Diversity of oral asaccharolytic Eubacterium species in periodontitis--identification of novel phylotypes representing uncultivated taxa

Oral asaccharolytic Eubacterium species are associated with periodontal disease and other oral infections. The aim of this study was to use a culture-independent molecular technique to determine the diversity of asaccharolytic Eubacterium species in subgingival plaque in periodontitis. An oligonucleotide PCR primer designed to amplify 16S rRNA genes of the oral asaccharolytic Eubacterium branch of the phylogenetic tree was constructed. This primer was used together with a universal primer in PCRs to amplify gene sequences directly from a single subgingival plaque sample. Fifty PCR products were purified by cloning, fully sequenced and subjected to molecular phylogenetic analysis. The sequences were assigned to four groups within a single lineage of the low G + C gram-positive bacteria. Group I (58% of the cloned sequences) was assigned to a branch that included Eubacterium nodatum, and Group II (22%) to a branch including Eubacterium infirmum. Group III (8%) was distinct from but related to E. infirmum at the species level, and Group IV (12%) was another novel taxon more distantly related to E. infirmum and E. nodatum.