Phylogenetic characterization of subgingival plaque associated with aggressive or chronic periodontitis

Background and aims: Most of the so far identified bacterial species and phylotypes in the human oral cavity can be assigned to one of 11 phyla of the domain Bacteria and within these to 18 different classes. However, more than 50% of the oral bacteria cannot yet be cultivated and information about their relevance with respect to oral health is scarce. The aim of this study was to investigate the composition of subgingival plaque associated with advanced periodontitis by quantitative microscopic single-cell analysis using a panel of rRNA probes with specificity to large taxonomic units (classes, genera, families). The approach extends the more conventional use of probes that are directed against species that have been implicated in earlier studies by culture analyses in the etiology of periodontal diseases. One of the focal question asked was whether it will be possible to detect nearly all bacteria present in subgingival pockets rather than the 20-30% that can be covered by species-specific identification assays. A secondary aim was the monitoring of the changes in biofilm composition caused by antibiotic treatment directed against Gram-negative anaerobes. Methods: Forty-five subgingival plaque samples from 16 patients suffering from advanced chronic or aggressive periodontitis were examined. Four patients underwent antibiotic treatment and were re-investigated 3-5 month after therapy. Fluorescent in situ hybridization (FISH) was used to investigate the prevalence and abundance of bacteria from 13 of the classes known to colonize subgingival pockets. To this end new 16S or 23S rDNA probes were designed, validated, and applied together with previously described probes in seeking as complete coverage as possible of the subgingival microbiota. This was accomplished either by using single probes, or by working with two or more complementary probes. Results: The mean number of identified bacteria from 10 of the 13 classes varied between 10 5 and 10 6 per ml sample. Much lower numbers, around 10 4 , were counted for Capnocytophaga , the only oral genus of the class Flavobacteria, whereas " -Proteobacteria and TM7 were present in only few samples at concentrations barely above the detection limit. The prevalence of members from the 11 other classes varied between 68% and 100% and exceeded 90% in seven of these taxonomic units. Overall, from the phylum of Synergistetes, especially from cluster A, which so far harbors only non-cultivable phylotypes. Among the ! -Proteobacteria detected by probe ! -42a a group of bacteria was frequently present, which could not be identified with our current set of phylogenetic probes. A similar result was obtained for bacteria belonging to the genus Prevotella , where much of the recognized diversity remained unexplainable by monitoring the most commonly studied Prevotella species. As expected the class of Bacteroidia with the major genera Porphyromonas , Tannerella and Prevotella accounted for a large number of the total subgingival biofilm bacteria. Spirochaetes detected by the two new probes Trep-G1-679 and Tlema738 with specificity for clusters l-lll and lV, respectively, were well represented in more than 80% of the samples. Among the Firmicutes bacteria streptococci and eubacteria occurred with similarly high prevalence and abundance as the spirochetes, whereas the peculiar Gram-negative group of the Negativicutes harboring primarily the Veillonella and Selenomonas species rarely reached very high densities. The phylum of TM7 was undetectable in almost all probes and seems unlikely that these still cultivated bacteria play a role in the disease of periodontitis as suggested in the literature. Similarly, " -Proteobacteria, apart from the Aggergatibacter actinomycetemcomitans and presumably a few other unidentified Pasteurellaceae species, were not detected. The data collected in this study show that the phyla-, class-, or family-specific probes are useful to analyze the composition of subgingival plaque associated with periodontitis. The number of microorganisms stained by the universal bacterial probe EUB338 and the number of bacteria labeled by all probes of non-overlapping specifity were nearly identical, which proves that it is possible to cover essentially the entire subgingival microbiota with probes to 11 major clusters of bacteria. A particular striking finding was the very high prevalence and density of large non-cultivable Synergistetes bacteria, especially of cluster A, raising important questions about a possible role of these bacteria in the periodontal inflammatory process.