Single-Cell Enumeration of an Uncultivated TM7 Subgroup in the

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

Whipple's disease and Tropheryma whippelii: secrets slowly revealed.

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Cross-talk in the gut

Modification of host signaling by the gut microbiota can influence weight gain and fat deposition

Modulation of the Host Interferon Response and ISGylation Pathway by B. pertussis Filamentous Hemagglutinin

Bordetella pertussis filamentous hemagglutinin (FHA) is a surface-associated and secreted protein that serves as a crucial adherence factor, and displays immunomodulatory activity in human peripheral blood mononuclear cells (PBMCs). In order to appreciate more fully the role of secreted FHA in pathogenesis, we analyzed FHA-induced changes in genome-wide transcript abundance in human PBMCs. Among the 683 known unique genes with greater than 3-fold change in transcript abundance following FHA treatment, 125 (18.3%) were identified as interferon (IFN)-regulated. Among the latter group were genes encoding several members of the IFN type I response, as well as 3 key components of the ISGylation pathway. Using real-time RT-PCR, we confirmed FHA-associated increases in transcript abundance for the genes encoding ubiquitin-like protein, ISG15, and its specific protease USP18. Western-blot analysis demonstrated the presence of both, free ISG15 and several ISGylated conjugates in FHA-stimulated PBMC lysates, but not in unstimulated cells. Intracellular FACS analysis provided evidence that monocytes and a natural killer-enriched cell population were the primary producers of ISG15 in PBMCs after FHA stimulation. Our data reveal previously-unrecognized effects of B. pertussis FHA on host IFN and ISGylation responses, and suggest previously-unsuspected mechanisms by which FHA may alter the outcome of the host-pathogen interaction

Cultivation of \u3cem\u3eTropheryma whipplei\u3c/em\u3e 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 nucleicacid 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 \u3c 0.01) or sites with severe periodontitis (0.29% ± 0.06%, P \u3c 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

Analysis of Conserved Non-rRNA Genes of \u3ci\u3eTropheryma whipplei\u3c/i\u3e

The causative agent of Whipple’s disease, Tropheryma whipplei, is a slow-growing bacterium that remains poorly-understood. Genetic characterization of this organism has relied heavily upon rRNA sequence analysis. Pending completion of a complete genome sequencing effort, we have characterized several conserved non-rRNA genes from T. whipplei directly from infected tissue using broad-range PCR and a genome-walking strategy. Our goals were to evaluate its phylogenetic relationships, and to find ways to expand the strain typing scheme, based on rDNA sequence comparisons. The genes coding for the ATP synthase beta subunit (atpD), elongation factor Tu (tuf), heat shock protein GroEL (groEL), beta subunit of DNA-dependent RNA polymerase (rpoB), and RNase P RNA (rnpB) were analyzed, as well as the regions upstream and downstream of the rRNA operon. Phylogenetic analyses with all nonrRNA marker molecules consistently placed T. whipplei within the class, Actinobacteria. The arrangement of genes in the atpD and rpoB chromosomal regions was also consistent with other actinomycete genomes. Tandem sequence repeats were found upstream and downstream of the rRNA operon, and downstream of the groEL gene. These chromosomal sites and the 16S-23S rRNA intergenic spacer regions were examined in the specimens of 11 patients, and a unique combination of tandem repeat numbers and spacer polymorphisms was found in each patient. These data provide the basis for a more discriminatory typing method for T. whipplei

Methanogenic \u3cem\u3eArchaea\u3c/em\u3e 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