Molecular Epidemiological Study of Periodontopathic Bacteria in Periodontal Diseases

对牙周病患者牙周病区分离的牙龈卟啉单胞菌(PG)和伴放线放线杆菌(AA)进行了生物分型、血清分型、核糖体分型和基因分型。这些分型方法及其细菌培养和分离既耗时又复杂。通过对有限数量的临床分离株的分析，很难完全阐明牙周病患者的微生物区系。本研究的目的是通过聚合酶链式反应(PCR)扩增出Pg和Aa的特异性抗原基因，并对其进行直接鉴定和基因分型。本研究选择外膜蛋白(OMP)基因和菌毛蛋白(FMP)基因分别作为PG和AA的特异性抗原基因。我们的特异性抗原基因的聚合酶链式反应方法可以直接和特异地检测斑块样本中的PG和AA。用聚合酶链式反应将Pg分为两种OMP基因(pga53和pga67)，用聚合酶链式反应-限制性片段长度多态性(RFLP)将AA分为4种FMP基因。我们调查了59例(145个位点)牙周病患者PG和AA基因的分布和患病率。大多数患者仅感染单一的PG和/或AA基因。在两个家庭(母亲-儿子-女儿)中，PG和AA的等位基因在家庭成员中分布相同。提示牙周病的发病和进展可能由PG和/或AA的克隆性基因所致，并可能在家族内传播。基于PG和AA特异性抗原基因的聚合酶链式反应-基因分型方法为牙周病的分子流行病学研究提供了有用的工具，为牙周病的传播和发病机制提供了新的认识。

Previous epidemiological studies of Porphyromonas gingivalis(Pg)and Actinobacillus actinomycetemcomitans(Aa)in periodontal diseases have performed to analyze their clinical isolates from diseased periodontal regions by biotyping, serotyping, ribotyping and genotyping. These typing methods following by their bacterial culture and isolation are time-consuming and complicated. It is difficult to fully elucidate the microflora of individulas with periodontals diseases by analysing the limited number of clinical isolates. The aim of this study was to examine the direct identification of Pg and Aa in subgingival palque samples and further their genotyping based on the specific antigen genes amplified by polymerase chain reaction(PCR). In this study, the outer membrane protein(OMP)gene and fimbrial protein(FMP)gene were selected as the specific antigen gene for Pg and Aa, respectively. Our PCR method for specific antigen genes could allow the direct and specific detection of Pg and Aa in plaque samples. Pg were classified into two OMP genotypes(pga53 and pga67)by PCR and Aa were differenciated into four FMP genotypes by PCR-restriction fragment-length polymorphism(RFLP). We examined the distribution and prevalence of Pg and Aa genotypes in 59 patients(145 sites)with periodontal diseases. Almost of the patients were infected with only single genotype of Pg and/or Aa. In the case of two families(mother-son-daughter), the identical genotypes of Pg and Aa were distributed among family members. These results indicated that periodontal diseases might be initiated and progressed by clonal genotype of Pg and/or Aa, which might be possibly transmitted within family. The PCR-genotyping method based on specific antigen genes of Pg and Aa would be useful tools for their molecular epidemiological study in periodontal diseases and provide new insights into their transmission and pathogenesis.