Microbial diversity in subgingival biofilm of subjects with aggressive periodontitis

Objectives: The purpose of this study was to determine the bacterial diversity in the subgingival plaque of subjects with aggressive periodontitis (AgP) by using culture-independent molecular methods based on ribosomal 16S rRNA gene cloning and by using the Checkerboard DNA-DNA hybridization technique. Methods:Twelve subjects with AgP and 30 periodontally healthy (PH) subjects were selected. Subgingival plaque samples were collected from 9 sites per subject for using in the Checkerboard DNA-DNA technique. For 16S cloning analysis, one sample per subject with PD>7mm of 10 subjects with AgP were selected. DNA was extracted and 16S rRNA gene amplified with universal primer pairs. Amplified genes were cloned, sequenced and identified by comparison with known 16S rRNA. sequences Results: Periodontal pathogens, such as Tannerella forsythia, Porphyromonas gingivalis and Treponema denticola were found in higher proportions AgP groups (p<0.001) than in PH subjects. Aggregatibacter (check spelling) actinomycetemcomitans was found in higher levels, proportions and prevalence in AgP than in PH subjects using genomic probes. As regard the 16S gene cloning analysis, 120 species were identified from 10 subjects and 1094 clones sequenced. Of these, 70 species were most prevalent. 57% of the species have not yet been grown in culture. Several species of Selenomonas and Streptococcus were found in high prevalence and proportion in all subjects. Overall, 50% of the clone libraries were represented by species of these two genera. Selenomonas sputigena, the species most commonly detected, was found in 9 of 10 subjects. Other species of Selenomonas were often present in high levels, including Selenomonas noxia, Selenomonas sp. EW084, Selenomonas sp. EW076, Selenomonas sp. FT050, Selenomonas sp. P2PA_80 and Selenomonas sp. strain GAA14. Conclusion: The subgingival microbiota of AgP markedly differed from PH subjects using genomic probes. Species other than A. actinomycetemcomitans, notably species of Selenomonas,may be associated with AgP.