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Background Methanobrevibacter smithii ( M. smithii ), the predominant methanogen in the human digestive tract, plays a key role in methane production. Despite its importance, the genomic diversity of M. smithii is poorly characterised, especially in extra-digestive sites such as the urinary and respiratory tracts, and the blood. Understanding this diversity would help unravel its potential role in human health and diseases. Results We report the genome of M. smithii strain U29, isolated from urine, expanding the known diversity of the species. The M. smithii U29 genome (scaffold level; 1̵822‒124-bp, 1745 protein-coding sequences) lacks Candidatus Nanopusillus sequences, unlike digestive tract strains. Comparative analysis has revealed a high similarity (99.86% ANI) with the reference M. smithii strain ATCC 35,061, although U29 contains 71 unique coding sequences including 12/71 (13 200-bp; 69% of the total extra-material size) with demonstrated Siphoviridae viral ancestry. Accordingly, M. smithii U29 has been identified as an intermediate M. smithii cell variant, exhibiting genomic traits potentially conferring adaptability to the urinary tract. Conclusion This study enhances our understanding of M. smithii genomic diversity and highlights the presence of viral sequences in the urinary tract M. smithii strain U29. These findings open up a hypothesis that viral integration may play a role in M. smithii mucosae colonisation and dynamics, underscoring the need for further investigations into the mechanisms underlying its tissue translocation and potential health implications.

The human endogenous intestinal microflora is an essential \"organ\" in providing nourishment, regulating epithelial development, and instructing innate immunity; yet, surprisingly, basic features remain poorly described. We examined 13,355 prokaryotic ribosomal RNA gene sequences from multiple colonic mucosal sites and feces of healthy subjects to improve our understanding of gut microbial diversity. A majority of the bacterial sequences corresponded to uncultivated species and novel microorganisms. We discovered significant intersubject variability and differences between stool and mucosa community composition. Characterization of this immensely diverse ecosystem is the first step in elucidating its role in health and disease.

The oral fluid microbiome comprises an important bacterial diversity, yet the presence of archaea has not been reported so far. In order to quest for the presence of methanogenic archaea (methanogens) in oral fluid, we used a polyphasic approach including PCR-sequencing detection, microscopic observation by fluorescence in-situ hybridization, isolation and culture, molecular identification and genotyping of methanogens in 200 oral fluid specimens. In the presence of negative controls, 64/200 (32%) prospectively analysed oral fluid specimens were PCR-positive for methanogens, all identified as Methanobrevibacter oralis by sequencing. Further, fluorescence in-situ hybridization detected methanogens in 19/48 (39.6%) investigated specimens; with morphology suggesting M. oralis in 10 cases and co-infecting Methanobrevibacter smithii in nine cases. M. oralis was cultured from 46/64 (71.8%) PCR-positive specimens and none of PCR-negative specimens; and one M. smithii isolate was co-cultured with M. oralis in one specimen. Multispacer Sequence Typing found one M. oralis genotype per specimen and a total of five different genotypes with 19/46 (41%) of isolates all belonging to spacer-type four. Statistical analyses showed a significant correlation between the PCR-detection of methanogens in oral fluid and tobacco smoking. These data indicate that M. oralis and M. smithii are oral fluid-borne methanogens in tobacco smokers. Both methanogens could be transmitted during intimate contacts such as mother-to-child contacts and kissing.

To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region ( E. coli 341–534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions.

Background The oral cavity of humans is inhabited by several hundreds of bacterial species and other microorganisms such as fungi and archaeal methanogens. Regarding methanogens, data have been obtained from oral cavity samples collected in Europe, America and Asia. There is no study published on the presence of methanogens in the oral cavity in persons living in Africa. The objective of our study was to bring new knowledge on the distribution of oral methanogens in persons living in Mali, Africa. Methods A total of 31 patients were included in the study during a 15-day collection period in September. Bacterial investigations consisted in culturing the bacteria in 5% sheep blood–enriched Columbia agar and PolyViteX agar plates. For archaeal research, we used various methods including culture, molecular biology and fluorescent in situ hybridization (FISH). Results Eight of 31 (26%) oral samples collected in eight patients consulting for stomatology diseases tested positive in polymerase chain-reaction (PCR)-based assays for methanogens including five cases of Methanobrevibacter oralis and one case each of Methanobrevibacter smithii , Methanobrevibacter massiliense and co-infection Methanobrevibacter oralis and Methanobrevibacter massiliense . Conclusions In this pilot study, we are reporting here the first characterization of methanogens in the oral cavity in eight patients in Mali. These methanogen species have already been documented in oral specimens collected from individuals in Europe, Asia, North America and Brazil.

Purpose Among irritable bowel syndrome (IBS) patients, breath methane producers overwhelmingly have constipation predominance (C-IBS). Although the most common methanogen in humans is Methanobrevibacter smithii , incidence and type of methanogenic bacteria in C-IBS patients are unknown. Methods By use of a questionnaire and lactulose breath testing, subjects with Rome II C-IBS and methane (>3 ppm) were selected ( n  = 9). The control group included subjects with IBS who had no breath methane ( n  = 10). Presence of bacterial DNA was assessed in a stool sample of each subject by quantitative-PCR using universal 16S rDNA primer. M. smithii was quantified by use of a specific rpoB gene primer. Results M. smithii was detected in both methane and non-methane subjects. However, counts and relative proportion of M. smithii were significantly higher for methane-positive than for methane-negative subjects (1.8 × 10 7  ± 3.0 × 10 7 vs 3.2 × 10 5  ± 7.6 × 10 5  copies/g wet stool, P  < 0.001; and 7.1 ± 6.3 % vs 0.24 ± 0.47 %, P  = 0.02 respectively). The minimum threshold of M. smithii resulting in positive lactulose breath testing for methane was 4.2 × 10 5  copies/g wet stool or 1.2 % of total stool bacteria. Finally, area-under-curve for breath methane correlated significantly with both absolute quantity and percentage of M. smithii in stool ( R  = 0.76; P  < 0.001 and R  = 0.77; P  < 0.001 respectively). Conclusions M. smithii is the predominant methanogen in C-IBS patients with methane on breath testing. The number and proportion of M. smithii in stool correlate well with amount of breath methane.

Non-lactating dairy cattle were transitioned to a high-concentrate diet to investigate the effect of ruminai pH suppression, commonly found in dairy cattle, on the density, diversity, and community structure of rumen methanogens, as well as the density of rumen protozoa. Four ruminally cannulated cows were fed a hay diet and transitioned to a 65% grain and 35% hay diet. The cattle were maintained on an high-concentrate diet for 3 weeks before the transition back to an hay diet, which was fed for an additional 3 weeks. Rumen fluid and solids and fecal samples were obtained prior to feeding during weeks 0 (hay), 1, and 3 (high-concentrate), and 4 and 6 (hay). Subacute ruminai acidosis was induced during week 1. During week 3 of the experiment, there was a significant increase in the number of protozoa present in the rumen fluid (P= 0.049) and rumen solids (P= 0.004), and a significant reduction in protozoa in the rumen fluid in week 6 (P=0.003). No significant effect of diet on density of rumen methanogens was found in any samples, as determined by real-time PCR. Clone libraries were constructed for weeks 0, 3, and 6, and the methanogen diversity of week 3 was found to differ from week 6. Week 3 was also found to have a significantly altered methanogen community structure, compared to the other weeks. Twenty-two unique 16S rRNA phylotypes were identified, three of which were found only during high-concentrate feeding, three were found during both phases of hay feeding, and seven were found in all three clone libraries. The genus Methanobrevibacter comprised 99% of the clones present. The rumen fluid at weeks 0, 3, and 6 of all the animals was found to contain a type A protozoal population. Ultimately, high-concentrate feeding did not significantly affect the density of rumen methanogens, but did alter methanogen diversity and community structure, as well as protozoal density within the rumen of nonlactating dairy cattle. Therefore, it may be necessary to monitor the rumen methanogen and protozoal communities of dairy cattle susceptible to depressed pH when methane abatement strategies are being investigated.

Background Pea fiber (PF) is a potential fibrous supplement in swine production. The influence of dietary PF on microbial community in the colon of pigs remains largely unexplored. Methanogens in the hindgut of monogastric animals play important roles in degradation of dietary fibers and efficient removal of microbial metabolic end product H 2 . Understanding the impact of dietary PF on the structure of colonic methanogens may help understand the mechanisms of microbe-mediated physiological functions of PF. This study investigated the influence of PF on the diversity and quantity and/or activity of colonic methanongens of piglets and finishing pigs. Four archaeal 16S rRNA clone libraries were constructed for piglets and finishers fed with control (Piglet-C and Finisher-C) or PF diet (Piglet-P and Finisher-P). Results There were 195, 190, 194 and 196 clones obtained from the library Piglet-C, Piglet-P, Finisher-C and Finisher-P, respectively, with corresponding 12, 11, 11 and 16 OTUs (operational taxonomic units). Significant differences of Shannon Index among the four libraries were found ( P  < 0.05). Libshuff analysis showed that the archaeal community structure among the four libraries were significantly different ( P  < 0.0001). The predominant methanogens shifted from Methanobrevibacter to Methanobrevibacter and Methanomassiliicoccus -like genus as a result of dietary PF. Supplementation of PF significantly increased the copy numbers of mcrA and dsrA genes ( P  < 0.05). Conclusions Alteration of methanogenic community structure may lead to functional transition from utilization of H 2 /CO 2 to employment of both H 2 /CO 2 and methanol/CO 2 . Quantification of three functional genes ( mcrA , dsrA and fhs ) of methanogens, sulfate-reducing bacteria (SRB) and acetogens revealed that dietary PF also increased the activity of methanogens and SRB,probably associated with increased proportion of Methanomassiliicoccus luminyensis -species. Further study is required to examine the interaction between specific methanogens and SRB during fermentation of dietary PF.

Background Tibetan sheep (TS) and Gansu Alpine Finewool sheep (GS) are both important plateau sheep raised and fed on the harsh Qinghai–Tibetan Plateau, China. Rumen methanogen and protozoal communities of plateau sheep are affected by their hosts and living environments, and play important roles in ruminant nutrition and greenhouse gas production. However, the characteristics, differences, and associations of these communities remain largely uncharacterized. Results The rumen methanogen and protozoal communities of plateau sheep were investigated by 16S/18S rRNA gene clone libraries. The predominant methanogen order in both sheep species was Methanobacteriales followed by Methanomassiliicoccales, which is consistent with those seen in global ruminants. However, the most dominant species was Methanobrevibacter millerae rather than Methanobrevibacter gottschalkii seen in most ruminants. Compared with GS and other ruminants, TS have more exclusive operational taxonomic units and a lower proportion (64.5%) of Methanobrevibacter . The protozoa were divided into Entodiniomorphida and Vestibuliferida, including nine genera and 15 species. The proportion of holotrich protozoa was much lower (1.1%) in TS than ordinary sheep. The most predominant genus was Entodinium (70.0%) in TS and Enoploplastron (48.8%) in GS, while the most common species was Entodinium furca monolobum (43.9%) and Enoploplastron triloricatum (45.0%) in TS and GS, respectively; Entodinium longinucleatum (22.8%) was only observed in TS. LIBSHUFF analysis indicated that the methanogen communities of TS were significantly different from those of GS, but no significant differences were found in protozoal communities. Conclusion Plateau sheep have coevolved with unique rumen methanogen and protozoal communities to adapt to harsh plateau environments. Moreover, the host appears to have a greater influence on rumen methanogen communities than on rumen protozoal communities. The observed associations of methanogens and protozoa, together with the findings of previous studies on methane emissions from ruminant livestock, revealed that the lower proportion of Methanobrevibacter and holotrich protozoa may be responsible for the lower methane emission of TS. These findings facilitate our understanding of the rumen microbial ecosystem in plateau sheep, and could help the development of new strategies to manipulate rumen microbes to improve productivity and reduce the emission of greenhouse gases.

The response of freshwater bacterial community to anthropogenic disturbance has been well documented, yet the studies of freshwater archaeal community are rare, especially in lotic environments. Here, we investigated planktonic and benthic archaeal communities in a human-perturbed watershed (Jiulong River Watershed, JRW) of southeast China by using Illumina 16S ribosomal RNA gene amplicon sequencing. The results of taxonomic assignments indicated that SAGMGC-1, Methanobacteriaceae , Methanospirillaceae , and Methanoregulaceae were the four most abundant families in surface waters, accounting for 12.65, 23.21, 18.58 and 10.97 % of planktonic communities, whereas Nitrososphaeraceae and Miscellaneous Crenarchaeotic Group occupied more than 49 % of benthic communities. The compositions of archaeal communities and populations in waters and sediments were significantly different from each other. Remarkably, the detection frequencies of families Methanobacteriaceae and Methanospirillaceae , and genera Methanobrevibacter and Methanosphaera in planktonic communities correlated strongly with bacterial fecal indicator, suggesting some parts of methanogenic Archaea may come from fecal contamination. Because soluble reactive phosphorus (SRP) and the ratio of dissolved inorganic nitrogen to SRP instead of nitrogen nutrients showed significant correlation with several planktonic Nitrosopumilus - and Nitrosotalea -like OTUs, Thaumarchaeota may play an unexplored role in biogeochemical cycling of river phosphorus. Multivariate statistical analyses revealed that the variation of α-diversity of planktonic archaeal community was best explained by water temperature, whereas nutrient concentrations and stoichiometry were the significant drivers of β-diversity of planktonic and benthic communities. Taken together, these results demonstrate that the structure of archaeal communities in the JRW is sensitive to anthropogenic disturbances caused by riparian human activities.