In search of an uncultured human-associated TM7 bacterium in the environment.

We have identified an environmental bacterium in the Candidate Division TM7 with ≥98.5% 16S rDNA gene homology to a group of TM7 bacteria associated with the human oral cavity and skin. The environmental TM7 bacterium (referred to as TM7a-like) was readily detectable in wastewater with molecular techniques over two years of sampling. We present the first images of TM7a-like cells through FISH technique and the first images of any TM7 as viable cells through the STARFISH technique. In situ quantification showed TM7 concentration in wastewater up to five times greater than in human oral sites. We speculate that upon further characterization of the physiology and genetics of the TM7a-like bacterium from environmental sources and confirmation of its genomic identity to human-associated counterparts it will serve as model organisms to better understand its role in human health. The approach proposed circumvents difficulties imposed by sampling humans, provides an alternative strategy to characterizing some diseases of unknown etiology, and renders a much needed understanding of the ecophysiological role hundreds of unique Bacteria and Archaea strains play in mixed microbial communities.

Phylometrics: a pipeline for inferring phylogenetic trees from a sequence relationship network perspective.

BACKGROUND: Comparative sequence analysis of the 16S rRNA gene is frequently used to characterize the microbial diversity of environmental samples. However, sequence similarities do not always imply functional or evolutionary relatedness due to many factors, including unequal rates of change and convergence. Thus, relying on top BLASTN hits for phylogenetic studies may misrepresent the diversity of these constituents. Furthermore, attempts to circumvent this issue by including a large number of BLASTN hits per sequence in one tree to explore their relatedness presents other problems. For instance, the multiple sequence alignment will be poor and computationally costly if not relying on manual alignment, and it may be difficult to derive meaningful relationships from the resulting tree. Analyzing sequence relationship networks within collective BLASTN results, however, reveal sequences that are closely related despite low rank. RESULTS: We have developed a web application, Phylometrics, that relies on networks of collective BLASTN results (rather than single BLASTN hits) to facilitate the process of building phylogenetic trees in an automated, high-throughput fashion while offering novel tools to find sequences that are of significant phylogenetic interest with minimal human involvement. The application, which can be installed locally in a laboratory or hosted remotely, utilizes a simple wizard-style format to guide the user through the pipeline without necessitating a background in programming. Furthermore, Phylometrics implements an independent job queuing system that enables users to continue to use the system while jobs are run with little or no degradation in performance. CONCLUSIONS: Phylometrics provides a novel data mining method to screen supplied DNA sequences and to identify sequences that are of significant phylogenetic interest using powerful analytical tools. Sequences that are identified as being similar to a number of supplied sequences may provide key insights into their functional or evolutionary relatedness. Users require the same basic computer skills as for navigating most internet applications.

jsPhyloSVG: a javascript library for visualizing interactive and vector-based phylogenetic trees on the web.

BACKGROUND: Many software packages have been developed to address the need for generating phylogenetic trees intended for print. With an increased use of the web to disseminate scientific literature, there is a need for phylogenetic trees to be viewable across many types of devices and feature some of the interactive elements that are integral to the browsing experience. We propose a novel approach for publishing interactive phylogenetic trees. METHODS/PRINCIPAL FINDINGS: We present a javascript library, jsPhyloSVG, which facilitates constructing interactive phylogenetic trees from raw Newick or phyloXML formats directly within the browser in Scalable Vector Graphics (SVG) format. It is designed to work across all major browsers and renders an alternative format for those browsers that do not support SVG. The library provides tools for building rectangular and circular phylograms with integrated charting. Interactive features may be integrated and made to respond to events such as clicks on any element of the tree, including labels. CONCLUSIONS/SIGNIFICANCE: jsPhyloSVG is an open-source solution for rendering dynamic phylogenetic trees. It is capable of generating complex and interactive phylogenetic trees across all major browsers without the need for plugins. It is novel in supporting the ability to interpret the tree inference formats directly, exposing the underlying markup to data-mining services. The library source code, extensive documentation and live examples are freely accessible at www.jsphylosvg.com.

Methanogenic Archaea and human periodontal disease.

Archaea have been isolated from the human colon, vagina, and oral cavity, but have not been established as causes of human disease. In this study, we reveal a relationship between the severity of periodontal disease and the relative abundance of archaeal small subunit ribosomal RNA genes (SSU rDNA) in the subgingival crevice by using quantitative PCR. Furthermore, the relative abundance of archaeal small subunit rDNA decreased at treated sites in association with clinical improvement. Archaea were harbored by 36% of periodontitis patients and were restricted to subgingival sites with periodontal disease. The presence of archaeal cells at these sites was confirmed by fluorescent in situ hybridization. The archaeal community at diseased sites was dominated by a Methanobrevibacter oralis-like phylotype and a distinct Methanobrevibacter subpopulation related to archaea that inhabit the gut of numerous animals. We hypothesize that methanogens participate in syntrophic relationships in the subgingival crevice that promote colonization by secondary fermenters during periodontitis. Because they are potential alternative syntrophic partners, our finding of larger Treponema populations sites without archaea provides further support for this hypothesis.

Single-cell enumeration of an uncultivated TM7 subgroup in the human subgingival crevice.

Specific oligonucleotide hybridization conditions were established for single-cell enumeration of uncultivated TM7 and IO25 bacteria by using clones expressing heterologous 16S rRNA. In situ analysis of human subgingival crevice specimens revealed that a greater proportion of samples from sites of chronic periodontitis than from healthy sites contained TM7 subgroup IO25. In addition, IO25 bacterial cells from periodontitis site samples were more abundant and fourfold longer than IO25 cells from healthy site samples.

Cultivation of Tropheryma whipplei from cerebrospinal fluid.

Whipple disease (WD) is a systemic disorder caused by the bacterium Tropheryma whipplei. Since the recognition of a bacterial etiology in 1961, many attempts have been made to cultivate this bacterium in vitro. It was eventually isolated, in 2000, from an infected heart valve, in coculture with human fibroblasts. Here we report the isolation of 2 new strains of T. whipplei from cerebrospinal fluid (CSF) of 2 patients with intestinal WD but no neurological signs or symptoms. One culture-positive specimen was obtained before treatment; the other was obtained 12 months after discontinuation of therapy, at a time of intestinal remission. In both cases, 15 passages of the cultures were completed over 17 months. Bacterial growth was measured by quantitative polymerase chain reaction, which suggested a generation time of 4 days. Staining with YO-PRO nucleic-acid dye showed characteristic rod-shaped bacteria arranged in chains. Fluorescent in situ hybridization with a T. whipplei-specific oligonucleotide probe, a broad-range bacterial probe, and a nonspecific nucleic-acid stain indicated that all visible bacteria were T. whipplei. Scanning electron microscopy and transmission electron microscopy showed both intracellular and extracellular bacteria. This first isolation of T. whipplei from CSF provides clear evidence of viable bacteria in the central nervous system in individuals with WD, even after prolonged antibiotic therapy.

Prevalence of bacteria of division TM7 in human subgingival plaque and their association with disease.

Members of the uncultivated bacterial division TM7 have been detected in the human mouth, but little information is available regarding their prevalence and diversity at this site. Human subgingival plaque samples from healthy sites and sites exhibiting various stages of periodontal disease were analyzed for the presence of TM7 bacteria. TM7 ribosomal DNA (rDNA) was found in 96% of the samples, and it accounted for approximately 0.3%, on average, of all bacterial rDNA in the samples as determined by real-time quantitative PCR. Two new phylotypes of this division were identified, and members of the division were found to exhibit filamentous morphology by fluorescence in situ hybridization. The abundance of TM7 rDNA relative to total bacterial rDNA was higher in sites with mild periodontitis (0.54% +/- 0.1%) than in either healthy sites (0.21% +/- 0.05%, P < 0.01) or sites with severe periodontitis (0.29% +/- 0.06%, P < 0.05). One division subgroup, the I025 phylotype, was detected in 1 of 18 healthy samples and 38 of 58 disease samples. These data suggest that this phylotype, and the TM7 bacterial division in general, may play a role in the multifactorial process leading to periodontitis.