Multilocus Sequence Analysis of Phylogroup 1 and 2 Oral Treponeme Strains.

More than 75 "species-level" phylotypes of spirochete bacteria belonging to the genus Treponema reside within the human oral cavity. The majority of these oral treponeme phylotypes correspond to as-yet-uncultivated taxa or strains of uncertain standing in taxonomy. Here, we analyze phylogenetic and taxonomic relationships between oral treponeme strains using a multilocus sequence analysis (MLSA) scheme based on the highly conserved 16S rRNA, pyrH, recA, and flaA genes. We utilized this MLSA scheme to analyze genetic data from a curated collection of oral treponeme strains (n = 71) of diverse geographical origins. This comprises phylogroup 1 (n = 23) and phylogroup 2 (n = 48) treponeme strains, including all relevant American Type Culture Collection reference strains. The taxonomy of all strains was confirmed or inferred via the analysis of ca. 1,450-bp 16S rRNA gene sequences using a combination of bioinformatic and phylogenetic approaches. Taxonomic and phylogenetic relationships between the respective treponeme strains were further investigated by analyzing individual and concatenated flaA (1,074-nucleotide [nt]), recA (1,377-nt), and pyrH (696-nt) gene sequence data sets. Our data confirmed the species differentiation between Treponema denticola (n = 41) and Treponema putidum (n = 7) strains. Notably, our results clearly supported the differentiation of the 23 phylogroup 1 treponeme strains into five distinct "species-level" phylotypes. These respectively corresponded to "Treponema vincentii" (n = 11), Treponema medium (n = 1), "Treponema sinensis" (Treponema sp. IA; n = 4), Treponema sp. IB (n = 3), and Treponema sp. IC (n = 4). In conclusion, our MLSA-based approach can be used to effectively discriminate oral treponeme taxa, confirm taxonomic assignment, and enable the delineation of species boundaries with high confidence. IMPORTANCE: Periodontal diseases are caused by persistent polymicrobial biofilm infections of the gums and underlying tooth-supporting structures and have a complex and variable etiology. Although Treponema denticola is strongly associated with periodontal diseases, the etiological roles of other treponeme species/phylotypes are less well defined. This is due to a paucity of formal species descriptions and a poor understanding of genetic relationships between oral treponeme taxa. Our study directly addresses these issues. It represents one of the most comprehensive analyses of oral treponeme strains performed to date, including isolates from North America, Europe, and Asia. We envisage that our results will greatly facilitate future metagenomic efforts aimed at characterizing the clinical distributions of oral treponeme species/phylotypes, helping investigators to establish a more detailed understanding of their etiological roles in periodontal diseases and other infectious diseases. Our results are also directly relevant to various polymicrobial tissue infections in animals, which also involve treponeme populations.

Effects of antioxidants on the microleakage of composite resin restorations after external tooth bleaching.

OBJECTIVE: To compare the effects of three antioxidants (sodium ascorbate, sodium ascorbate combined with a surfactant, and catalase) on the microleakage of composite resin restorations after external tooth bleaching with 10% carbamide peroxide. MATERIALS AND METHODS: Buccal cavities were prepared on the surface of 60 intact premolars, which were randomly divided into six groups. All cavities were filled with composite resin. In group 1, teeth were not bleached; in group 2, cavities were filled immediately after bleaching; in group 3, cavities were delay-filled for 3 weeks; in group 4, cavities were treated with sodium ascorbate after bleaching and then filled; in group 5, cavities were treated with sodium ascorbate combined with surfactant after bleaching and then filled; and in group 6, cavities were treated with catalase after bleaching and then filled. Microleakage of the restorations was measured from sections using a stereomicroscope. RESULTS: Group 1 displayed the least amount of microleakage, while group 2 showed the greatest amount of microleakage (P < 0.05). Groups 3 and 4 were similar to group 2 (P > 0.05). However, groups 5 and 6 showed a significantly lower amount of microleakage compared to group 2 (P < 0.05). CONCLUSION: Microleakage increased significantly after external bleaching with 10% carbamide peroxide, and decreased when the bleached teeth were treated with sodium ascorbate combined with Tween(®) 80, or with catalase. Catalase was more effective in decreasing microleakage, while delayed filling or treatment with sodium ascorbate alone did not effectively decrease the microleakage.

Comparative analysis of oral treponemes associated with periodontal health and disease.

BACKGROUND: Periodontal diseases, such as periodontitis, are chronic inflammatory infections affecting the gingivae (gums), underlying connective tissues and bone that support the teeth. Oral treponemes (genus Treponema) are widely-considered to play important roles in periodontal disease etiology and pathogenesis; however, precise relationships remain to be fully established. METHODS: A 16S rRNA clone library-based approach was used to comprehensively characterize and compare the diversity of treponeme taxa present in subgingival plaque sampled from periodontitis patients (n = 10) versus periodontitis-free controls (n = 10). 16S rRNA gene sequences were assigned to operational taxonomic units (OTUs) using a 99% identity cut-off A variety of taxonomy (OTU) and phylogeny-based statistical approaches were used to compare populations of treponeme OTUs present in both subject groups. RESULTS: A total of 615 plasmid clones containing ca. 1500 bp Treponema 16S rRNA gene sequences were obtained; 365 from periodontitis subjects, 250 from periodontitis-free controls. These were assigned to 110 treponeme OTUs. 93 OTUs were detected in the periodontitis subjects (mean 9.3 ± 5.2 OTUs per subject; range 9-26), and 43 OTUs were detected in controls (mean 4.3 ± 5.9 OTUs per subject; range 3-20). OTUs belonging to oral treponeme phylogroups 1-7 were detected in both subject sets. Phylogroup 1 treponemes had the highest levels of OTU richness (diversity) and clonal abundance within both subject groups. Levels of OTU richness and clonal abundance of phylogroup 2 treponemes were significantly higher in the periodontitis subjects (Mann Whitney U-test, p < 0.001). Both OTU-based and phylogeny-based analyses clearly indicated that there were significant differences in the composition of treponeme communities present in periodontitis versus control subjects. The detection frequency of five OTUs showed a statistically-significant correlation with disease status. The OTU (8P47) that corresponded to the type strain of Treponema denticola had the strongest association with periodontitis (p < 0.01). CONCLUSIONS: Higher levels of treponeme taxon richness and clonal abundance were associated with periodontitis. However, our results clearly indicated that subjects free from clinical symptoms of periodontal disease also contained highly diverse populations of treponeme bacteria within their subgingival microbiota. Our data supports the hypothesis that specific treponeme taxa are associated with periodontal disease.

Multilocus sequence analysis of Treponema denticola strains of diverse origin.

BACKGROUND: The oral spirochete bacterium Treponema denticola is associated with both the incidence and severity of periodontal disease. Although the biological or phenotypic properties of a significant number of T. denticola isolates have been reported in the literature, their genetic diversity or phylogeny has never been systematically investigated. Here, we describe a multilocus sequence analysis (MLSA) of 20 of the most highly studied reference strains and clinical isolates of T. denticola; which were originally isolated from subgingival plaque samples taken from subjects from China, Japan, the Netherlands, Canada and the USA. RESULTS: The sequences of the 16S ribosomal RNA gene, and 7 conserved protein-encoding genes (flaA, recA, pyrH, ppnK, dnaN, era and radC) were successfully determined for each strain. Sequence data was analyzed using a variety of bioinformatic and phylogenetic software tools. We found no evidence of positive selection or DNA recombination within the protein-encoding genes, where levels of intraspecific sequence polymorphism varied from 18.8% (flaA) to 8.9% (dnaN). Phylogenetic analysis of the concatenated protein-encoding gene sequence data (ca. 6,513 nucleotides for each strain) using Bayesian and maximum likelihood approaches indicated that the T. denticola strains were monophyletic, and formed 6 well-defined clades. All analyzed T. denticola strains appeared to have a genetic origin distinct from that of 'Treponema vincentii' or Treponema pallidum. No specific geographical relationships could be established; but several strains isolated from different continents appear to be closely related at the genetic level. CONCLUSIONS: Our analyses indicate that previous biological and biophysical investigations have predominantly focused on a subset of T. denticola strains with a relatively narrow range of genetic diversity. Our methodology and results establish a genetic framework for the discrimination and phylogenetic analysis of T. denticola isolates, which will greatly assist future biological and epidemiological investigations involving this putative 'periodontopathogen'.

The microfloral analysis of secondary caries biofilm around Class I and Class II composite and amalgam fillings.

BACKGROUND: Secondary caries is responsible for 60 percent of all replacement restorations in the typical dental practice. The diversity of the bacterial sources and the different types of filling materials could play a role in secondary caries. The aim of this study was to determine and compare the microbial spectrum of secondary caries biofilms around amalgam and composite resin restorations. METHODS: Clinical samples were collected from freshly extracted teeth diagnosed with clinical secondary caries. Samples were categorized into four groups according to the types of restoration materials and the classification of the cavity. Biofilms were harvested from the tooth-restoration interface using a dental explorer and after dilution were incubated on special agars. The bacteria were identified using the biochemical appraisal system. Statistical calculations were carried out using SPSS11.5 software to analyze the prevalence of the bacteria involved in secondary caries. RESULTS: Samples from a total of four groups were collected: two groups were collected from amalgam restorations, each had 21 samples from both Class I and Class II caries; and the other two groups were from composite resin restorations, each had 13 samples from both class I and class II caries. Our results showed: (1) Anaerobic species were dominant in both restoration materials. (2) In terms of the types of individual bacteria, no significant differences were found among the four groups according to the geometric mean of the detected bacteria (P > 0.05). However, there were significant differences among the detected bacteria within each group (P < 0.05). The composition of each bacterium had no statistical difference among the four groups (P > 0.05), but showed significant differences among the detected bacteria in each group (P < 0.05). (3) Among the four groups, there were no significant differences for the detection rate of each bacterium (P > 0.05), however, the detection rate of each bacterium within each group was statistically different among the detected bacteria (P < 0.05). CONCLUSIONS: The proportion of obligatory anaerobic species was much greater than the facultative anaerobic species in the biofilm of secondary caries. Statistically, the materials of restoration and the location of secondary caries did not show any significant effects on the composition of the microflora.