In vitro interactions between Blautia hydrogenotrophica, Desulfovibrio piger and Methanobrevibacter smithii under hydrogenotrophic conditions.

Methanogens, reductive acetogens and sulfate-reducing bacteria play an important role in disposing of hydrogen in gut ecosystems. However, how they interact with each other remains largely unknown. This in vitro study cocultured Blautia hydrogenotrophica (reductive acetogen), Desulfovibrio piger (sulfate reducer) and Methanobrevibacter smithii (methanogen). Results revealed that these three species coexisted and did not compete for hydrogen in the early phase of incubations. Sulfate reduction was not affected by B. hydrogenotrophica and M. smithii. D. piger inhibited the growth of B. hydrogenotrophica and M. smithii after 10 h incubations, and the inhibition on M. smithii was associated with increased sulfide concentration. Remarkably, M. smithii growth lag phase was shortened by coculturing with B. hydrogenotrophica and D. piger. Formate was rapidly used by M. smithii under high acetate concentration. Overall, these findings indicated that the interactions of the hydrogenotrophic microbes are condition-dependent, suggesting their interactions may vary in gut ecosystems.

Culture- and metagenomics-enabled analyses of the Methanosphaera genus reveals their monophyletic origin and differentiation according to genome size.

The genus Methanosphaera is a well-recognized but poorly characterized member of the mammalian gut microbiome, and distinctive from Methanobrevibacter smithii for its ability to induce a pro-inflammatory response in humans. Here we have used a combination of culture- and metagenomics-based approaches to expand the representation and information for the genus, which has supported the examination of their phylogeny and physiological capacity. Novel isolates of the genus Methanosphaera were recovered from bovine rumen digesta and human stool, with the bovine isolate remarkable for its large genome size relative to other Methanosphaera isolates from monogastric hosts. To substantiate this observation, we then recovered seven high-quality Methanosphaera-affiliated population genomes from ruminant and human gut metagenomic datasets. Our analyses confirm a monophyletic origin of Methanosphaera spp. and that the colonization of monogastric and ruminant hosts favors representatives of the genus with different genome sizes, reflecting differences in the genome content needed to persist in these different habitats.

Methanogens in humans: potentially beneficial or harmful for health.

Methanogens are anaerobic prokaryotes from the domain archaea that utilize hydrogen to reduce carbon dioxide, acetate, and a variety of methyl compounds into methane. Earlier believed to inhabit only the extreme environments, these organisms are now reported to be found in various environments including mesophilic habitats and the human body. The biological significance of methanogens for humans has been re-evaluated in the last few decades. Their contribution towards pathogenicity has received much less attention than their bacterial counterparts. In humans, methanogens have been studied in the gastrointestinal tract, mouth, and vagina, and considerable focus has shifted towards elucidating their possible role in the progression of disease conditions in humans. Methanoarchaea are also part of the human skin microbiome and proposed to play a role in ammonia turnover. Compared to hundreds of different bacterial species, the human body harbors only a handful of methanogen species represented by Methanobrevibacter smithii, Methanobrevibacter oralis, Methanosphaera stadtmanae, Methanomassiliicoccus luminyensis, Candidatus Methanomassiliicoccus intestinalis, and Candidatus Methanomethylophilus alvus. Their presence in the human gut suggests an indirect correlation with severe diseases of the colon. In this review, we examine the current knowledge about the methanoarchaea in the human body and possible beneficial or less favorable interactions.

Alterations of the human gut Methanobrevibacter smithii as a biomarker for inflammatory bowel diseases.

It is hypothesized that direct and indirect homeostasis between gut microbiota plays a key role in different intestine disorders. Archaea methanogens, an ancient domain of single-celled organism, are major archaea in the digestive system. Recent evidence has shown that the variable prevalence of methanogens in different individuals could have certain effects on inflammatory bowel diseases (IBD). We aimed to assess the prevalence of Methanobrevibacter smithii between Iranian patients suffering from IBD and healthy control subjects. Stool DNA extracts from 47 healthy controls and 61 IBD patients were investigated. Quantitative real time PCR was performed for detecting Mbb. smithii load. We found a significantly decreased the Mbb. smithii load between IBD patients and healthy subjects. It is assumed that there is a reverse association between Mbb. smithii bacterial load and susceptibility to IBD, and this association could be extended to IBD patients in remission as we found that Mbb. smithii bacterial load is markedly higher among healthy subjects in comparison to IBD patients.

Methanobrevibacter smithii, a methanogen consistently colonising the newborn stomach.

Methanobrevibacter smithii is the main human methanogen almost always found in the digestive tract of adults. Yet, the age at which M. smithii establishes itself as part of the developing intestinal microflora remains unknown. In order to gain insight into this, we developed a polyphasic approach, including microscopic observation by fluorescence in situ hybridisation, polymerase chain reaction (PCR) sequencing detection, identification and culture, to isolate and genotype M. smithii in one-day-old newborns' gastric juice specimens. In the presence of negative controls, 50/50 (100%) prospectively analysed newborn gastric juice specimens were PCR-positive for methanogens, all identified as M. smithii by sequencing. We succeeded in cultivating M. smithii in 35/50 (70%) newborn gastric juice specimens, while 15/50 specimens remained sterile. Further, M. smithii was observed by direct microscopic investigation using fluorescence in situ hybridisation. Multispacer sequence typing found one of seven different genotypes per specimen, these genotypes having all been previously described in adult human stools. Methanobrevibacter smithii is an early inhabitant of the human stomach, colonising the gastric mucosa just after birth, and the mother's gut microbiota is a probable source of colonisation.

Assessing Methanobrevibacter smithii and Clostridium difficile as not conventional faecal indicators in effluents of a wastewater treatment plant integrated with sludge anaerobic digestion.

Wastewater treatment plants (WWTP) are an important source of surface water contamination by enteric pathogens, affecting the role of environmental water as a microbial reservoir. We describe the release to the environment of certain anaerobes of human and environmental concern. The work was focused on emerging microbial targets. They are tracing, by RT-qPCR, on WWTP effluents, both liquid and solid, when an anaerobic digestion step is included. The focus is placed on Clostridium spp. with the specific quantification of Clostridium perfringens, as typical bioindicator, and Clostridium difficile, as emerging pathogen not only confined into nosocomial infection. Moreover methanogens were quantified for their involvement in the anaerobic digestion, and in particular on Methanobrevibacter smithii as major methanogenic component of the human gut microbiome and as not conventional faecal indicator. In the water samples, a reduction, statistically significant, in all microbial targets was observed (p < 0.01), 2 log for the total bacteria, 1.4 log for the Clostridium spp. and M. smithii, 1 log for total methanogens, C. perfringens and C. difficile. The AD process contribute to a significant change in microbial levels into the sludge for total bacteria and total methanogens (p < 0.01), both when the input sludge are primary and secondary, while for the presence of Clostridium spp. and C. difficile there was not a significant change. The produced data are innovative showing which is the diffusion of such anaerobic microorganisms throughout the WWTP and opening a discussion on the implementation of possible techniques for a more efficient microbial removal from effluents, particularly bio-solids, to reduce the potential release of pathogens into the environment.

Irritable Bowel Syndrome, Particularly the Constipation-Predominant Form, Involves an Increase in Methanobrevibacter smithii, Which Is Associated with Higher Methane Production.

BACKGROUND/AIMS: Because Methanobrevibacter smithii produces methane, delaying gut transit, we evaluated M. smithii loads in irritable bowel syndrome (IBS) patients and healthy controls (HC). METHODS: Quantitative real-time polymerase chain reaction for M. smithii was performed on the feces of 47 IBS patients (Rome III) and 30 HC. On the lactulose hydrogen breath test (LHBT, done for 25 IBS patients), a fasting methane result ≥10 ppm using 10 g of lactulose defined methane-producers. RESULTS: Of 47, 20 had constipation (IBS-C), 20 had diarrhea (IBS-D) and seven were not sub-typed. The M. smithii copy number was higher among IBS patients than HC (Log105.4, interquartile range [IQR; 3.2 to 6.3] vs 1.9 [0.0 to 3.4], p<0.001), particularly among IBS-C compared to IBS-D patients (Log106.1 [5.5 to 6.6] vs 3.4 [0.6 to 5.7], p=0.001); the copy number negatively correlated with the stool frequency (R=-0.420, p=0.003). The M. smithii copy number was higher among methane-producers than nonproducers (Log106.4, IQR [5.7 to 7.4] vs 4.1 [1.8 to 5.8], p=0.001). Using a receiver operating characteristic curve, the best cutoff for M. smithii among methane producers was Log106.0 (sensitivity, 64%; specificity, 86%; area under curve [AUC], 0.896). The AUC for breath methane correlated with the M. smithii copy number among methane producers (r=0.74, p=0.008). Abdominal bloating was more common among methane producers (n=9/11 [82%] vs 5/14 [36%], p=0.021). CONCLUSIONS: Patients with IBS, particularly IBS-C, had higher copy numbers of M. smithii than HC. On LHBT, breath methane levels correlated with M. smithii loads.

The intestinal archaea Methanosphaera stadtmanae and Methanobrevibacter smithii activate human dendritic cells.

The methanoarchaea Methanosphaera stadtmanae and Methanobrevibacter smithii are known to be part of the indigenous human gut microbiota. Although the immunomodulatory effects of bacterial gut commensals have been studied extensively in the last decade, the impact of methanoarchaea in human's health and disease was rarely examined. Consequently, we studied and report here on the effects of M. stadtmanae and M. smithii on human immune cells. Whereas exposure to M. stadtmanae leads to substantial release of proinflammatory cytokines in monocyte-derived dendritic cells (moDCs), only weak activation was detected after incubation with M. smithii. Phagocytosis of M. stadtmanae by moDCs was demonstrated by confocal microscopy as well as transmission electronic microscopy (TEM) and shown to be crucial for cellular activation by using specific inhibitors. Both strains, albeit to different extents, initiate a maturation program in moDCs as revealed by up-regulation of the cell-surface receptors CD86 and CD197 suggesting additional activation of adaptive immune responses. Furthermore, M. stadtmanae and M. smithii were capable to alter the gene expression of antimicrobial peptides in moDCs to different extents. Taken together, our findings strongly argue that the archaeal gut inhabitants M. stadtmanae and M. smithii are specifically recognized by the human innate immune system. Moreover, both strains are capable of inducing an inflammatory cytokine response to different extents arguing that they might have diverse immunomodulatory functions. In conclusion, we propose that the impact of intestinal methanoarchaea on pathological conditions involving the gut microbiota has been underestimated until now.

The positive effect of exogenous hemin on a resistance of strict anaerobic archaeon Methanobrevibacter arboriphilus to oxidative stresses.

Methanogenic archaeon Methanobrevibacter arboriphilus (strains AZ and DH1), which is a strict anaerobic microorganism not able to synthesize heme, possessed a very high catalase activity in the presence of 20-50 µM hemin in a growth medium. We investigated the effect of various oxidative stresses (hydrogen peroxide and oxygenation) on M. arboriphilus cells grown on the standard nutrient medium supplemented with 0.1 % yeast extract, and on the same medium supplemented with hemin. It was demonstrated that 30 µM hemin had a very significant positive effect on the resistance of M. arboriphilus strains to H(2)O(2) and O(2) stresses because of 30- to 40-fold increase of heme catalase activity. Thus, hydrogen peroxide (0.6-1.2 mM) or oxygen (3-5 %) had a strong negative impact on low-catalase cultures grown in the hemin-free standard medium, whereas the presence of 30 µM hemin in the medium results in a high yield of biomass even under conditions of four times stronger H(2)O(2) and two times stronger O(2) stresses. The intracellular catalase activity reached a high level in 30-60 min after hemin was added to the nutrient medium, but the activity already increased about 5-7-fold in 6 min after hemin addition. Our experimental data suggest that exogenous hemin provides an effective antioxidative defense in representatives of the genus Methanobrevibacter, specially playing an important role in the puromycin-insensitive formation of the active heme-containing catalase from presynthesized apoenzyme and heme.

A humanized gnotobiotic mouse model of host-archaeal-bacterial mutualism.

Our colons harbor trillions of microbes including a prominent archaeon, Methanobrevibacter smithii. To examine the contributions of Archaea to digestive health, we colonized germ-free mice with Bacteroides thetaiotaomicron, an adaptive bacterial forager of the polysaccharides that we consume, with or without M. smithii or the sulfate-reducing bacterium Desulfovibrio piger. Whole-genome transcriptional profiling of B. thetaiotaomicron, combined with mass spectrometry, revealed that, unlike D. piger, M. smithii directs B. thetaiotaomicron to focus on fermentation of dietary fructans to acetate, whereas B. thetaiotaomicron-derived formate is used by M. smithii for methanogenesis. B. thetaiotaomicron-M. smithii cocolonization produces a significant increase in host adiposity compared with monoassociated, or B. thetaiotaomicron-D. piger biassociated, animals. These findings demonstrate a link between this archaeon, prioritized bacterial utilization of polysaccharides commonly encountered in our modern diets, and host energy balance.

Development of methods for the detection and quantification of 7alpha-dehydroxylating clostridia, Desulfovibrio vulgaris, Methanobrevibacter smithii, and Lactobacillus plantarum in human feces.

BACKGROUND: Mounting evidence suggests a relationship between bacterial metabolism of certain dietary and endogenous factors and the development of colorectal cancer. Deoxycholic acid (DCA) is a well studied co-carcinogen and bio-transformation product of 7alpha-dehydroxylating Clostridia. H2S is a cytotoxic metabolite produced primarily by sulfate-reducing bacteria (SRB). The production of methane indicates low levels of active SRB. Lactic acid bacteria (LAB) have received attention recently due to their putative anti-tumor properties. METHOD: Human stool was spiked with pure cultures of bacteria and diluted in several enriched media. Each dilution titer was analyzed for the presence of the organism by PCR and biochemical assays. Duplicate stool aliquots were stored under various conditions for a 1 month period at -20 degrees C to test viability and detection. RESULTS: Growth and enumeration of each spiked organism was confirmed by PCR and biochemical assays. The combination of bead beating and chemical lysis steps produced the greatest DNA yields. PCR assays detected as low as 75 fg target DNA. The ability to detect Methanobrevibacter smithii, and Desulfovibrio vulgaris by either PCR or biochemical assay declined significantly after storage at -20 degrees C for 1 month. CONCLUSIONS: Accurate detection and quantification of each bacterium using the described methods resulted when stool was processed immediately after collection. Storage of some members of the gut flora results in decrease in or loss of viability.

Quantitative immunologic analysis of the methanogenic flora of digestors reveals a considerable diversity.

To determine which methanogens occur in digestors, we performed a quantitative immunologic analysis of a variety of samples. A comprehensive panel of calibrated polyclonal antibody probes of predefined specificity spectra was used. This allowed precise identification of bacteria by antigenic fingerprinting. A considerable diversity of methanogens was uncovered, much larger than previously reported, encompassing at least 14 strains of 11 species. Strategies were developed to measure the load of any given methanogen in a sample and to compare samples quantitatively. Two methanogens were found to predominate which were antigenically closely related with either Methanobacterium formicicum MF or Methanobrevibacter arboriphilus AZ. Fundamental data, probes, and methods are now available to monitor methanogenic subpopulations during digestor operation and thus learn about their respective roles and predictive significance.