Molecular analysis of bacteria in periodontitis: evaluation of clone libraries, novel phylotypes and putative pathogens

Subgingival plaque samples were obtained from 26 subjects with advanced periodontal lesions. Bacterial diversity was analysed by amplification of the 16S rRNA genes with two different primer sets, and subsequent cloning and sequencing. A total of 578 sequences was analysed after the exclusion of chimeras. The authors found 148 phylotypes with the clone library 27f/519r (number of clones n=322; overage, C=66%) and 75 phylotypes with the clone library 515f/1525r (n = 256; C=84%). Comparative sequence analysis revealed that 70% of all of the analysed sequences showed a similarity of at least 99% to sequences deposited in public databases. The classes Actinobacteria, Bacilli, Bacteroidetes, Clostridia, Deferribacteres, Flavobacteria, Fusobacteria, Mollicutes, Spirochaetes and all classes of the Proteobacteria were represented. Sequences that were at least 99% identical to Porphyromonas gingivalis, Filifactor alocis and Treponema socranskii were present in at least one-third of the patients. Libraries generated with the two PCR primer pairs differed significantly in their representation of the families Porphyromonadaceae, Prevotellaceae, Fusobacteriaceae, Eubacteriaceae, Streptococcaceae and Acidaminococcaceae. A total of 14 sequences exhibited less than 97% identity to sequences published previously and were assigned to six different families within the phyla Bacteroidetes and Firmicutes. Twelve of 20 putative pathogens were recovered, which were recently proposed to be associated with periodontitis.