Microarray analysis of the microflora of root caries in elderly.

The present study used a new 16S rRNA-based microarray with probes for over 300 bacterial species to better define the bacterial profiles of healthy root surfaces and root caries (RC) in the elderly. Supragingival plaque was collected from 20 healthy subjects (Controls) and from healthy and carious roots and carious dentin from 21 RC subjects (Patients). Collectively, 179 bacterial species and species groups were detected. A higher bacterial diversity was observed in Controls than in Patients. Lactobacillus casei/paracasei/rhamnosus and Pseudoramibacter alactolyticus were notably associated with most RC samples. Streptococcus mutans was detected more frequently in the infected dentin than in the other samples, but the difference was not significant. Actinomyces was found more frequently in Controls. Thus, species other than Actinomyces and S. mutans may play a role as pathogens of RC. The results from this study were in general agreement with those of our previous study based on 16S rRNA gene sequencing.

The diversity of periodontal spirochetes by 16S rRNA analysis.

The purpose of this study was to examine the diversity of spirochetes in the subgingival pocket of multiple subjects with a range of periodontal conditions, including two healthy, one adult periodontitis, three acute necrotizing ulcerative gingivitis, eight refractory periodontitis, and one human immunodeficiency virus (HIV) periodontitis. The 16S rRNA genes of spirochetes in plaque were amplified by polymerase chain reaction using spirochete selective primers. Over 500 clones were sequenced and subjected to phylogenetic analysis. The sequences clustered into the 10 known cultivated Treponema species and into 47 as-yet-uncultivated Treponema species. Most of these Treponema species were identified from multiple clones and subjects. The human periodontal pocket harbors a highly diverse treponeme population. Of the cultivated species, Treponema denticola, Treponema maltophilum and Treponema sp. Smibert-3 were most commonly encountered in diseased subjects but rarely in healthy subjects. Molecular tools based on the sequence data from this study will allow determination of the prevalence of cultivable and uncultivable treponemes in oral diseases.

Bacterial diversity in human subgingival plaque.

The purpose of this study was to determine the bacterial diversity in the human subgingival plaque by using culture-independent molecular methods as part of an ongoing effort to obtain full 16S rRNA sequences for all cultivable and not-yet-cultivated species of human oral bacteria. Subgingival plaque was analyzed from healthy subjects and subjects with refractory periodontitis, adult periodontitis, human immunodeficiency virus periodontitis, and acute necrotizing ulcerative gingivitis. 16S ribosomal DNA (rDNA) bacterial genes from DNA isolated from subgingival plaque samples were PCR amplified with all-bacterial or selective primers and cloned into Escherichia coli. The sequences of cloned 16S rDNA inserts were used to determine species identity or closest relatives by comparison with sequences of known species. A total of 2,522 clones were analyzed. Nearly complete sequences of approximately 1,500 bases were obtained for putative new species. About 60% of the clones fell into 132 known species, 70 of which were identified from multiple subjects. About 40% of the clones were novel phylotypes. Of the 215 novel phylotypes, 75 were identified from multiple subjects. Known putative periodontal pathogens such as Porphyromonas gingivalis, Bacteroides forsythus, and Treponema denticola were identified from multiple subjects, but typically as a minor component of the plaque as seen in cultivable studies. Several phylotypes fell into two recently described phyla previously associated with extreme natural environments, for which there are no cultivable species. A number of species or phylotypes were found only in subjects with disease, and a few were found only in healthy subjects. The organisms identified only from diseased sites deserve further study as potential pathogens. Based on the sequence data in this study, the predominant subgingival microbial community consisted of 347 species or phylotypes that fall into 9 bacterial phyla. Based on the 347 species seen in our sample of 2,522 clones, we estimate that there are 68 additional unseen species, for a total estimate of 415 species in the subgingival plaque. When organisms found on other oral surfaces such as the cheek, tongue, and teeth are added to this number, the best estimate of the total species diversity in the oral cavity is approximately 500 species, as previously proposed.