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      A new methanogen “ Methanobrevibacter massiliense” isolated in a case of severe periodontitis

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          Abstract

          Background

          A few methanogens have been previously recovered from periodontitis lesions, yet their repertoire may not be completed. We recovered a previously unreported methanogen species in this situation.

          Case presentation

          A 64-year-old Caucasian woman was diagnosed with chronic, severe generalized periodontitis. In the presence of negative controls, an 18-month culture of periodontal pockets in anaerobe Hungate tube yielded “ Methanobrevibacter massiliense” and Pyramidobacter piscolens.

          Conclusions

          This case report provides evidence of the symbiotic strategy deployed by the methanogens and the anaerobes, and reports the first culture of a new methanogen, “ M. massiliense”.

          Electronic supplementary material

          The online version of this article (10.1186/s13104-017-2980-3) contains supplementary material, which is available to authorized users.

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          Most cited references 17

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          Methanomassiliicoccus luminyensis gen. nov., sp. nov., a methanogenic archaeon isolated from human faeces.

          During attempts to obtain novel, human-associated species of the domain Archaea, a coccoid micro-organism, designated strain B10(T), was isolated in pure culture from a sample of human faeces collected in Marseille, France. On the basis of its phenotypic characteristics and 16S rRNA and mcrA gene sequences, the novel strain was classified as a methanogenic archaeon. Cells of the strain were non-motile, Gram-staining-positive cocci that were approximately 850 nm in diameter and showed autofluorescence at 420 nm. Cells were lysed by 0.1% (w/v) SDS. With hydrogen as the electron donor, strain B10(T) produced methane by reducing methanol. The novel strain was unable to produce methane when hydrogen or methanol was the sole energy source. In an atmosphere containing CO(2), strain B10(T) could not produce methane from formate, acetate, trimethylamine, 2-butanol, 2-propanol, cyclopentanol, 2-pentanol, ethanol, 1-propanol or 2,3-butanediol. Strain B10(T) grew optimally with 0.5-1.0% (w/v) NaCl, at pH 7.6 and at 37 °C. It required tungstate-selenite for growth. The complete genome of the novel strain was sequenced; the size of the genome was estimated to be 2.05 Mb and the genomic DNA G+C content was 59.93 mol%. In phylogenetic analyses based on 16S rRNA gene sequences, the highest sequence similarities (98.0-98.7%) were seen between strain B10(T) and several uncultured, methanogenic Archaea that had been collected from the digestive tracts of a cockroach, a chicken and mammals. In the same analysis, the non-methanogenic 'Candidatus Aciduliprofundum boonei' DSM 19572 was identified as the cultured micro-organism that was most closely related to strain B10(T) (83.0% 16S rRNA gene sequence similarity). Each of the three treeing algorithms used in the analysis of 16S rRNA gene sequences indicated that strain B10(T) belongs to a novel order that is distinct from the Thermoplasmatales. The novel strain also appeared to be distinct from Methanosphaera stadtmanae DSM 3091(T) (72.9% 16S rRNA gene sequence similarity), another methanogenic archaeon that was isolated from human faeces and can use methanol in the presence of hydrogen. Based on the genetic and phenotypic evidence, strain B10(T) represents a novel species of a new genus for which the name Methanomassiliicoccus luminyensis gen. nov., sp. nov. is proposed. The type strain of the type species is B10(T) ( = DSM 24529(T) = CSUR P135(T)).
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            The mcrA gene as an alternative to 16S rRNA in the phylogenetic analysis of methanogen populations in landfill.

            Inferred amino acid sequences of the methyl coenzyme-M reductase (mcrA) gene from five different methanogen species were aligned and two regions with a high degree of homology flanking a more variable region were identified. Analysis of the DNA sequences from the conserved regions yielded two degenerate sequences from which a forward primer, a 32-mer, and a reverse primer, a 23-mer, could be derived for use in the specific PCR-based detection of methanogens. The primers were successfully evaluated against 23 species of methanogen representing all five recognized orders of this group of Archaea, generating a PCR product between 464 and 491 bp. Comparisons between the mcrA and 16S small subunit rRNA gene sequences using PHYLIP demonstrated that the tree topologies were strikingly similar. Methods were developed to enable the analysis of methanogen populations in landfill using the mcrA gene as the target. Two landfill sites were examined and 63 clones from a site in Mucking, Essex, and 102 from a site in Odcombe, Somerset, were analysed. Analysis revealed a far greater diversity in the methanogen population within landfill material than has been seen previously.
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              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. Methodology/Principal Findings 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/Significance 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.
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                Author and article information

                Contributors
                tt.hong.huynh@gmail.com
                marion.pignoly@ap-hm.fr
                michel.drancourt@univ-amu.fr
                Gerard.aboudharam@univ-amu.fr
                Journal
                BMC Res Notes
                BMC Res Notes
                BMC Research Notes
                BioMed Central (London )
                1756-0500
                1 December 2017
                1 December 2017
                2017
                : 10
                Affiliations
                [1 ]ISNI 0000 0001 2176 4817, GRID grid.5399.6, UFR Odontologie, , Aix-Marseille Université, ; 27, Boulevard Jean Moulin, Marseille Cedex 5, France
                [2 ]ISNI 0000 0001 2176 4817, GRID grid.5399.6, URMITE, CNRS, UMR 7278, IRD 198, IHU Méditerranée-Infection, , Aix-Marseille Université, ; Marseille, France
                Article
                2980
                10.1186/s13104-017-2980-3
                5710069
                29191236
                © The Author(s) 2017

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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                Case Report
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                © The Author(s) 2017

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