不同fimA基因型牙龈卟啉单胞菌在青春期龈炎患者中的分布

目的:分析不同fimA基因型牙龈卟啉单胞菌(Porphyromonas gingivalis,P.gingivalis)在青春期龈炎患者和青春期牙周健康者中的分布。方法:收集51例青春期龈炎患者和46例青春期牙周健康者的龈下菌斑,采用16SrRNA PCR检测P.gingivalis,并根据各fimA基因型的特异引物,用PCR检测不同fimA基因型菌株的分布。结果:龈炎组各fimA基因型P.gingivalis的总检出率:Ⅰ型34.2%,Ⅱ型55.3%,Ⅳ型18.4%,Ⅲ型和Ⅴ型未检出;另有7例(占18.4%)未分出型别。健康组各fimA基因型P.gingivalis的总检出率:Ⅰ型14.3%,Ⅱ型85.7%,Ⅲ型14.3%,Ⅳ型28.6%,Ⅴ型未检出;另有2例(占14.3%)未分出型别。结论:青春期龈炎和青春期牙周健康者龈下菌斑中的P.gingivalis存在fimA基因多态性。fimA基因Ⅱ型是其主要存在的基因型,其次是Ⅰ型和Ⅳ型,Ⅲ型和Ⅴ型较少或不能检出。     

龈下菌斑中牙龈卟啉单胞菌牙龈素基因片段的检测

目的：检测慢性牙周炎患者龈下菌斑中牙龈卟啉单胞菌（Porphyromonas gingivalis,P.gingivalis）的2个基因片段kgp-cd和rgpB-cd,探讨2个基因片段的存在和缺失与牙周临床指标之间的关系.方法：选择慢性牙周炎患者龈下菌斑84个,对P.gingivalis阳性的龈下菌斑样本进行kgp-cd和rgpB-cd基因片段检测;根据2个基因片段的有无,将P.gingivalis分为A型和B型,采用SPSS11.5统计软件包,用t检验和x2检验分析不同基因型P.gingivalis与牙周临床指标的关系.结果：A型P.gingivalis在慢性牙周炎中的检出率高于B型（P＜0.05）,分别为85.29%和14.71%.不同基因型P.gingivalis引起的牙周袋探诊深度和牙龈出血倾向存在显著性差异（PD：t=2.85,P＜0.05;BOP：P＜0.05）.结论：kgp-cd和rgpB-cd基因与P.gingivalis的致病性有关.     

不同牙周状态下龈下菌斑中牙龈卟啉单胞菌kgp基因差异研究

目的：探讨不同kgp基因型P.gingivalis在慢性牙周炎发生发展中的作用。方法：选择不同牙周状态下的龈下菌斑样本104个,根据kgp—cd基因序列的不同用限制性片断长度多态性分析的方法将P．gingivalis分为4个基因型。结果：慢性牙周炎患者病变重部位和病变轻部位检出率最高的分别为kgpcdⅡ型（56．25％）和Ⅰ型（51．72％）,对照组仅检出kgp--cdJ型（25％）和Ⅲ型（75％）。结论：kgpcdⅡ型P．gingivalis可能与慢性牙周炎的关系最密切,在致病过程中起主要作用,而kgp—cdⅠ型可能与慢性牙周炎无关,为健康人群或牙周健康部位的定置菌。     

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。     

牙龈卟啉单胞菌的牙龈蛋白酶K活性与青春期龈炎的临床相关性

目的：检测并比较青春期龈炎患者的龈下菌斑中牙龈卟啉单胞菌（P.gingivalis）临床分离株的分泌蛋白和菌体蛋白中牙龈蛋白酶K（Kgp）的活性,探讨Kgp在青春期龈炎中的临床意义。方法：受试对象为36例14～17岁青春期龈炎患者,检测并记录受检者的各牙周临床指标GI、SBI、PD的测值,取龈下菌斑进行P.gingivalis的分离培养,16S rRNA聚合酶链反应（PCR）法鉴定。将P.gingivalis临床分离株复苏,在对数生长期末提取分泌蛋白和菌体蛋白,用N-p-硝基苯胺乙酸盐分析Kgp的氨基酸溶解活性。采用SPSS11.0软件包,分泌蛋白与菌体蛋白的Kgp活性比较用t检验;Kgp活性与各牙周临床指标之间的相关关系用秩相关检验分析,P〈0.05具有显著性差异。结果：青春期龈炎的P.gingivalis临床分离株的分泌蛋白中Kgp的活性高于菌体蛋白（P〈0.01）,Kgp的活性与GI、SBI、PD之间有正相关关系,统计学上有显著性差异（P〈0.05）。结论：Kgp酶活性的高低与青春期龈炎的严重程度有关。     

维吾尔族慢性牙周炎中牙龈卟啉单胞菌菌fimA毒力基因型的分布

目的：分析维吾尔族慢性牙周炎患者龈下菌斑中牙龈卟啉单胞菌菌毛fimA毒力基因型的分布情况。方法：收集52例维吾尔族慢性牙周炎患者的龈下菌斑，采用16SrRNA PCR法检测牙龈卟啉单胞菌，并根据菌毛fima毒力基因型的特异引物，用聚合酶链反应（PCR）检测Ⅱ型fimA和Ⅳ型fima菌株的分布。结果：16SrRNA PCR法检测牙龈卟啉单胞菌在龈下菌斑中阳性检出率是76．9％。牙周袋PPD〉6mm位点龈下菌斑标本的P．gingivalis检出率高于4〈PPD≤6mm采样的位点，2组差异有统计学意义（P〈0．05）。牙龈卟啉单胞菌菌毛．fimA毒力基因型在牙龈卟啉单胞菌感染者的检出率分别是：Ⅱ fimA型为37．5％，ⅣfimA型为22．5％。结论：维吾尔族慢性牙周炎患者龈下菌斑中牙龈卟啉单胞菌有较高的检出率。牙龈卟啉单胞菌存在fima毒力基因多态性。     

成人牙周健康状况与fimA基因型牙龈卟啉单胞菌的相关性

目的分析不同fimA基因型牙龈卟啉单胞菌（P.gingivalis）在牙周健康人群和慢性牙周炎人群中的分布,探讨不同fimA基因型P.gingivalis与成人牙周状况的相关关系。方法收集牙周健康组（136例）和慢性牙周炎组（115例）的龈下菌斑样本,采用16S rRNA PCR法检测P.gingivalis,并根据各fimA基因型（Ⅰ～Ⅴ和Ⅰb）的特异性引物检测不同fimA基因型P.gingivalis菌株的分布,计算OR值和95%可信区间。结果牙周健康组和慢性牙周炎组龈下菌斑样本中P.gingivalis阳性率分别为22.1%和81.7%,多数样本中只检测到1种fimA基因型。牙周健康组中ⅠfimA型的检出率最高（占66.7%）;慢性牙周炎组中则为ⅡfimA基因型（占43.6%）,其次为Ⅳ和Ⅰb fimA基因型。慢性牙周炎的发生与P.gingivalis的关系密切（OR=16.36）,Ⅰ、Ⅰb、Ⅱ、Ⅲ、Ⅳ、ⅤfimA基因型P.gingivalis与慢性牙周炎相关性的OR值分别为0.97、13.26、36.62、4.57、22.86、1.19;ⅡfimA基因型P.gingivalis与慢性牙周炎的相关性最强,其次为Ⅳ和Ⅰb型。结论P.gingivalis菌株的fimA基因型存在差异,特异性fimA基因型P.gingivalis可能与成人慢性牙周炎的发生关系密切。     

青春期龈炎龈下菌斑中牙龈卟啉单胞菌的检测

目的:检测青春期龈炎患者龈下菌斑中牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)的存在情况,探讨青春期Pg与牙龈健康状况的关系。方法:14～17岁青春期龈炎患者及牙龈健康者各36例,采用16SrRNA聚合酶链反应(polymerase chain reaction,PCR)检测2组受检者的龈下菌斑样本中Pg的存在,并用电泳凝胶成像分析软件检测各电泳条带的平均灰度值,计算Pg的相对含量;同时检测并记录受检者的各牙周指数测值,观察其与Pg相对含量的相关性。数据用SPSS11.5软件包作统计学分析。2组Pg阳性检出率的比较采用χ2检验;Pg的相对含量以及Pg阳性检出者与阴性检出者的牙龈炎严重度的比较采用Wilcoxon秩和检验;Pg的相对含量与各牙周指数之间的相关关系采用Spearman相关分析。结果:青春期龈炎组与牙龈健康组的龈下菌斑中Pg的阳性检出率分别为47.22%和25.00%,两者之间具有显著性差异(P<0.05);青春期龈炎组与牙龈健康组的Pg在龈下菌斑中的平均相对含量分别为48.02%和21.46%,统计学上有高度显著性差异(P<0.01);在青春期龈炎组Pg阳性个体中,Pg的相对含量与牙龈指数(gingivalindex,GI)、龈沟出血指数(sulcusbleedingindex,SBI)、探诊深度(probedepth,PD)数值的高低呈正相关;青春期龈炎组中,Pg阳性检出者的GI、SBI数值高于阴性检出者,两者之间具有高度显著性差异(P<0.01)。结论:青春期个体的龈下菌斑中有Pg的定植,且与牙龈健康状况有密切关系。     

不同fimA基因型牙龈卟啉单胞菌在牙周健康人群中的分布

目的调查不同fimA基因型牙龈卟啉单胞菌 (P.gingivalis)在牙周健康人群中的分布情况。方法收集136例牙周健康者的龈下菌斑样本,采用16S rRNAPCR法检测P.gingivalis;并根据各fimA基因型特异性引物,用PCR法检测不同fimA基因型P.gingivalis的分布。 结果136例牙周健康者的龈下菌斑样本中携带P.gingivalis的阳性率为22.1%。大多数受检者龈下菌斑中只检测到1种fimA基因型 P.gingivalis菌株(80.0%);5例样本检出了2种fimA型P.gingivalis(16.7%),且均为ⅠfimA型与其它fimA 型P.gingivalis的联合检出。各fimA型P.gingivalis的检出情况:Ⅰ型(66.7%)、Ⅰb型(6.7%)、Ⅱ型(6.7%)、 Ⅲ型(10%)、Ⅳ型(6.7%)、Ⅴ型(16.7%)。Ⅰ型的检出率明显高于其它各型,差异有统计学意义(P<0.05);其它各fimA型 P.gingi-valis间的检出差异均无统计学意义。结论本研究条件下ⅠfimA型P.gingivalis在牙周健康人群中检出例数最高,提示:我 国牙周状况不同人群携带的P.gingivalis菌株fimA基因型可能存在差异。?     

不同fimA基因型牙龈卟啉单胞菌在慢性牙周炎患者中的分布

目的　分析不同fimA基因型牙龈卟啉单胞菌(P.gingivalis)在慢性牙周炎患者中的分布状况。方法　收集101例慢性牙周炎患者的龈下菌斑,采用常规培养法和16S rRNA PCR检测P.gingivalis,并根据各fimA基因型的特异引物,用聚合酶链反应(PCR)检测不同fimA基因型菌株的分布。结果　16S rRNA PCR检测P.gingivalis阳性检出率为88·1%。大多数受检牙龈下菌斑中只检测出一种fimA基因型菌株(65·1%),各fimA基因型的总检出率: ⅠfimA为24·7%;ⅡfimA为43·8%;ⅢfimA为15·7%;ⅣfimA为40·4%;VfimA为3·4%。结论　慢性牙周炎患者龈下菌斑中的牙龈卟啉单胞菌存在fimA基因多态性,ⅡfimA和ⅣfimA基因型P.gingivalis菌株与慢性牙周炎的发生发展关系密切。     

牙龈卟啉单胞菌及其牙龈蛋白酶K对青少年牙龈健康的影响

目的：分析牙龈卟啉单胞菌（P. gingivalis）及牙龈蛋白酶K（Kgp）对青少年牙龈健康的影响。方法???收集12~17岁青少年牙龈正常组（50例）、牙龈炎症指数Ⅰ级组（25例）和牙龈炎症指数Ⅱ级组（32例）的3组龈沟液标本,应用16S?rDNA?PCR技术检测各样本中的P. gingivalis及Kgp。3组P. gingivalis和Kgp阳性检出率的比较采用χ2检验;P. gingivalis和Kgp的相对含量与牙龈炎症指数之间的关系采用Spearman相关分析。结果???P. gingivalis在牙龈炎症指数Ⅰ级组的检出率大于牙龈正常组,牙龈炎症指数Ⅱ级组的检出率大于牙龈炎症指数Ⅰ级组和牙龈正常组,牙龈炎症指数Ⅰ级组、Ⅱ级组与牙龈正常组间有明显差异（P<0.01）,牙龈炎症指数Ⅰ级组与Ⅱ级组间差异具有统计学意义（P<0.05）。Kgp在牙龈炎症指数Ⅰ级组的检出率大于牙龈正常组,牙龈炎症指数Ⅱ级组的检出率大于牙龈炎症指数Ⅰ级组和牙龈正常组,牙龈炎症指数Ⅰ级组与牙龈正常组间差异具有统计学意义（P<0.05）,牙龈炎症指数Ⅱ级组与牙龈正常组间有明显差异（P<0.01）。P. gingivalis和Kgp的检出率随着牙龈炎症指数的增加而升高。结论???P. gingivalis和Kgp与青少年牙龈健康密切相关。     

Infection by Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans, and antibody responses at different ages in humans

This study examined the serum IgG and IgM responses against Porphyromonas gingivalis and 3 serotypes of Actinobacillus actinomycetemcomitans , and the correlations of these responses with age and homologous infection. A total of 90 individuals were included in this study: 40 subjects with gingivitis, 40 periodontally healthy subjects, and 10 adult periodontitis subjects. The subjects in the gingivitis and periodontally healthy groups were divided into 4 stages based on their physiological age: early childhood, school age, puberty, and adult. In the gingivitis group, there was a positive correlation between increase in age and increase in serum IgG antibody levels against P. gingivalis until puberty. However, no statistically significant difference was found between the puberty stage and the adult stage. The average level of IgG antibodies against A. actinomycetemcomitans in the school age gingivitis group was significantly higher than that in the early childhood gingivitis group for all serotypes (p < 0.01). In serotype c, IgG antibody levels in the school age gingivitis group were significantly higher than in the early childhood gingivitis group or the adult gingivitis group (p < 0.01). With both P. gingivalis and A. actinomycetemcomitans , positive correlations between elevated IgG level and infections by these microorganisms were found in the puberty gingivitis and adult periodontitis groups.     

Prevalence of Porphyromonas gingivalis fimA genotypes in Japanese children

Porphyromonas gingivalis FimA fimbriae have been classified into 6 genotypes (types I-V and Ib) based on the diversity of the fimA genes encoding the fimbrial subunits. We investigated the prevalence of fimA genotype in Japanese children. Dental plaque specimens were obtained from 400 subjects (age; 2 to 15 years), including 134 with healthy gingiva, 239 with gingivitis and 27 with periodontitis, and then analyzed by polymerase chain reaction. P. gingivalis was detected in 1.5%, 10.0% and 29.6% of these subjects, respectively. Significant differences were observed with regard to P. gingivalis infection among the groups [chi-squared analysis: gingivitis vs. healthy, P < 0.01, odds ratio (OR) = 7.4; periodontitis vs. healthy, P < 0.001, OR = 27.8]. In P. gingivalis-positive subjects with periodontitis, the most prevalent fimA types were type Ib/type II combination (37.5%) and type IV (37.5%), followed by type II (25.0%), while type IV (33.3%) and type II (29.2%) were most often detected in those with gingivitis. Our results suggest that the presence of P. gingivalis is associated with periodontal diseases, and that the type II, IV and Ib/II combination are the most common among fimA genotypes.     

Prevalence of fimA genotypes of Porphyromonas gingivalis and other periodontal bacteria in a Spanish population with chronic periodontitis

Objectives: The aim of this study was to determine the prevalence of the different fimA genotypes of Porphyromonas gingivalis in adult Spanish patients with chronic periodontitis, patients with gingivitis and periodontally healthy subjects, and the relationship between these genotypes and other periodontopathogenic bacteria. Study design: Samples of subgingival plaque were taken from 86 patients (33 with chronic periodontitis, 16 with gingivitis, and 37 periodontally healthy) in the course of a full periodontal examination. PCR was employed to determine the presence of the 6 fimA genotypes of Porphyromonas gingivalis (I-V and Ib) and of Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Treponema denticola. Results: Porphyromonas gingivalis fimA genotypes II and Ib were present in significantly higher percentages in periodontal patients (39.4% and 12.1% respectively) than in healthy or gingivitis subjects. The prevalence of Tannerella forsythia, Treponema denticola, and Porphyromonas gingivalis fimA genotype IV was significantly higher in the group that presented bleeding greater than 30%. A positive correlation was found between Porphyromonas gingivalis fimA genotype IV and Treponema denticola. Conclusions: A strong association between Porphyromonas gingivalis fimA genotypes II and Ib and chronic periodontitis exists in the Spanish population. The most prevalent genotype in periodontal patients is II. Key words:Periodontitis, Porphyromonas gingivalis, fimA genotype, periodontal bacteria, polymerase chain reaction.     

Clonal analysis of Porphyromonas gingivalis by the arbitrarily primed polymerase chain reaction

Genetic analysis of Porphyromonas gingivalis strains may distinguish between virulent and nonvirulent strains and also may be used to trace individual strains in epidemiological studies. The present study examined the utility of the arbitrarily primed polymerase chain reaction for genotypic fingerprinting of P. gingivalis. DNA was extracted according to conventional methods. Ten-base oligonucleotide primers with arbitrary sequences were used with the polymerase chain reaction to amplify P. gingivalis genomic DNA. The amplification products were analyzed by agarose gel electrophoresis. The primer GACCGCTTGT grouped 73 P. gingivalis strains into 23 genotypes, including 16 genotypes containing a single strain each. The primer AGGGGTCTTG identified 45 different genotypes, 33 of which contained a single strain. P. gingivalis strains ATCC 332771^T and 381 belonged to the same genotype. Likewise, strains W50 and W83 were of the same genetic clone. The present study indicates that the arbitarily primed polymerase chain reaction represents a valuable and easy method for clonal analysis of P. gingivalis.     

青春期龈炎龈下菌斑中牙龈卟啉单胞菌的牙龈蛋白酶K的表达

目的:检测并比较青春期龈炎的牙龈卟啉单胞菌(P.gingivalis)临床分离株的分泌蛋白和菌体蛋白中牙龈蛋白酶K(Kgp)的表达强度,揭示Kgp与青春期龈炎之间可能存在的致病关系。方法:受试对象为14～17岁青春期龈炎患者36例,检测并记录受检者的各牙周指数GI、SBI和PD测值,取龈下菌斑进行P.gin-givalis的分离培养,16SrRNAPCR法鉴定。将P.gingivalis临床分离株于对数生长期末提取分泌蛋白和菌体蛋白,用抗KgpN-末端IgG亚基的单克隆抗体进行Westernblot检测,采用SPSS11.0软件包,秩相关检验分析Kgp的表达强度与各牙周指数之间的相关关系。结果:青春期龈炎的P.gingivalis临床分离株的分泌蛋白和菌体蛋白中,KgpN-末端IgG亚基的表达强度与各牙周指数数值的高低有正相关关系,统计学上有显著性差异(P<0.01)。结论:Kgp对青春期龈炎有一定的致病作用。     

Identification of the genes associated with a virulent strain of Porphyromonas gingivalis using the subtractive hybridization technique

Background/aims:  Porphyromonas gingivalis , a major etiological organism implicated in periodontal disease, can be classified into virulent and avirulent strains. Our aim was to identify a gene for the virulence of P .  gingivalis .
Methods:  The subtractive hybridization technique was employed to identify the genes specific to P .  gingivalis W83, a virulent strain. In this study, P. gingivalis W83 was used as the tester strain, and P .  gingivalis ATCC 33277 was the driver strain. The prevalence of W83-specific genes was determined by Southern blot analysis of several P. gingivalis strains.
Results:  We obtained 575 colonies using the subtractive hybridization technique. From among these, 26 DNA fragments were subjected to a homology search using the BLAST program. Compared with strain ATCC 33277, strain W83 contained 12 unique clones. The specificities of the isolated DNA fragments were analyzed among four P. gingivalis strains by Southern blot analysis. Five genes showed specificity for strain W83 compared with strain ATCC 33277. All five genes were also identified in strain W50.
Conclusions:  The subtractive hybridization technique was effective in screening the two strains for specific DNA sequences, some of which might be responsible for determining virulence. The results suggested that several genes specific to strain W83 were associated with its virulence. Further analysis of these DNA fragments will provide important information on the pathogenesis of virulent P .  gingivalis strains.     

The prevalence and pathogenic differences of Porphyromonas gingivalis fimA genotypes in patients with aggressive periodontitis

BACKGROUND: The fimA gene, which encodes fimbrillin (FimA), is found in Porphyromonas gingivalis and has been classified into six genotypes based on nucleotide sequence. P. gingivalis that possesses the type II fimA gene is prevalent in adult periodontitis. OBJECTIVES: The aim of this study was to investigate the prevalence of P. gingivalis fimA genotypes in Japanese aggressive periodontitis patients, and to examine their virulence. METHODS: Subgingival plaque samples were obtained from 223 sites in 18 aggressive periodontitis patients and 95 sites in 22 periodontally healthy young adults. Actinobacillus actinomycetemcomitans, P. gingivalis and Tannerella forsythensis detection, determination of the fimA genotype in P. gingivalis, and the quantification of P. gingivalis were analyzed by polymerase chain reaction (PCR) methods. The proteolytic activities of the P. gingivalis fimA type I and fimA type II were also examined. RESULTS: In aggressive periodontitis patients, the most prevalent fimA genotype was the type II (46.7%), followed by the type Ib and type I, whereas in healthy subjects, the type I fimA was the only genotype detected. The number of P. gingivalis pathogens was the greatest in the type I fimA positive sites, and the frequency of coexisting A. actinomycetemcomitans and T. forsythensis was highest in the type II fimA positive sites in the aggressive periodontitis patients. Both the arginine-specific cysteine proteinase (Arg-gingipain) and lysine-specific cysteine proteinase (Lys-gingipain) activity of the P. gingivalis fimA type I strain were significantly higher than those of the fimA type II strains. CONCLUSIONS: These results suggest that differences in virulence exist among different fimA genotypes. Coadherence with other pathogens in P. gingivalis fimA type II-associated aggressive periodontitis and quantitative increases in P. gingivalis in fimA type I-associated aggressive periodontitis are related to this virulence.