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


RESULTS
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.


CONCLUSIONS
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.
Dridi
Bédis
B
Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes UMR CNRS 6236 IRD 3R198, IFR 48, Faculté de Médecine, Université de la Méditerranée and Pôle des Maladies Infectieuses, Assistance Publique-Hôpitaux de Marseille, Marseille, France.
Henry
Mireille
M
El Khéchine
Amel
A
Raoult
Didier
D
Drancourt
Michel
M
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002648:Child; D002675:Child, Preschool; D019641:DNA, Archaeal / Q000032:analysis*; D005243:Feces; D005260:Female; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D007239:Infections / Q000453:epidemiology* / Q000382:microbiology*; D007422:Intestines / Q000382:microbiology*; D008297:Male; D017013:Methanobacteriaceae / Q000378:metabolism*; D041581:Methanobrevibacter / Q000378:metabolism*; D008828:Microbiological Techniques; D008875:Middle Aged; D012336:RNA, Ribosomal, 16S / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D019641:DNA, Archaeal; D012336:RNA, Ribosomal, 16S