Dataset Information

Relative abundance of Bacteroides spp. in stools and wastewaters as determined by hierarchical oligonucleotide primer extension.

ABSTRACT: A molecular method, termed hierarchical oligonucleotide primer extension (HOPE), was used to determine the relative abundances of predominant Bacteroides spp. present in fecal microbiota and wastewaters. For this analysis, genomic DNA in feces of healthy human adults, bovines, and swine and in wastewaters was extracted and total bacterial 16S rRNA genes were PCR amplified and used as the DNA templates for HOPE. Nineteen oligonucleotide primers were designed to detect 14 Bacteroides spp. at different hierarchical levels (domain, order, cluster, and species) and were arranged into and used in six multiplex HOPE reaction mixtures. Results showed that species like B. vulgatus, B. thetaiotaomicron, B. caccae, B. uniformis, B. fragilis, B. eggerthii, and B. massiliensis could be individually detected in human feces at abundances corresponding to as little as 0.1% of PCR-amplified 16S rRNA genes. Minor species like B. pyogenes, B. salyersiae, and B. nordii were detected only collectively using a primer that targeted the B. fragilis subgroup (corresponding to approximately 0.2% of PCR-amplified 16S rRNA genes). Furthermore, Bac303-related targets (i.e., most Bacteroidales) were observed to account for 28 to 44% of PCR-amplified 16S rRNA genes from human fecal microbiota, and their abundances were higher than those detected in the bovine and swine fecal microbiota and in wastewaters by factors of five and two, respectively. These results were comparable to those obtained by quantitative PCR and to those reported previously from studies using whole-cell fluorescence hybridization and 16S rRNA clone library methods, supporting the conclusion that HOPE can be a sensitive, specific, and rapid method to determine the relative abundances of Bacteroides spp. predominant in fecal samples.

SUBMITTER: Hong PY

PROVIDER: S-EPMC2394900 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Relative abundance of Bacteroides spp. in stools and wastewaters as determined by hierarchical oligonucleotide primer extension.

Hong Pei-Ying PY Wu Jer-Horng JH Liu Wen-Tso WT

Applied and environmental microbiology 20080314 9

A molecular method, termed hierarchical oligonucleotide primer extension (HOPE), was used to determine the relative abundances of predominant Bacteroides spp. present in fecal microbiota and wastewaters. For this analysis, genomic DNA in feces of healthy human adults, bovines, and swine and in wastewaters was extracted and total bacterial 16S rRNA genes were PCR amplified and used as the DNA templates for HOPE. Nineteen oligonucleotide primers were designed to detect 14 Bacteroides spp. at diffe ...[more]

PMID: 18344347

Similar Datasets

Project description:BackgroundPatients with pelvic malignancies often receive radiosensitising chemotherapy with radiotherapy to improve survival; however, this is at the expense of increased normal tissue toxicity, particularly in elderly patients. Here, we explore if an alternative, low-cost, and non-toxic approach can achieve radiosensitisation in mice transplanted with human bladder cancer cells. Other investigators have shown slower growth of transplanted tumours in mice fed high-fibre diets. We hypothesised that mice fed a high-fibre diet would have improved tumour control following ionising radiation (IR) and that this would be mediated through the gut microbiota.ResultsWe investigated the effects of four different diets (low-fibre, soluble high-fibre, insoluble high-fibre, and mixed soluble/insoluble high-fibre diets) on tumour growth in immunodeficient mice implanted with human bladder cancer flank xenografts and treated with ionising radiation, simultaneously investigating the composition of their gut microbiomes by 16S rRNA sequencing. A significantly higher relative abundance of Bacteroides acidifaciens was seen in the gut (faecal) microbiome of the soluble high-fibre group, and the soluble high-fibre diet resulted in delayed tumour growth after irradiation compared to the other groups. Within the soluble high-fibre group, responders to irradiation had significantly higher abundance of B. acidifaciens than non-responders. When all mice fed with different diets were pooled, an association was found between the survival time of mice and relative abundance of B. acidifaciens. The gut microbiome in responders was predicted to be enriched for carbohydrate metabolism pathways, and in vitro experiments on the transplanted human bladder cancer cell line suggested a role for microbial-generated short-chain fatty acids and/or other metabolites in the enhanced radiosensitivity of the tumour cells.ConclusionsSoluble high-fibre diets sensitised tumour xenografts to irradiation, and this phenotype was associated with modification of the microbiome and positively correlated with B. acidifaciens abundance. Our findings might be exploitable for improving radiotherapy response in human patients.

Project description:Nonenzymatic RNA primer extension by activated mononucleotides has long served as a model for the study of prebiotic RNA copying. We have recently shown that the rate of primer extension is greatly enhanced by the formation of an imidazolium-bridged dinucleotide between the incoming monomer and a second, downstream activated monomer. However, the rate of primer extension is further enhanced if the downstream monomer is replaced by an activated oligonucleotide. Even an unactivated downstream oligonucleotide provides a modest enhancement in the rate of reaction of a primer with a single activated monomer. Here we study the mechanism of these effects through crystallographic studies of RNA complexes with the recently synthesized nonhydrolyzable substrate analog, guanosine 5'-(4-methylimidazolyl)-phosphonate (ICG). ICG mimics 2-methylimidazole activated guanosine-5'-phosphate (2-MeImpG), a commonly used substrate in nonenzymatic primer extension experiments. We present crystal structures of primer-template complexes with either one or two ICG residues bound downstream of a primer. In both cases, the aryl-phosphonate moiety of the ICG adjacent to the primer is disordered. To investigate the effect of a downstream oligonucleotide, we transcribed a short RNA oligonucleotide with either a 5'-ICG residue, a 5'-phosphate or a 5'-hydroxyl. We then determined crystal structures of primer-template complexes with a bound ICG monomer sandwiched between the primer and each of the three downstream oligonucleotides. Surprisingly, all three oligonucleotides rigidify the ICG monomer conformation and position it for attack by the primer 3'-hydroxyl. Furthermore, when GpppG, an analog of the imidazolium-bridged intermediate, is sandwiched between an upstream primer and a downstream helper oligonucleotide, or covalently linked to the 5'-end of the downstream oligonucleotide, the complex is better preorganized for primer extension than in the absence of a downstream oligonucleotide. Our results suggest that a downstream helper oligonucleotide contributes to the catalysis of primer extension by favoring a reactive conformation of the primer-template-intermediate complex.

Project description:DNA methylation age (DNAm age, epigenetic clock) is a novel and promising biomarker of aging. It is calculated from the methylation fraction of specific cytosine phosphate guanine sites (CpG sites) of genomic DNA. Several groups have proposed epigenetic clock algorithms and these differ mostly regarding the number and location of the CpG sites considered and the method used to assess the methylation status. Most epigenetic clocks are based on a large number of CpGs, e.g. as measured by DNAm microarrays. We have recently evaluated an epigenetic clock based on the methylation fraction of seven CpGs that were determined by methylation-sensitive single nucleotide primer extension (MS-SNuPE). This method is more cost-effective when compared to array-based technologies as only a few CpGs need to be examined. However, there is only little data on the correspondence in epigenetic age estimation using the 7-CpG clock and other algorithms. To bridge this gap, in this study we measured the 7-CpG DNAm age using two methods, via MS-SNuPE and via the MethylationEPIC array, in a sample of 1,058 participants of the Berlin Aging Study II (BASE-II), assessed as part of the GendAge study. On average, participants were 75.6 years old (SD: 3.7, age range: 64.9-90.0, 52.6% female). Agreement between methods was assessed by Bland-Altman plots. DNAm age was highly correlated between methods (Pearson's r = 0.9) and Bland-Altman plots showed a difference of 3.1 years. DNAm age by the 7-CpG formula was 71.2 years (SD: 6.9 years, SNuPE) and 68.1 years (SD: 6.4 years, EPIC array). The mean of difference in methylation fraction between methods for the seven individual CpG sites was between 0.7 and 13 percent. To allow direct conversion of DNAm age obtained from both methods we developed an adjustment formula with a randomly selected training set of 529 participants using linear regression. After conversion of the Illumina data in a second and independent validation set, the adjusted DNAm age was 71.44 years (SD: 6.1 years, n = 529). In summary, we found the results of DNAm clocks to be highly comparable. Furthermore, we developed an adjustment formula that allows for direct conversion of DNAm age estimates between methods and enables one singular clock to be used in studies that employ either the Illumina or the SNuPE method.

Dataset Information

Relative abundance of Bacteroides spp. in stools and wastewaters as determined by hierarchical oligonucleotide primer extension.

Publications

Relative abundance of Bacteroides spp. in stools and wastewaters as determined by hierarchical oligonucleotide primer extension.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets