and the association of the prtH gene of B. forsythus with periodontal disease. A total of 160 subgingival plaque samples were assayed with the polymerase chain reaction method using oligonucleotide primers targeting the prtH and the 16S rDNA genes of B. forsythus. Primers targeting the 16S rDNA gene of B. forsythus were used to determine the occurrence of the bacteria in the subgingival plaque samples at baseline. At baseline, B. forsythus was detected in 78 out of 86 (91%) diseased sites and 33 out of 74 (45%) healthy sites studied. Among the 86 diseased sites examined, 73 sites (85%) were colonized by the bacteria with the prtH genotype. In sites of the periodontally healthy, 7 out of 73 (10%) possessed B. forsythus with the prtH genotype. The results obtained suggested strong association of the prtH gene of B. forsythus with adult periodontitis. Although this bacterial species was detected from about half of the periodontally healthy samples, only a fraction of these subjects possess the bacteria strain with the prtH genetic subtype. We propose the use of the prtH gene as an alternative to the more widely used 16S rDNA gene of B. forsythus, for a more accurate determination of the prevalence of periodontal health and disease in epidemiological studies and clinical screening.     

Bacteroides forsythus ATCC43037菌体蛋白与人类唾液酸性富脯蛋白的相互作用

目的：探讨福赛类杆菌与人类唾液富脯蛋白相互作用的蛋白分子。方法：Western—blot方法。将人工合成唾液富脯蛋白用生物素标记，福赛类杆菌全菌蛋白凝胶电泳，半干转移至纤维膜上，观察二者的相互作用。结果：富脯蛋白能与分子量为85KD、65KD、60KD、以及49KD的福赛类杆菌蛋白发生结合。结论：福赛类杆菌存在与人类唾液富脯蛋白相互结合的粘附素。     

Porphyromonas gingivalis, Prevotella intermedia and Bacteroides forsythus in plaque subjacent to bridge pontics

Abstract. This study examined the distribution of P. gingivalis, P. intermedia and B. forsythus in plaque on metallic and porcelain pontics adjacent to healthy and inflamed mucosa. Subpontic plaque was collected from 33 inflamed and 31 healthy sites. Plaque suspension was incubated with specific rabbit antisera to P. gingivalis (FDC 381), P. intermedia (ATCC 25261) and B. forsythus (FDC 335), and the labelled cells disclosed with fluorescein-labelled goat-anti-rabbit IgG by indirect immunofluorescence microscopy. Mean proportions of P. gingivalis, P. intermedia , and B. forsythus at inflamed sites were 0.60±0.75, 2.48±2.28, and 0.44±0.64% respectively, and at healthy sites 0.21±0.43, 1.27±1.05, and 0.15±0.18% respectively. These differences were statistically significant. Almost all sites were positive for P. intermedia , whereas only 12/31 healthy and 21/33 inflamed sites were positive for P. gingivalis. 18/31 healthy and 28/33 inflamed sites were positive for B. forsythus. P. intermedia was recovered in higher proportions from metallic pontics adjacent to inflamed sites (MI) than healthy sites (MH) or porcelain pontics next to inflamed (PI) or healthy sites (PH), P. gingivalis is was recovered in higher proportions from MI than PH. We conclude that both the nature of the pontic material and the health status of the mucosa affect the composition of the associated microbiota.     

Interbacterial binding among strains of pathogenic and commensal oral bacterial species

Strong interspecies adherence was demonstrated among the periodontal pathogens Treponerna denticola, Bacteroides forsythus and Porphyromonas gingivalis , and between these pathogens and the commensal plaque organism Streptococcus crista. Adherence showed specificity and demonstrated saturation binding kinetics. Binding between B. forsythus and P. gingivalis appeared to be a unimodal protein-protein interaction. Binding between the other organisms was at least bi-modal involving interactions between combinations of proteins and carbohydrates with a variety of sugar specificities. Salivary molecules prevented adherence between T. denticola and S. crista , and serum eliminated B. forsythus binding to P. gingivalis. All other interactions occurred to some degree in the presence of serum and saliva. Such interbacterial binding interactions may be important in the establishment of periodontopathic plaque.     

The prevalence of BANA-hydrolyzing periodontopathic bacteria in smokers

Smoking has been identified as a risk factor for development of periodontal disease and a strong indicator for treatment failure in periodontal patients. This study examined 172 patients categorized as current smokers (n=55), previous smokers (n=38) or individuals that had never smoked (n=79). A total of 670 interproximal plaques collected with a wooden toothpick were analyzed for hydrolysis of the synthetic trypsin substrate benzoyl-DL-arginine naphthylamide (BANA). About 95% of the BANA hydrolysis by plaque is due to the presence of one or more of the periodontopathogens, P. gingivalis, T. denticola or B. forsythus. Gingival health was measured using the papillary bleeding score (PBS). Current smokers had less gingival bleeding than previous smokers or those who had never smoked (20% versus 41% and 25%, respectively). Plaque removed from non-bleeding sites in current smokers were 11x more likely to have a positive BANA reaction when compared to plaque removed from non-bleeding sites in individuals who never smoked. A significant positive relationship exists between smoking and colonization by the BANA periodontopathogens. Smoking may select for these periodontopathic species in the plaque and may be one reason why smoking is a risk factor in periodontal disease development.