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International audience; Orthopedic prosthesis infection must be medically managed after appropriate microbiological documentation. While bacteria and fungi are acknowledged to be causative opportunistic pathogens in this situation, the potential role of methanogens in orthopedic prosthesis infections is still unknown. In a retrospective study, a total of 100 joint and bone samples collected from 25 patients were screened by specific PCR assays for the detection of methanogens. PCR-positive samples were observed by autofluorescence, electron microscopy and tentatively cultured under specific culture conditions. Methanogens were detected by quantitative PCR in 4/100 samples, in the presence of negative controls. Sequencing identified Methanobrevibacter oralis in two cases, Methanobrevibacter smithii in one case and Methanobrevibacter wolinii in one case. Microscopic methods confirmed molecular findings and bacterial culture yielded two strains of Staphylococcus aureus, one strain of Staphylococcus epidermidis and one strain of Proteus mirabilis. These unprecedented data highlight the presence of methanogens in joint and bone samples of patients also diagnosed with bacterial orthopedic prosthesis infection, questioning the role of methanogens as additional opportunistic co-pathogens in this situation.

Orthopedic prosthesis infection must be medically managed after appropriate microbiological documentation. While bacteria and fungi are acknowledged to be causative opportunistic pathogens in this situation, the potential role of methanogens in orthopedic prosthesis infections is still unknown. In a retrospective study, a total of 100 joint and bone samples collected from 25 patients were screened by specific PCR assays for the detection of methanogens. PCR-positive samples were observed by autofluorescence, electron microscopy and tentatively cultured under specific culture conditions. Methanogens were detected by quantitative PCR in 4/100 samples, in the presence of negative controls. Sequencing identified Methanobrevibacter oralis in two cases, Methanobrevibacter smithii in one case and Methanobrevibacter wolinii in one case. Microscopic methods confirmed molecular findings and bacterial culture yielded two strains of Staphylococcus aureus, one strain of Staphylococcus epidermidis and one strain of Proteus mirabilis. These unprecedented data highlight the presence of methanogens in joint and bone samples of patients also diagnosed with bacterial orthopedic prosthesis infection, questioning the role of methanogens as additional opportunistic co-pathogens in this situation.

Reduction in greenhouse gas emission from beef production is essential to the survival of the beef industry from environmental and social-economic perspectives. There are different systems available to measure methane from animals, but they are expensive, not easily accessible, and not suitable for large-scale methane measurements on the farm. Therefore exploring indicator traits, which are easy to measure, cost-effective, and suitable for large-scale measurement, are recommended. The objectives of this study were to examine the diversity of fecal methanogen profile among efficient and inefficient beef heifers on pasture and investigate methanogen profile as a possible proxy to predict methane emission in beef cattle consuming a forage diet. Forty pregnant (1st trimester) heifers previously classified for postweaning residual feed intake adjusted for off-test back fat (RFIfat; 20 high and 20 low) were included in this study. To determine individual pasture grazing intake, heifers were dosed with 1 kg of C32 labeled pellets once per day from Day 0 to Day 12, and fecal samples were collected twice daily from Day 8 to Day 15. Fecal samples from Days 8, 10, and 12 were analyzed for their methanogen profile. Animals were monitored individually for methane and carbon dioxide production using a GreenFeed Emissions Monitoring system. Total methanogen population and methanogenic community diversity of fecal samples were not different (P > 0.1) between low and high RFIfat groups, as measured by quantitative PCR and α- and β-diversity indices. However, both groups had a different methanogen profile; the relative abundance of Methanobrevibacter wolinii and relatives were higher (P

MethanobrevibacterandMethanosphaeraspecies represent some of the most prevalent methanogenic archaea in the gastrointestinal tract of animals and humans and play an important role in this environment. The aim of this study was to identify genomic features that are shared or specific for members of each genus with a special emphasis of the analysis on the assimilation of nitrogen and acetate and the utilization of methanol and ethanol for methanogenesis. Here, draft genome sequences ofMethanobrevibacter thaueristrain DSM 11995T,Methanobrevibacter woeseistrain DSM 11979T, andMethanosphaera cuniculistrain 4103Tare reported and compared to those of 16 otherMethanobrevibacterandMethanosphaeragenomes, including genomes of the 13 currently available types of strains of the two genera. The comparative genome analyses indicate that among other genes, the absence of molybdopterin cofactor biosynthesis is conserved inMethanosphaeraspecies but reveals also that the three species share a core set of more than 300 genes that distinguishes the genusMethanosphaerafrom the genusMethanobrevibacter. Multilocus sequence analysis shows that the genusMethanobrevibactercan be subdivided into clades, potentially new genera, which may display characteristic specific metabolic features. These features include not only the potential ability of nitrogen fixation and acetate assimilation in a clade comprised ofMethanobrevibacterspecies from the termite gut andMethanobrevibacter arboriphilusstrains but also the potential capability to utilize ethanol and methanol in a clade comprisingMethanobrevibacter woliniistrain DSM 11976T,Mbb.sp. AbM4, andMbb. boviskoreanistrain DSM 25824T.

An in vivo study was conducted to compare the enteric methane emissions and diversity of ruminal methanogens in cattle and buffaloes kept in the same environment and fed on the same diet. Six cattle and six buffaloes were fed on a similar diet comprising Napier (Pennisetum purpureum) green grass and concentrate in 70:30. After 90 days of feeding, the daily enteric methane emissions were quantified by using the SF6 technique and ruminal fluid samples from animals were collected for the diversity analysis. The daily enteric methane emissions were significantly greater in cattle as compared to buffaloes; however, methane yields were not different between the two species. Methanogens were ranked at different taxonomic levels against the Rumen and Intestinal Methanogen-Database. The archaeal communities in both host species were dominated by the phylum Euryarchaeota; however, Crenarchaeota represented Methanobacteriales were most prominent and their proportion did not differ between the two hosts. Methanomicrobiales and Methanomassillicoccales constituted the second largest group of methanogens in cattle and buffaloes, respectively. Methanocellales (Methanocella arvoryza) were exclusively detected in the buffaloes. At the species level, Methanobrevibacter gottschalkii had the highest abundance (55–57%) in both the host species. The relative abundance of Methanobrevibacter wolinii between the two hosts differed significantly. Methanosarcinales, the acetoclastic methanogens were significantly greater in cattle than the buffaloes. It is concluded that the ruminal methane yield in cattle and buffaloes fed on the same diet did not differ. With the diet used in this study, there was a limited influence (

The primary objective of this study was to investigate the effect of dietary fiber on methanogenic diversity and community composition in the hindgut of indigenous Chinese Lantang gilts to explain the unexpected findings reported earlier that Lantang gilts fed low-fiber diet (LFD) produced more methane than those fed high-fiber diet (HFD). In total, 12 Lantang gilts (58.7±0.37 kg) were randomly divided into two dietary groups (six replicates (pigs) per group) and fed either LFD (NDF=201.46 g/kg) or HFD (NDF=329.70 g/kg). Wheat bran was the main source of fiber for the LFD, whereas ground rice hull (mixture of rice hull and rice bran) was used for the HFD. Results showed that the methanogens in the hindgut of Lantang gilts belonged to four known species (Methanobrevibacter ruminantium, Methanobrevibacter wolinii, Methanosphaera stadtmanae and Methanobrevibacter smithii), with about 89% of the methanogens belonging to the genus Methanobrevibacter. The 16S ribosomal RNA (rRNA) gene copies of Methanobrevibacter were more than three times higher (P0.05) was observed in 16S rRNA gene copies of Fibrobacter succinogenes between the two dietary groups, and 18S rRNA gene copies of anaerobic fungi in gilts fed LFD were lower than (P

The β-1, 3-glucan from yeast has been extensively examined for its immuno-enhancing effects in animals. However, investigation on the relationship among β-glucan, gut microbiota and immune-modulating effects remains limited particularly in pigs. Considering the critical roles of gut methanogens in the microbial fermentation, energy metabolism and disease resistance, we investigated the phylogenetic diversity of methanogens from fermented cultures of porcine colonic digesta with (G) or without (N) yeast β-glucan based on sequences of the archaeal 16S rRNA gene. A total of 145 sequences in the G library were assigned into 8 operational taxonomic units (OTUs) with the majority of sequences (114/145) related to strains ＂Methanobrevibacter millerae＂ or ＂Methanobrevibacter gottschalkii＂ with high identities ranging from 97.9 to 98.6%, followed by 23 sequences to ＂Methanobrevibacter ruminantium＂, 2 sequences to ＂Methanobrevibacter smithii＂ and one sequence to ＂Methanobrevibacter wolinii＂. The 142 sequences in the N library were assigned to 2 OTUs with most sequences (127/142) related to strains ＂M. millerae＂ or ＂M. gottschalkii＂ with sequence identities ranging from 97.9 to 98.5%, and 15 sequences related to ＂M. gottschalkii＂ with 97.9% identity. Shannon diversity index showed that the G library exhibited significantly higher archaeal diversity (P＜0.05) and Libshuff analysis indicated the differences in the community structure between the two libraries were significant (P＜0.0001). In conclusion, the current study provides evidence that addition of yeast β-glucan significantly increased the diversity of methanogens in ＂in vitro＂ fermented porcine colonic digesta.

Methanobrevibacter sp. AbM4 was originally isolated from the abomasal contents of a sheep and was chosen as a representative of the Methanobrevibacter wolinii clade for genome sequencing. The AbM4 genome is smaller than that of the rumen methanogen M. ruminantium M1 (2.0 Mb versus 2.93 Mb), encodes fewer open reading frames (ORFs) (1,671 versus 2,217) and has a lower G+C percentage (29% versus 33%). Overall, the composition of the AbM4 genome is very similar to that of M1 suggesting that the methanogenesis pathway and central metabolism of these strains are highly similar, and both organisms are likely to be amenable to inhibition by small molecule inhibitors and vaccine-based methane mitigation technologies targeting these conserved features. The main differences compared to M1 are that AbM4 has a complete coenzyme M biosynthesis pathway and does not contain a prophage or non-ribosomal peptide synthase genes. However, AbM4 has a large CRISPR region and several type I and type II restriction-modification system components. Unusually, DNA-directed RNA polymerase B' and B'' subunits of AbM4 are joined, a feature only previously observed in some thermophilic archaea. AbM4 has a much reduced complement of genes encoding adhesin-like proteins which suggests it occupies a ruminal niche different from that of M1.

Three methanogen 16S rRNA gene clone libraries were constructed from liquid (LM), solid (SM) and epithelium (EM) fractions taken from the rumen of Jinnan cattle in China. After the amplification by PCR using methanogen-specific primers Met86F and Met1340R, equal quantities of PCR products from the same fractions from each of the four cattle were mixed together and used to construct the three libraries. Sequence analysis showed that the 268 LM clones were divided into 35 phylotypes with 18 sequences of phylotypes affiliated with the genus Methanobrevibacter (84.3% of clones). The 135 SM clones were divided into 19 phylotypes with 11 phylotypes affiliated with the genus Methanobrevibacter (77.8%). The 267 EM clones were divided into 33 phylotypes with 15 phylotypes affiliated with the genus Methanobrevibacter (77.2%). Clones closely related to Methanomicrobium mobile and Methanobrevibacter wolinii were only found in the LM library, and those to Methanobrevibacter ruminantium and Methanobrevibacter gottschalkii only in the SM library. LM library comprised 12.4% unidentified euryarchaeal clones, SM library 23.7% and EM library 25.5%, respectively. Five phylotypes (accession number: EF055528 and EF055531–EF055534) did not belong to the Euryarchaeota sequences we had known. One possible new genus (represented by phylotype E17, accession number EF055528) belonging to Methanobacteriaceae was identified from EM library. Quantitative real-time PCR for the first time revealed that epithelium fraction had significantly higher density of methanogens, with methanogenic mcrA gene copies (9.95 log 10 (copies per gram of wet weight)) than solid (9.26, P < 0.01) and the liquid (8.44, P < 0.001). The three clone libraries also appeared different in Shannon index (EM library 2.12, LM library 2.05 and SM library 1.73). Our results showed that there were apparent differences in the methanogenic diversity and abundance in the three different fractions within the rumen of Jinnan cattle, with Methanobrevibacter species predominant in all the three libraries and with epithelium fraction having more unknown species and higher density of methanogens.

Formal nomenclature is proposed for five methanogens, isolated from horse, pig, cow, goose and sheep faeces, that represent four novel species of the genus Methanobrevibacter. The four species, Methanobrevibacter gottschalkii sp. nov., Methanobrevibacter thaueri sp. nov., Methanobrevibacter woesei sp. nov. and Methanobrevibacter wolinii sp. nov., are distinguished from each other by a lack of genomic DNA reassociation and from previously described members of the genus on the basis of differences in the sequences of the 16S rRNA genes.

Three strictly anaerobic, methanogenic strains JH1T, JH4 and JH8 were isolated from rumen of the Korean native cattle (HanWoo; Bos taurus coreanae) in South Korea. The colonies were circular, opaque, and slightly yellowish. Phylogenetic analyses of 16S rRNA gene and mcrA (encoding α subunit of methyl-coenzyme M reductase) sequences confirmed the affiliation of the novel strains with the Methanobacteriales , and Methanobrevibacter wolinii SHT was the most closely related species. The 16S rRNA gene and mcrA sequence similarities between strains JH1T, JH4 and JH8 and M. wolinii SHT were 96.2 and 89.0 % respectively, and DNA–DNA hybridization of the isolates and M. wolinii DSM 11976T showed a 20 % reassociation. Strain JH1T exhibited 92 % DNA–DNA relatedness with strains JH4 and JH8, and their 16S rRNA gene and mcrA sequences were identical. Cells stained Gram-positive and were non-motile rods, 1.5–1.8 µm long and 0.6 µm wide. The strains were able to use H2/CO2 and formate. The optimum temperature and pH ranges for growth were 37–40 °C and pH 6.5–7.0. The DNA G+C content of strain JH1T was 28 mol%. Based on data from this study using a polyphasic approach, the three strains represent a novel species of genus Methanobrevibacter , for which the name Methanobrevibacter boviskoreani sp. nov. is proposed. The type strain is JH1T ( = KCTC 4102T = JCM 18376T).

Microbial consortia in a two-phase, anaerobic bioreactor using a mixture of fruit and vegetable wastes were established. Bacterial and archaeal communities obtained by a culture-independent approach based on single strand conformation polymorphism analysis of total 16S rDNA showed the adaptation of the microflora to the process parameters. Throughout the 90 d of the study, the species composition of the bacterial community changed significantly. Bacterial 16S rDNA showed at least 7 different major species with a very prominent one corresponding to a Megasphaera elsdenii whereas bacterial 16S rDNA of a methanization bioreactor showed 10 different major species. After two weeks, Prevotella ruminicola became major and its dominance increased continuously until day 50. After an acid shock at pH 5, the 16S rDNA archaeal patterns in the acidogenic reactor showed two major prominent species corresponding to Methanosphaera stadtmanii and Methanobrevibacter wolinii, a hydrogenotrophic bacterium.

Background Methane emissions by methanogen from livestock ruminants have significantly contributed to the agricultural greenhouse gas effect. It is worthwhile to compare methanogen from “energy-saving” animal (yak) and normal animal (cattle) in order to investigate the link between methanogen structure and low methane production. Results Diversity of methanogens from the yak and cattle rumen was investigated by analysis of 16S rRNA gene sequences from rumen digesta samples from four yaks (209 clones) and four cattle (205 clones) from the Qinghai-Tibetan Plateau area (QTP). Overall, a total of 414 clones (i.e. sequences) were examined and assigned to 95 operational taxonomic units (OTUs) using MOTHUR, based upon a 98% species-level identity criterion. Forty-six OTUs were unique to the yak clone library and 34 OTUs were unique to the cattle clone library, while 15 OTUs were found in both libraries. Of the 95 OTUs, 93 putative new species were identified. Sequences belonging to the Thermoplasmatales-affiliated Linage C (TALC) were found to dominate in both libraries, accounting for 80.9% and 62.9% of the sequences from the yak and cattle clone libraries, respectively. Sequences belonging to the Methanobacteriales represented the second largest clade in both libraries. However, Methanobrevibacter wolinii (QTPC 110) was only found in the cattle library. The number of clones from the order Methanomicrobiales was greater in cattle than in the yak clone library. Although the Shannon index value indicated similar diversity between the two libraries, the Libshuff analysis indicated that the methanogen community structure of the yak was significantly different than those from cattle. Conclusion This study revealed for the first time the molecular diversity of methanogen community in yaks and cattle in Qinghai-Tibetan Plateau area in China. From the analysis, we conclude that yaks have a unique rumen microbial ecosystem that is significantly different from that of cattle, this may also help to explain why yak produce less methane than cattle.