Complete Genome Sequence of Human Oral Phylogroup 1 Treponema sp. Strain OMZ 804 (ATCC 700766), Originally Isolated from Periodontitis Dental Plaque.

Host-associated treponeme bacteria play etiological roles in human and animal soft tissue infections. Treponema sp. strain OMZ 804 (ATCC 700766) was isolated from dental plaque sampled from a patient with periodontitis in Switzerland in 1994. We report here the complete genome sequence of its 2.98-Mb circular chromosome.

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.

Complete Genome Sequence for Treponema sp. OMZ 838 (ATCC 700772, DSM 16789), Isolated from a Necrotizing Ulcerative Gingivitis Lesion.

The oral treponeme bacterium Treponema sp. OMZ 838 was originally isolated from a human necrotizing ulcerative gingivitis (NUG) lesion. Its taxonomic status remains uncertain. The complete genome sequence length was determined to be 2,708,067 bp, with a G+C content of 44.58%, and 2,236 predicted coding DNA sequences (CDS).

Complete Genome Sequence of the Oral Spirochete Bacterium Treponema putidum Strain OMZ 758T (ATCC 700334T).

The oral spirochete bacterium Treponema putidum inhabits human periodontal niches. The complete genome sequence of the OMZ 758(T) (ATCC 700334(T)) strain of this species was determined, revealing a 2,796,913-bp chromosome, with a G+C content of 37.30% and a single plasmid (pTPu1; 3,649 bp) identical to pTS1 from Treponema denticola.

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'.

Bacteriocin immunity proteins play a role in quorum-sensing system regulated antimicrobial sensitivity of Streptococcus mutans UA159.

OBJECTIVE: Antimicrobial sensitivity of Streptococcus mutans can be modulated by putative bacteriocin immunity proteins. Bacteria use a quorum sensing (QS) system to regulate physiological activities including bacteriocin production, antimicrobial response, and biofilm formation. QS system of S. mutans is dependent on competence stimulating peptide (CSP), whose precursor is encoded by comC. However, whether bacteriocin immunity proteins play a role in QS system regulated S. mutans antimicrobial sensitivity is still unknown. We hypothesize that bacteriocin immunity proteins encoded by immA and immB play roles in QS regulated antimicrobial sensitivity in S. mutans UA159. DESIGN: In this study, sensitivity of S. mutans UA 159 comC mutant in planktonic and biofilm states to clinically used antimicrobials was investigated by the plate count method and confocal laser scanning microscopy. The effect of immA and immB inactivation on S. mutans antimicrobial sensitivity was studied. The expression of immA and immB in S. mutans comC mutant before and after chlorhexidine (CHX) treatment was also examined. RESULTS: It was found that comC, immA and immB mutation resulted in enhanced antimicrobial sensitivity to sodium fluoride (NaF), CHX and ampicillin (AMP) in planktonic states. After 2% CHX treatment, the live/dead cell ratio in comC mutant and wild strain biofilms decreased 67% and 39% (P<0.05). The expression of immA and immB was up-regulated in wild strain after CHX treatment, while the up-regulation of immB was largely inhibited in comC mutant (P<0.05). CONCLUSIONS: These findings suggest that the effect of S. mutans UA159 comC mutation on antimicrobial sensitivity can be due, in part, to attenuation of the expression of the bacteriocin immunity proteins related genes.

[Effects of immA and immB coding putative bacteriocin immunity proteins on the antimicrobial sensitivity in planktonic Streptococcus mutans and biofilm formation].

OBJECTIVE: To investigate the effects of putative bacteriocin immunity proteins on the growth mode of Streptococcus mutans (Sm). To observe the differences of antimicrobial sensitivity in planktonic Sm wild-type strains and mutant strains caused by the inactivation of bacteriocin immunity proteins and their influence on the biofilm formation. METHODS: Sm wild-type strains (WT) and its knockout mutants defective in immA and immB (ΔimmA(-) and ΔimmB(-) mutants) coding putative bacteriocin immunity proteins were cultured in brain heart infusion (BHI) and selected by erythromycin at the concentration of 10 mg/L. Optical density was detected by spectrophotometer every hour and growth curve was drawn. WT, ΔimmA(-) and ΔimmB(-) mutants were treated with ampicillin (0.04, 0.05, 0.06, 0.07, 0.08 mg/L), sodium fluoride (50, 100, 150, 200, 250 mg/L) and sodium hypochlorite (0.078%, 0.156%, 0.313%, 0.625%, 1.250%) for 24 hours. Optical density was detected by multifunctional micro plate reader. WT and the mutants were cultured in MBEC(TM) P&G Assay for 24 hours. The minimum biofilm eradication concentration (MBEC) of chlorhexidine against Sm was determined by serial dilution method. Confocal laser scanning microscopy (CLSM) was used to visualize the biofilm architecture, depth and ratio of live to dead bacteria. RESULTS: Growth curve showed that it took about 3 hours to reach exponential phase and about 7 hours to stationary phase for WT, while 4 hours to exponential phase and 8 hours to stationary phase for mutants. Optical density of mutants were lower than WT in the presence of various antimicrobial agents (P < 0.01). In 0.06 mg/L ampicillin group, optical density value of WT, ΔimmA(-) and ΔimmB(-) mutants were 0.334 ± 0.016, 0.027 ± 0.016 and 0.047 ± 0.018. In 150 mg/L sodium fluoride group, optical density value of WT and mutants were 0.254 ± 0.018, 0.129 ± 0.011 and 0.167 ± 0.010. In 0.313% sodium hypochlorite group, optical density value of WT and mutants were 0.467 ± 0.008, 0.017 ± 0.006 and 0.050 ± 0.006. The MBEC of chlorhexidine against Sm WT, ΔimmA(-) and ΔimmB(-) mutants were 6.25, 1.57, and 3.13 mg/L. The results by CLSM showed a noticeable difference in biofilm architecture. The depth of WT biofilm was higher than the mutants biofilm (P < 0.01). The ratio of live to dead bacteria of WT biofilm was higher than ΔimmA(-) mutants in all layers (P < 0.05) and ΔimmB(-) mutants in the outer and intermedium layer (P < 0.01). There is no significant different between the inner layers of WT and ΔimmB(-) mutants (P = 0.191). CONCLUSIONS: Putative bacteriocin immunity proteins have influence on the growth mode of Sm. The antimicrobial sensitivity of planktonic Sm can be up-regulated by the inactivation of immA or immB. The MBEC of chlorhexidine against ΔimmA(-) and ΔimmB(-) mutants is lower than WT. The inactivation of immA or immB affects the biofilm formation.