High prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae detected in the human gut using an improved DNA detection protocol

The low and variable prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae DNA in human stool contrasts with the paramount role of these methanogenic Archaea in digestion processes. We hypothesized that this contrast is a consequence of the inefficiencies of current protocols for ar...

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Published in:PloS one 2009-09, Vol.4 (9), p.e7063-e7063
Main Authors: Dridi, Bédis, Henry, Mireille, El Khéchine, Amel, Raoult, Didier, Drancourt, Michel
Format: Article
Language:English
Subjects:
Adolescent
Adult
Aged
Aged, 80 and over
Archaea
Bacteria
Carbon dioxide
Child
Child, Preschool
Deoxyribonucleic acid
Digestive system
Digestive tract
DNA
DNA, Archaeal - analysis
E coli
Endopeptidase K
Feces
Female
Gastrointestinal tract
Gene Expression Regulation, Bacterial
Gene sequencing
Genes
Genomes
Humans
Infant
Infant, Newborn
Infection - epidemiology
Infection - microbiology
Infectious Diseases
Intestine
Intestines - microbiology
Male
Methanobacteriaceae - metabolism
Methanobrevibacter - metabolism
Methanobrevibacter smithii
Methanogenic archaea
Methanogenic bacteria
Methanosphaera stadtmanae
Microbiological Techniques
Microbiology/Applied Microbiology
Microbiology/Medical Microbiology
Middle Aged
Phylogenetics
Polymerase chain reaction
Proteinase
Reverse Transcriptase Polymerase Chain Reaction - methods
RNA
RNA, Ribosomal, 16S - metabolism
RpoB protein
rRNA 16S
Tuberculosis
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creator Dridi, Bédis
Henry, Mireille
El Khéchine, Amel
Raoult, Didier
Drancourt, Michel
description The low and variable prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae DNA in human stool contrasts with the paramount role of these methanogenic Archaea in digestion processes. We hypothesized that this contrast is a consequence of the inefficiencies of current protocols for archaeon DNA extraction. We developed a new protocol for the extraction and PCR-based detection of M. smithii and M. stadtmanae DNA in human stool. Stool specimens collected from 700 individuals were filtered, mechanically lysed twice, and incubated overnight with proteinase K prior to DNA extraction using a commercial DNA extraction kit. Total DNA was used as a template for quantitative real-time PCR targeting M. smithii and M. stadtmanae 16S rRNA and rpoB genes. Amplification of 16S rRNA and rpoB yielded positive detection of M. smithii in 95.7% and M. stadtmanae in 29.4% of specimens. Sequencing of 16S rRNA gene PCR products from 30 randomly selected specimens (15 for M. smithii and 15 for M. stadtmanae) yielded a sequence similarity of 99-100% using the reference M. smithii ATCC 35061 and M. stadtmanae DSM 3091 sequences. In contrast to previous reports, these data indicate a high prevalence of the methanogens M. smithii and M. stadtmanae in the human gut, with the former being an almost ubiquitous inhabitant of the intestinal microbiome.
doi_str_mv 10.1371/journal.pone.0007063
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We hypothesized that this contrast is a consequence of the inefficiencies of current protocols for archaeon DNA extraction. We developed a new protocol for the extraction and PCR-based detection of M. smithii and M. stadtmanae DNA in human stool. Stool specimens collected from 700 individuals were filtered, mechanically lysed twice, and incubated overnight with proteinase K prior to DNA extraction using a commercial DNA extraction kit. Total DNA was used as a template for quantitative real-time PCR targeting M. smithii and M. stadtmanae 16S rRNA and rpoB genes. Amplification of 16S rRNA and rpoB yielded positive detection of M. smithii in 95.7% and M. stadtmanae in 29.4% of specimens. Sequencing of 16S rRNA gene PCR products from 30 randomly selected specimens (15 for M. smithii and 15 for M. stadtmanae) yielded a sequence similarity of 99-100% using the reference M. smithii ATCC 35061 and M. stadtmanae DSM 3091 sequences. 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We hypothesized that this contrast is a consequence of the inefficiencies of current protocols for archaeon DNA extraction. We developed a new protocol for the extraction and PCR-based detection of M. smithii and M. stadtmanae DNA in human stool. Stool specimens collected from 700 individuals were filtered, mechanically lysed twice, and incubated overnight with proteinase K prior to DNA extraction using a commercial DNA extraction kit. Total DNA was used as a template for quantitative real-time PCR targeting M. smithii and M. stadtmanae 16S rRNA and rpoB genes. Amplification of 16S rRNA and rpoB yielded positive detection of M. smithii in 95.7% and M. stadtmanae in 29.4% of specimens. Sequencing of 16S rRNA gene PCR products from 30 randomly selected specimens (15 for M. smithii and 15 for M. stadtmanae) yielded a sequence similarity of 99-100% using the reference M. smithii ATCC 35061 and M. stadtmanae DSM 3091 sequences. In contrast to previous reports, these data indicate a high prevalence of the methanogens M. smithii and M. stadtmanae in the human gut, with the former being an almost ubiquitous inhabitant of the intestinal microbiome.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>19759898</pmid><doi>10.1371/journal.pone.0007063</doi><tpages>e7063</tpages><oa>free_for_read</oa></addata></record>
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subjects Adolescent
Adult
Aged
Aged, 80 and over
Archaea
Bacteria
Carbon dioxide
Child
Child, Preschool
Deoxyribonucleic acid
Digestive system
Digestive tract
DNA
DNA, Archaeal - analysis
E coli
Endopeptidase K
Feces
Female
Gastrointestinal tract
Gene Expression Regulation, Bacterial
Gene sequencing
Genes
Genomes
Humans
Infant
Infant, Newborn
Infection - epidemiology
Infection - microbiology
Infectious Diseases
Intestine
Intestines - microbiology
Male
Methanobacteriaceae - metabolism
Methanobrevibacter - metabolism
Methanobrevibacter smithii
Methanogenic archaea
Methanogenic bacteria
Methanosphaera stadtmanae
Microbiological Techniques
Microbiology/Applied Microbiology
Microbiology/Medical Microbiology
Middle Aged
Phylogenetics
Polymerase chain reaction
Proteinase
Reverse Transcriptase Polymerase Chain Reaction - methods
RNA
RNA, Ribosomal, 16S - metabolism
RpoB protein
rRNA 16S
Tuberculosis
title High prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae detected in the human gut using an improved DNA detection protocol
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