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This study evaluated the breath CH4excretion and concentration ofM. smithiiin intestinal microbiota of schoolchildren from 2 slums. One hundred and eleven children from a slum near a sanitary landfill, 35 children of a slum located away from the sanitary landfill, and 32 children from a high socioeconomic level school were included in the study. Real-time PCR was performed to quantify theM. smithii nifHgene and it was present in the microbiota of all the participating children, with higherP<0.05concentrations in those who lived in the slum near the landfill (3.16×107 CFU/g of feces), comparing with the children from the slum away from the landfill (2.05×106 CFU/g of feces) and those from the high socioeconomic level group (3.93×105 CFU/g of feces). The prevalence of children who present breath methane was 53% in the slum near the landfill, 31% in the slum further away from the landfill and, 22% in the high socioeconomic level group. To live near a landfill is associated with higher concentrations ofM. smithiiin intestinal microbiota, comparing with those who live away from the landfill, regardless of their socioeconomics conditions.

The risk of decompression sickness (DCS) was modulated by varying the biochemical activity used to eliminate some of the hydrogen (H2) stored in the tissues of pigs (19.4 ± 0.2 kg) during hyperbaric exposures to H2. Treated pigs ( n = 16) received intestinal injections of Methanobrevibacter smithii, a microbe that metabolizes H2to water and CH4. Surgical controls ( n = 10) received intestinal injections of saline, and an additional control group ( n = 10) was untreated. Pigs were placed in a chamber and compressed to 24 atm abs (20.6–22.9 atm H2). After 3 h, the pigs were decompressed and observed for symptoms of DCS for 1 h. Pigs with M. smithii had a significantly lower ( P < 0.05) incidence of DCS (44%; 7/16) than all controls (80%; 16/20). The DCS risk decreased with increasing activity of microbes injected (logistic regression, P < 0.05). Thus the supplemental tissue washout of the diluent gas by microbial metabolism was inversely correlated with DCS risk in a dose-dependent manner in this pig model.

Background: The archaeon Methanobrevibacter smithii is regarded as part of the indigenous microflora of the human intestine but may be connected with pathological conditions. The microbe is extremely oxygen intolerant and is not detectable by anaerobic culture techniques for bacteria. Accordingly, to date quantitative antimicrobial susceptibility data of human isolates are missing. Methods: The anoxic Hungate technique and a three-step culture procedure using media supplemented with antibiotics were applied to isolate M. smithii from randomly selected human feces. The minimum inhibitory concentrations (MICs) of 15 isolates and the reference strain DSM 861 were determined using a broth macrodilution test resembling the procedure for testing anaerobic bacteria. Results: The 16 strains were highly resistant (MICs >64 mg/l) against penicillin G, cephalothin, vancomycin, streptomycin, gentamicin, ciprofloxacin, and clindamycin. Metronidazole inhibited the strains at MICs between 0.5 and 64 mg/l. Conclusions: Multiple-antibiotic-resistant Methanoarchaea occur in the human gut. They may be selected during therapy with common antibacterial agents and may be eliminated by the application of metronidazole.

The population of methanogens in the sheep rumen microbial ecosystem was studied by using 16S rDNA cloning analysis, epifluorescence microscopy (which detects autofluorescence of a specific cofactor F420 in methanogens) and the 16S rRNA-targeted in situ hybridization technique. The 16S rDNA clone libraries were constructed by PCR amplification with an Archaea-specific primer set and partial sequencing of the clonal 16S rDNAs was done. Phylogenetic analysis indicated that the clones were affiliated with Methanomicrobium ruminantium and mobile, Methanobrevibacter smithii. Epifluorescence microscopy (F420 autofluorescence) and in situ hybridization by using a newly designed M. mobile-specific 16S rRNA-targeted oligonucleotide probe found that methanogens accounted for approximately 3.6% of total ruminal microorganisms and approximately 54% of the total methanogens were M. mobile.

The phylogeny of methanogenic archaea associated with ciliate protozoa in a sheep rumen was investigated. Ruminal ciliate protozoa were exhaustively washed and mixtures of genomic DNA extracted. Archaea-specific nested PCR amplification was conducted with the ciliate genomic mixture. The resultant small subunit (16S) ribosomal RNA gene (ssu rDNA) was cloned into Escherichia coli JM 109. Many methanogens were still observed on and/or in ciliate cells by fluorescent microscopy even after exhaustive washing with buffer. Partial sequences of ssu rDNA close to Methanobrevibacter smithii were dominant in the retrieved sequences. RFLP analyses on the retrieved sequences revealed the absence of Methanobrevibacter ruminantium in the protozoal preparation. The association of Methanobrevibacter spp. with ruminal ciliate protozoa was demonstrated by the isolation of archaeal ssu rDNA phylogenetically close to that of M. smithii.

Mice were immunized with bovine serum albumin (BSA) entrapped within archaeosomes (i.e. liposomes) composed of the total polar lipids (TPL) from the two methanogenic archaea common to the human digestive tract. Methanobrevibacter smithii archaeosomes boosted serum anti-BSA antibody to titers comparable to those achieved with Freund’s adjuvant, whereas Methanosphaera stadtmanae archaeosomes were relatively poor adjuvants. An explanation for this difference was sought by analysis of the polar lipid composition of each archaeobacterium. Fast atom bombardment mass spectrometry and NMR analyses of the purified lipids revealed a remarkable similarity in the ether lipid structures present in each TPL extract. However, the relative amounts of each lipid species varied dramatically. The phospholipid fraction in M. stadtmanae TPL was dominated by archaetidylinositol (50 mol% of TPL) and the glycolipid fraction by β-Glcp-(1,6)-β-Glcp-(1,1)-archaeol (36 mol%), whereas in M. smithii extracts, both caldarchaeol and archaeol lipids containing a phosphoserine head group were relatively abundant. Liposomes prepared from purified archaetidylinositol and from M. stadtmanae TPL supplemented with increasing amounts of phosphatidylserine elicited poor humoral responses to encapsulated BSA. A dramatic loss in the adjuvanticity of M. smithii archaeosomes was seen upon incorporation of 36 mol% of the uncharged lipid diglucosyl archaeol and, to a lesser extent, of 50 mol% of archaetidylinositol. Interestingly, the relative rates of uptake of M. smithii and M. stadtmanae archaeosomes by phagocytic cultures in vitro were similar. Thus, the lipid composition may influence archaeosome adjuvanticity, particularly a high diglucosyl archaeol and/or archaetidyl inositol content, resulting in a low adjuvant activity.

Comparative 16S rRNA gene sequence and genomic DNA reassociation analyses were used to assess the phylogenetic relationships of Methanobrevibacter fecal isolates. The 16S rRNA gene sequences of Methanobrevibacter smithii strain PS and the human fecal isolates B181 and ALI were essentially identical, and their genomic DNA reassociated at values greater than 94%. The analysis of 16S rRNA sequences of the horse, pig, cow, rat, and goose fecal isolates confirm that they are members of the genus Methanobrevibacter. They had a high degree of sequence similarity (97–98%) with the 16S rRNA gene of M. smithii, indicating that they share a common line of descent. The 16S rRNA genes of the horse and pig isolates had 99.3% sequence similarity. Sequence analysis of the 16S rRNA gene of the sheep fecal isolate showed that it formed a separate line of descent in the genus Methanobrevibacter. Genomic DNA reassociation studies indicate that the horse, pig, cow, and goose fecal isolates represent at least three new species. The horse and pig isolates were the only animal isolates that had > 70% genomic DNA reassociation and represent strains of a single species. The cow, goose, and sheep isolates had little or no genomic DNA reassociation with M. smithii or with each other. The relationship of the rat isolate to the other animal isolates was not determined. An evaluation of the relationship of 16S rRNA gene sequence similarity and genomic DNA reassociation of Methanobrevibacter and other methanogenic archaea indicated that genomic DNA reassociation studies are necessary to establish that two methanogenic organisms belong to the same species.

International audience; Genus and species level analysis is the best way to characterize alterations in the human gut microbiota that are associated with obesity, because the clustering of obese and lean microbiotas increases with the taxonomic depth of the analysis. Bifidobacterium genus members have been associated with a lean status, whereas different Lactobacillus species are associated both with a lean and an obese status. OBJECTIVES AND METHODS: We analyzed the fecal concentrations of Bacteroidetes, Firmicutes, Methanobrevibacter smithii, the genus Lactobacillus, five other Lactobacillus species previously linked with lean or obese populations, Escherichia coli and Bifidobacterium animalis in 263 individuals, including 134 obese, 38 overweight, 76 lean and 15 anorexic subjects to test for the correlation between bacterial concentration and body mass index (BMI). Of these subjects, 137 were used in our previous study. FINDINGS: Firmicutes were found in 498.5%, Bacteroidetes in 67%, M. smithii in 64%, E. coli in 51%, Lactobacillus species between 17 and 25% and B. animalis in 11% of individuals. The fecal concentration of Lactobacillus reuteri was positively correlated with BMI (coefficient ¼ 0.85; 95% confidence interval (CI) 0.12–0.58; P ¼ 0.02) in agreement with what was reported for Lactobacillus sakei. As reported, B. animalis (coefficient ¼ À 0.84; 95% CI À 1.61 to À 0.07; P ¼ 0.03) and M. smithii (coefficient ¼ À 0.43, 95% CI À 0.90 to 0.05; P ¼ 0.08) were negatively associated with the BMI. Unexpectedly, E. coli was found here for the first time to negatively correlate with the BMI (coefficient ¼ À 1.05; 95% CI À 1.60 to À 0.50; Po0.001). CONCLUSION: Our findings confirm the specificity of the obese microbiota and emphasize the correlation between the concentration of certain Lactobacillus species and obesity.

A decade ago a hypothesis was proposed that, similar to the Bacteria and Eukarya, the archaeal domain of life might harbor certain species capable of causing disease (Cavicchioli et al., 2003; Eckburg et al., 2003). Now, a decade later, it is time to revisit the topic in the light of new data available. The last decade witnessed a massive use of molecular ecology tools in clinical microbiology, and these data can be inspected for the potential involvement of the Archaea in various infectious diseases in humans and animals. Unlike the Bacteria, the diversity of the Archaea in the human body is substantially lower, including representatives of only one phylum, Euryarchaeota. The phylum includes three species: Methanobrevibacter smithii, found mostly in the gut and vagina (Miller and Wolin, 1982; Belay et al., 1990); Methanosphaera stadtmanae, found mostly in the gut (Miller and Wolin, 1985); and M. oralis, found mostly in the oral cavity (Belay et al., 1988; Ferrari et al., 1994). Compared to bacteria, the relative abundance of archaea is also much lower (Miller and Wolin, 1982), although the recent improvements in cultivation and DNA detection may help to estimate these numbers more accurately (Dridi et al., 2009; Khelaifia et al., 2013). The first work suggesting an association of the Archaea with human gastrointestinal disease was published in 1985 (McKay et al., 1985). Patients with Crohn’s disease, ulcerative colitis and primary pneumatosis intestinalis displayed a significantly lower incidence of methane excretion compared to healthy subjects. On the contrary, patients with diverticulosis showed a significantly increased incidence of methanogens compared to control (Weaver et al., 1986). Since then, there have been a number of studies, with more recent ones using molecular ecology markers, such as the 16S rRNA and mcrA genes, which have confirmed these two initial observations. That is, in conditions characterized by extended transit time in the intestine, the incidence, and rate of methane production are higher (Fiedorek et al., 1990; Pimentel et al., 2003; Chatterjee et al., 2007), while the diarrheal conditions of human gastrointestinal disease result in the opposite trend with lower incidences of methanogenic archaea and lower rates of methane production (McKay et al., 1985; Scanlan et al., 2008). Chemotherapy-induced diarrhea in cancer patients has also resulted in the decrease of methanogenic archaea in parallel with the loss of beneficial bacteria (Stringer et al., 2013). Indeed, experimental interventions to increase the intestinal transit rate have resulted in the decline of fecal methanogens and methane production, while the opposite effect has been observed in the loperamide treatment group (Lewis and Cochrane, 2007). Recent reassessments of the role of methane production among patients with gastrointestinal disturbances have clearly associated elevated methane production with alterations in intestinal motility, such as constipation, but not with other conditions (Attaluri et al., 2010; Furnari et al., 2012). Thus, a pathogenic link with methanogens is unlikely (Di Stefano and Corazza, 2010), and their involvement in gastrointestinal disease is presently uncorroborated. From the ecological/system biology viewpoint the numbers and the methane production by archaea is more likely a reflection of syntrophic relationships in the gut where the local environment, depending on a number of factors, may favor hydrogen channeling through alternative mechanisms of hydrogen disposal, such as methanogenesis, sulfate reduction, or acetogenesis. In this regard, alternative generation of highly toxic hydrogen sulfide as a result of sulfate reduction in the gut may impose much higher health risks (Medani et al., 2011; Carbonero et al., 2012) compared to more inert methane. The best-studied cases of a potential involvement of the Archaea in human pathology, however, are linked to periodontal disease. Methanobrevibacter oralislike phylotypes, for example, have been detected by PCR in up to 36% of periodontitis patients (Lepp et al., 2004). In another study, five cases of apical periodontitis out of 20 total have been found positive for the Archaea (Vianna et al., 2006). The follow up work by the same group has greatly contributed to our understanding of the role played by hydrogenotrophic microorganisms in periodontal disease (Vianna et al., 2008). Compared to periodontitis patients, the supragingival plaque of healthy subjects harbors a lower total microbial load, and the hydrogenotrophic group is represented exclusively by acetogenic bacteria, also at lower numbers. On the contrary, the subgingival plaque from periodontitis patients harbors a larger number of total bacteria, and the hydrogenotrophic group includes methanogenic archaea and sulphate-reducing bacteria (SRB) in addition to acetogenic bacteria. The latter two groups are absent in healthy control subjects but present in 65% of periodontitis patients, alone or in combination (Vianna et al., 2008). It needs to be noted here that the presence of SRB in human saliva can be detected in 30% of subjects and, among other oral and systemic conditions, the only statistically significant association of the SRB carriage is with periodontitis (Heggendorn et al., 2013). Although the proportion of hydrogenotrophs in periodontal disease is below 1% of the total microbiota (Vianna et al., 2008), the hydrogen sink they provide may

A number of PCR-based methods for detecting human fecal material in environmental waters have been developed over the past decade, but these methods have rarely received independent comparative testing in large multi-laboratory studies. Here, we evaluated ten of these methods (BacH, BacHum-UCD, Bacteroides thetaiotaomicron (BtH), BsteriF1, gyrB, HF183 endpoint, HF183 SYBR, HF183 Taqman(®), HumM2, and Methanobrevibacter smithii nifH (Mnif)) using 64 blind samples prepared in one laboratory. The blind samples contained either one or two fecal sources from human, wastewater or non-human sources. The assay results were assessed for presence/absence of the human markers and also quantitatively while varying the following: 1) classification of samples that were detected but not quantifiable (DNQ) as positive or negative; 2) reference fecal sample concentration unit of measure (such as culturable indicator bacteria, wet mass, total DNA, etc); and 3) human fecal source type (stool, sewage or septage). Assay performance using presence/absence metrics was found to depend on the classification of DNQ samples. The assays that performed best quantitatively varied based on the fecal concentration unit of measure and laboratory protocol. All methods were consistently more sensitive to human stools compared to sewage or septage in both the presence/absence and quantitative analysis. Overall, HF183 Taqman(®) was found to be the most effective marker of human fecal contamination in this California-based study.

Background The PCR-based detection of archaea DNA in human specimens relies on efficient DNA extraction. We previously designed one such protocol involving only manual steps. In an effort to reduce the workload involved, we compared this manual protocol to semi-automated and automated protocols for archaea DNA extraction from human specimens. Findings We tested 110 human stool specimens using each protocol. An automated protocol using the EZ1 Advanced XL extractor with the V 1.066069118 Qiagen DNA bacteria card and the EZ1® DNA Tissue Kit (Qiagen, Courtaboeuf, France) yielded 35/110 (32%) positives for the real-time PCR detection of the Methanobrevibacter smithii 16S rRNA gene, with average Ct values of 36.1. A semi-automated protocol combining glass-powder crushing, overnight proteinase K digestion and lysis in the buffer from the EZ1 kit yielded 90/110 (82%) positive specimens (P = 0.001) with an average Ct value of 27.4 (P = 0.001). The manual protocol yielded 100/110 (91%) positive specimens (P = 0.001) with an average Ct value of 30.33 (P = 0.001). However, neither the number of positive specimens nor the Ct values were significantly different between the manual protocol and the semi-automated protocol (P > 0.1 and P > 0.1). Conclusion Proteinase K digestion and glass powder crushing dramatically increase the extraction yield of archaea DNA from human stools. The semi-automated protocol described here was more rapid than the manual protocol and yielded significantly more archaeal DNA. It could be applied for extracting total stool DNA for further PCR amplification.

Aims The relationship between land use (undeveloped, cattle grazing, urban), faecal indicator bacteria (FIB) levels and microbial source tracking (MST) marker detection was investigated in lakes created following phosphate mining. Methods and Results Faecal coliforms and enterococci were cultured, and MST markers were detected by PCR [Methanobrevibacter smithii, human polyomaviruses (HPyVs), ruminant, human (HF183) and general Bacteroidales]. FIB levels varied significantly by sampling date and were correlated with antecedent rainfall. FIB levels varied with land use category only in the case of faecal coliform levels in sediments of urban lakes, which were significantly greater than those in undeveloped or cattle-impacted lakes. Ruminant Bacteroidales were detected consistently in cattle-impacted lakes (57%) and rarely in other lakes. HPyVs was the only human source marker detected. Conclusions Rainfall was more strongly associated with FIB levels than land use category. The detection frequency of only the ruminant MST marker was associated with land use. Significance and Impact of Study Microbial source tracking (MST) can fine-tune the assessment of human health risk from recreational use of inland waters, particularly when similar FIB levels but different surrounding land use and probable impacts exist.

The human gut microbiome plays an influential role in maintaining human health and it is a potential target for prevention and treatment of disease. Genome-scale metabolic models (GEMs) can provide an increased understanding of the mechanisms behind the effects of diet, the genotype-phenotype relationship and microbial robustness. Here we reconstructed GEMs for three key species, (Bacteroidesthetaiotamicron, Eubacteriumrectale and Methanobrevibactersmithii) as relevant representatives of three main phyla in the human gut (Bacteroidetes, Firmicutes and Euryarchaeota). We simulated the interactions between these three bacteria in different combinations of gut ecosystems and compared the predictions with the experimental results obtained from colonization of germ free mice. Furthermore, we used our GEMs for analyzing the contribution of each species to the overall metabolism of the gut microbiota based on transcriptome data and demonstrated that these models can be used as a scaffold for understanding bacterial interactions in the gut.

Recent advances in parallel genomic processing and computational mapping have been applied to the native human microbial environment to provide a new understanding of the role of the microbiome in health and disease. In particular, studies of the distal gut microbiome have proposed that changes in gut microbiota are related to obesity, the metabolic syndrome, and Western diet. We examined the changes in the distal gut microbiome composition as it relates to the lean and obese phenotypes, particularly after surgical weight loss. A PubMed search of publications from January 1, 2005, through December 31, 2012, used the search terms weight, obesity, microbiome, and bariatric surgery. We included studies that provided information on subjects' weight and/or body mass index and a formal assessment of the microbiome. Certain bacteria, specifically the archaeon Methanobrevibacter smithii, have enhanced ability to metabolize dietary substrate, thereby increasing host energy intake and weight gain. With weight loss, there is a decrease in the ratio of Firmicutes to Bacteroidetes phyla. One major finding from microbial sequencing analyses after Roux-en-Y gastric bypass is the comparative overabundance of Proteobacteria in the distal gut microbiome, which is distinct from the changes seen in weight loss without Roux-en-Y gastric bypass. This review provides the practicing surgeon with (1) an update on the state of a rapidly innovating branch of clinical bioinformatics, specifically, the microbiome; (2) a new understanding of the microbiome changes after Roux-en-Y gastric bypass and weight loss; and (3) a basis for understanding further clinical applications of studies of the distal gut microbiome, such as in Crohn disease, ulcerative colitis, and infectious colitis.

This study was aimed at evaluating the host specificity and host sensitivity of two bovine feces-associated bacterial (BacCow-UCD and cowM3) and one viral [bovine adenovirus (B-AVs)] microbial source tracking (MST) markers by screening 130 fecal and wastewater samples from 10 target and nontarget host groups in southeast Queensland, Australia. In addition, 36 water samples were collected from a reservoir and tested for the occurrence of all three bovine feces-associated markers along with fecal indicator bacteria (FIB), Campylobacter spp., Escherichia coli O157, and Salmonella spp. The overall host specificity values of the BacCow-UCD, cowM3, and B-AVs markers to differentiate between bovine and other nontarget host groups were 0.66, 0.88, and 1.00, respectively (maximum value of 1.00). The overall host sensitivity values of these markers, however, in composite bovine wastewater and individual bovine fecal DNA samples were 0.93, 0.90, and 0.60, respectively (maximum value of 1.00). Among the 36 water samples tested, 56%, 22%, and 6% samples were PCR positive for the BacCow-UCD, cowM3, and B-AVs markers, respectively. Among the 36 samples tested, 50% and 14% samples were PCR positive for the Campylobacter 16S rRNA and E. coli O157 rfbE genes, respectively. Based on the results, we recommend that multiple bovine feces-associated markers be used if possible for bovine fecal pollution tracking. Nonetheless, the presence of the multiple bovine feces-associated markers along with the presence of potential zoonotic pathogens indicates bovine fecal pollution in the reservoir water samples. Further research is required to understand the decay rates of these markers in relation to FIB and zoonotic pathogens.

Aims To develop and test a fluorescence in situ hybridization (FISH) based technique and to identify and quantify simultaneously those methanogenic populations colonizing Entodinium spp. in the rumen of cows fed different forages. Methods and Results New FISH probes targeting protozoal Entodinium spp. were designed and used together with FISH probes for methanogens in the cow rumen. The composition and relative abundance of methanogenic populations colonizing Entodinium simplex-, E. caudaum- and Entodinium furca-related populations were similar. Methanogens including Methanobrevibacter thaueri, Methanobrevibacter millerae and Methanobrevibacter smithii, and members of Methanomicrobium and Methanosphaera were generally the predominant colonizers of protozoa, regardless of the forage fed to cattle. Individual animals appeared to differ in which ruminal methanogenic populations colonized each of the individual Entodinium spp. Conclusions Simultaneous FISH probing is shown here to be a reliable and effective approach to investigate the dynamics of symbiotic relationships between ruminal protozoa and methanogens at a single cell level. Phylogenetically closely related Entodinium spp. were colonized by similar methanogenic populations regardless of the forage fed. Significance and Impact of the Study This is the first report of the methanogenic archaeal populations that specifically colonize Entodinium spp. as identified using simultaneous FISH probing.

Lovastatin, a natural byproduct of some fungi, is able to inhibit HMG-CoA (3-hydroxy-3methyl glutaryl CoA) reductase. This is a key enzyme involved in isoprenoid synthesis and essential for cell membrane formation in methanogenic Archaea. In this paper, experiments were designed to test the hypothesis that lovastatin secreted byAspergillus terreusin fermented rice straw extracts (FRSE) can inhibit growth and CH4production inMethanobrevibacter smithii(a test methanogen). By HPLC analysis, 75% of the total lovastatin in FRSE was in the active hydroxyacid form, andin vitrostudies confirmed that this had a stronger effect in reducing both growth and CH4production inM. smithiicompared to commercial lovastatin. Transmission electron micrographs revealed distorted morphological divisions of lovastatin- and FRSE-treatedM. smithiicells, supporting its role in blocking normal cell membrane synthesis. Real-time PCR confirmed that both commercial lovastatin and FRSE increased (P<0.01) the expression of HMG-CoA reductase gene (hmg). In addition, expressions of other gene transcripts inM. smithii. with a key involvement in methanogenesis were also affected. Experimental confirmation that CH4production is inhibited by lovastatin inA. terreus-fermented rice straw paves the way for its evaluation as a feed additive for mitigating CH4production in ruminants.

In a simple laboratory model system containing co-cultures of cellulolytic bacteria from landfills and Methanobrevibacter smithii, methanogenesis was optimal at 37°C. Although paper solubilization was greatest in cultures containing Eubacterium strain LF 9, methanogenesis was greatest in cultures containing Eubacterium strain LF 11. Coating filter paper with printer's ink reduced methanogenesis. Cultures grown on newspaper produced less methane than those grown on filter paper. However, the rates of methane production from these substrates were similar when calculated in relation to microbial biomass present.

Methane produced by the methanoarchaeonMethanobrevibacter smithii(M. smithii) has been linked to constipation, irritable bowel syndrome with constipation (IBS-C), and obesity. Lovastatin, which demonstrates a cholesterol-lowering effect by the inhibition of HMG-CoA reductase, may also have an anti-methanogenesis effect through direct inhibition of enzymes in the archaeal methanogenesis pathway. We conducted protein-ligand docking experiments to evaluate this possibility. Results are consistent with recent clinical findings.METHODS: F420-dependent methylenetetrahydromethanopterin dehydrogenase (mtd), a key methanogenesis enzyme with a known sequence but no tertiary protein structural information, was modeled for two different methanogenic archaea:M. smithiiandMethanopyrus kandleri. Once protein models were developed, ligand-binding sites were identified. Multiple ligands and their respective protonation, isomeric and tautomeric representations were docked into each site, including F420-coenzyme (natural ligand), lactone and β-hydroxyacid forms of lovastatin and simvastatin, and other co-complexed ligands found in related crystal structures.RESULTS: 1) Generally, for each modeled site the lactone form of the statins had more favorable site interactions compared to F420; 2) The statin lactone forms generally had the most favorable docking scores, even relative to the native template PDB ligands; and 3) The statin β-hydroxyacid forms had less favorable docking scores, typically scoring in the middle with some of the F420 tautomeric forms. Consistent with these computational results were those from a recent phase II clinical trial (NCT02495623) with a proprietary, modified-release lovastatin-lactone (SYN-010) in patients with IBS-C, which showed a reduction in symptoms and breath methane levels, compared to placebo.CONCLUSION: The lactone form of lovastatin exhibits preferential binding over the native-F420 coenzyme ligandin silicoand thus could inhibit the activity of the keyM. smithiimethanogenesis enzymemtdin vivo. Statin lactones may thus exert a methane-reducing effect that is distinct from cholesterol lowering activity, which requires HMGR inhibition by statin β-hydroxyacid forms.

Four fistulated adult Murrah buffaloes were fed on a basal diet consisting of wheat straw and concentrate mixture in a 4 × 4 Latin square design to study the effects of feeding plants containing secondary metabolites on rumen metabolites and methanogen diversity. The four groups were Control (no additive), Mix-1 (ajwain oil and lemon grass oil in a 1 : 1 ratio @ 0.05% of dry matter intake), Mix-2 (garlic and soapnut in a 2 : 1 ratio @ 2% of dry matter intake) and Mix-3 (garlic, soapnut, harad and ajwain in a 2 : 1 : 1 : 1 ratio @ 1% of dry matter intake). In each phase of 30 days’ duration, after 19 days of feeding, rumen liquor was sampled for two consecutive days at 0, 2, 4, 6 and 8 h post-feeding, whereas rumen content was sampled at 0 h feeding. The pH of the rumen liquor was recorded at every collection and then the rumen liquor of every collection was pooled day-wise and animal-wise. These pooled samples were used for estimation of rumen metabolites like ammonia, lactic acid and volatile fatty acids. Microscopic counting of protozoa was done in both 0 h and pooled samples of rumen liquor. Rumen contents collected from different locations of rumen were processed for enzyme estimation. The rumen contents were squeezed and the liquid portion was used for DNA isolation, which was further processed to determine methanogen diversity. Daily intake of feed was similar (P > 0.05) in all the four groups. The ammonia-N concentration and ciliate protozoa population were reduced significantly in the treatment groups supplemented with additives. Rumen pH, lactic acid, volatile fatty acids and enzyme activities were not affected (P > 0.05) by feeding of any of these additives. Methanogenic diversity comparison was made between the Control and Mix-1 group. The basic local alignment search tool (BLAST) analysis of the 133 (44 from the Control group and 89 from the Mix-1 group) sequences showed similarity of the sequences of rumen archaea by up to 97% to the known sequences of rumen methanogens. The sequences with minimum length of 750 bp were selected for phylogenetic analysis. Per cent identity of these sequences with that of the available nearest neighbour as calculated by MEGA 5.03 software showed identity of the clones in the range of 88–97%. The clones were similar with Methanobrevibacter smithii ATCC 35061, uncultured Methanobrevibacter sp. clone MEME95 and M. ruminantium M1. Overall, feeding of any of these feed additives to fistulated buffaloes did not affect feed intake, rumen pH, or rumen metabolites except ammonia and enzyme profile. Methanogen diversity showed the possibility of Methanobrevibacter as the major methanogen in buffalo rumen liquor.

Background Campylobacter jejuni infection is a leading cause of gastroenteritis and post infectious irritable bowel syndrome (PI-IBS). Unanswered questions include the role of cytokines, effects on gut flora, and why IBS is not more prevalent in countries with higher gastroenteritis rates. Therefore, we determined the effects of early and repeat C. jejuni infections on post infectious phenotypes, gut flora, and cytokine levels in a rat model of functional bowel and microbial changes. Methods Sprague-Dawley rats were gavaged with 108 cfu C. jejuni as juveniles and again as adults (J+/A+), as adults only (J-/A+), or vehicle (controls). Stool consistency during acute colonization, post infectious stool wet weight, total bacteria and Methanobrevibacter smithii levels in bowel segments, and ileal cytokines were evaluated. Key Results C. jejuni colonization was longer for first exposures as juveniles (43.4 ± 1.7 days) vs. adults (30.4 ± 3.5 days) (P

Methanogenic community structure, methane production (CH 4 ), and volatile fatty acid (VFA) profiles were investigated in Swedish dairy cows fed a diet with a forage/concentrate ratio of 500/500 or 900/100 g/kg of dry matter (DM) of total DM intake (DMI). The rumen methanogenic population was evaluated using terminal restriction fragment length polymorphism (T-RFLP) analysis, 16S rRNA gene libraries, and quantitative real-time PCR (qRT-PCR). Mean CH 4 yields did not differ ( P > 0.05) between diets, being 16.9 and 20.2 g/kg DMI for the 500/500 and 900/100 diets, respectively. The T-RFLP analysis revealed that populations differed between individual cows and that each individual population responded differently to the diets. The 16S rRNA gene libraries revealed that Methanobrevibacter spp. dominated for both diets. CH 4 production was positively correlated with a dominance of sequences representing T-RFs related to Methanobrevibacter thaueri , Methanobrevibacter millerae , and Methanobrevibacter smithii relative to Methanobrevibacter ruminantium and Methanobrevibacter olleyae . Total numbers of methanogens and total numbers of Methanobacteriales were significantly higher with the 500/500 diet ( P < 0.0004 and P < 0.002, respectively). However, no relationship was found between CH 4 production and total number of methanogens. No differences were seen in total VFA, propionic acid, or acetic acid contents, but the molar proportion of butyric acid in the rumen was higher for the 500/500 diet than for the 900/100 diet ( P < 0.05). Interestingly, the results also revealed that a division of the identified methanogenic species into two groups, suggested in the work of King et al. (E. E. King, R. P. Smith, B. St-Pierre, and A. D. G. Wright, Appl. Environ. Microbiol. 77: 5682–5687, 2011), increased the understanding of the variation in CH 4 production between different cows.

Several bacteria, viruses, and parasites cause diarrhea as coinfecting pathogens. We designed a DNA microarray comprising 60-bp probes spotted 194 times for the multiplex detection of 33 enteropathogenic bacteria and seven enteropathogenic viruses, and the archaeon Methanobrevibacter smithii was used as an internal positive control. Nine pathogen-free stool specimens were used as negative controls. One of these control specimens was further spiked with Salmonella enterica as a positive control. The microarray was then tested with 40 pathological stool specimens, comprising S. enterica (n = 30), Campylobacter jejuni (n = 4), pathogenic Escherichia coli (n = 2), and adenovirus (n = 4). M. smithii was detected in 47/49 (95.9%) specimens, no pathogen was detected in negative controls and S. enterica was identified in the S. enterica-spiked positive control. The overall specificity was 100% and the overall sensitivity was 97.5% because one S. enterica sample was missed by the microarray. The multiplexed detection of C. jejuni spiked into an adenovirus-positive stool sample gave positive results, with fluorescence values of 14.3 and 9.1, respectively. These data indicate that using the protocol developed in this article, the DNA array allows for the multiplexed detection of some enteropathogens in stool samples.

As members of the indigenous human microbiota found on several mucosal tissues, Methanobrevibacter smithii and Methanosphaera stadtmanae are exposed to the effects of antimicrobial peptides (AMPs) secreted by these epithelia. Although antimicrobial and molecular effects of AMPs on bacteria are well described, data for archaea are not available yet. Besides, it is not clear whether AMPs affect them as the archaeal cell envelope differs profoundly in terms of chemical composition and structure from that of bacteria. The effects of different synthetic AMPs on growth of M. smithii , M. stadtmanae , and Methanosarcina mazei were tested using a microtiter plate assay adapted to their anaerobic growth requirements. All three tested methanoarchaea were highly sensitive against derivatives of human cathelicidin, of porcine lysin, and a synthetic antilipopolysaccharide peptide (Lpep); however, sensitivities differed markedly among the methanoarchaeal strains. The potent AMP concentrations affecting growth were below 10 μM, whereas growth of Escherichia coli WBB01 was not affected at peptide concentrations up to 10 μM under the same anaerobic growth conditions. Atomic force microscopy and transmission electron microscopy revealed that the structural integrity of the methanoarchaeal cells is destroyed within 4 h after incubation with AMPs. The disruption of the cell envelope of M. smithii , M. stadtmanae , and M. mazei within a few minutes of exposure was verified by using LIVE/DEAD staining. Our results strongly suggest that the release of AMPs by eukaryotic epithelial cells is a potent defense mechanism targeting not only bacteria, but also methanoarchaea.

Human and ecosystem health can be damaged by fecal contamination of recreational waters. Microbial source tracking (MST) can be used to specifically detect domestic sewage containing human waste, thereby informing both risk assessment and remediation strategies. Previously, an inter-laboratory collaboration developed standardized PCR methods for a bacterial, an archaeal, and a viral indicator of human sewage. Here we present results for two subsequent years of field testing in fresh and salt water by five laboratories across the U.S. Gulf Coast (two in Florida and one each in Mississippi, Louisiana and Texas) using common standard operating procedures (SOPs) developed previously. Culturable enterococci were enumerated by membrane filtration, and PCR was used to detect three MST markers targeting domestic sewage: human-associated Bacteroides (HF183), Methanobrevibacter smithii and human polyomaviruses BK and JC (HPyVs). Detection of sewage markers in surface waters was significantly associated with higher enterococci levels and with exceedance of the recreational water quality standard in four or three regions, respectively. Sewage markers were frequently co-detected in single samples, e.g., M. smithii and HF183 were co-detected in 81% of Louisiana samples, and HPyVs and M. smithii were co-detected in over 40% of southwest Florida and Mississippi samples. This study demonstrates the robustness and inter-laboratory transferability of these three markers for the detection of pollution from domestic sewage in the waters impacting the Gulf of Mexico over a coastal range of over 1000 miles.

The diversity of fecal methanogens of Erhualian (obese type) and Landrace (lean type) pigs was examined using separate 16S rRNA gene libraries for each breed. A total of 763 clones were analyzed; 381 from the Erhualian library and 382 from the Landrace library were identified belonging to the genus Methanobrevibacter. Others were identified belonging to the genus Methanosphaera. The two libraries showed significant differences in diversity (P<0.05) and composition (P<0.0001). Only two operational taxonomic units (OTUs) were found in both libraries, whereas six OTUs were found only in the Erhualian library and 23 OTUs were found only in the Landrace library. Real-time PCR showed that the abundance of fecal methanogens in Landrace pigs was significantly higher than that in Erhualian pigs (P<0.05). Results showed that the Landrace pig (lean) harbored a greater diversity and higher numbers of methanogenmcrAgene copies than the Erhualian pig (obese). These differences may be related to the fatness or leanness in these two pig breeds. The results provide new leads for further investigations on the fat storage of pigs or even humans.

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.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.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, P0.001; 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; P0.001 and R = 0.77; P0.001 respectively).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.

Campylobacter spp. are the leading cause of gastroenteritis worldwide. Most human infections result from contaminated food; however, infections are also caused by recreational waterway contamination. Campylobacter culture is technically challenging and enumeration by culture-based methods is onerous. Thus, we employed qPCR to quantify Campylobacter spp. in fresh- and marine-water samples, raw sewage and animal feces. Multiplex PCR determined whether Campylobacter jejuni or C. coli, most commonly associated with human disease, were present in qPCR-positive samples. Campylobacters were detected in raw sewage, and in feces of all avian and mammalian species tested. Campylobacter-positive concentrations ranged from 68 to 2.3 × 106 cells per 500 mL. Although C. jejuni and C. coli were rare in waterways, they were prevalent in sewage and feces. Campylobacter-specific qPCR screening of environmental waters did not correlate with the regulatory EPA method 1600 (Enterococcus culture), nor with culture-independent, molecular-based microbial source tracking indicators, such as human polyomavirus, human Bacteroidales and Methanobrevibacter smithii. Our results suggest that neither the standard EPA method nor the newly proposed culture-independent methods are appropriate surrogates for Campylobacter contamination in water. Thus, assays for specific pathogens may be necessary to protect human health, especially in waters that are contaminated with sewage and animal feces.

Methanoarchaea have an outstanding capability to methylate numerous metal(loid)s therefore producing toxic and highly mobile derivatives. Here, we report that the production of methylated bismuth species by the methanoarchaeumMethanobrevibacter smithii, a common member of the human intestine, impairs the growth of members of the beneficial intestinal microbiota at low concentrations. The bacteriumBacteroides thetaiotaomicron, which is of great importance for the welfare of the host due to its versatile digestive abilities and its protective function for the intestine, is highly sensitive against methylated, but not against inorganic, bismuth species. The level of methylated bismuth species produced by the methanoarchaeumM. smithiiin a coculture experiment causes a reduction of the maximum cell density ofB. thetaiotaomicron. This observation suggests that the production of methylated organometal(loid) species in the human intestine, caused by the activity of methanoarchaea, may affect the health of the host. The impact of the species to reduce the number of the physiological intestinal microbiota brings an additional focus on the potentially harmful role of methanoarchaea in the intestine of a higher organism.

International audience; Background: Obesity is associated with increased health risk and has been associated with alterations in bacterial gut microbiota, with mainly a reduction in Bacteroidetes, but few data exist at the genus and species level. It has been reported that the Lactobacillus and Bifidobacterium genus representatives may have a critical role in weight regulation as an anti-obesity effect in experimental models and humans, or as a growth-promoter effect in agriculture depending on the strains. Objectives and methods: To confirm reported gut alterations and test whether Lactobacillus or Bifidobacterium species found in the human gut are associated with obesity or lean status, we analyzed the stools of 68 obese and 47 controls targeting Firmicutes, Bacteroidetes, Methanobrevibacter smithii, Lactococcus lactis, Bifidobacterium animalis and seven species of Lactobacillus by quantitative PCR (qPCR) and culture on a Lactobacillus-selective medium. Findings: In qPCR, B. animalis (odds ratio (OR) 0.63; 95% confidence interval (CI) 0.39-1.01; P = 0.056) and M. smithii (OR = 0.76; 95% CI 0.59-0.97; P = 0.03) were associated with normal weight whereas Lactobacillus reuteri (OR = 1.79; 95% CI 1.03-3.10; P = 0.04) was associated with obesity. Conclusion: The gut microbiota associated with human obesity is depleted in M. smithii. Some Bifidobacterium or Lactobacillus species were associated with normal weight (B. animalis) while others (L. reuteri) were associated with obesity. Therefore, gut microbiota composition at the species level is related to body weight and obesity, which might be of relevance for further studies and the management of obesity. These results must be considered cautiously because it is the first study to date that links specific species of Lactobacillus with obesity in humans. International Journal of Obesity (2012) 36, 817-825; doi:10.1038/ijo.2011.153; published online 9 August 2011

Bacteria and Archaea are evolutionarily and biochemically distinct domains found together in many environments. Robust ‘universal’ PCR primer sets targeting both the bacterial 16S rRNA gene and the type I chaperonin gene have been established. However, ‘universal’ PCR primers for Archaea are currently limited to the 16S rRNA gene. We investigated the type II chaperonin (known as the thermosome, TF55, CCT or TCP-1) as a potential universal target (UT) for Archaea. Reproducible amplification of thermosome gene sequences from all major phyla tested was achieved through the application of a mixture or ‘cocktail’ of two forward and two reverse primers. Phylogenies based on the ∼750-bp thermosome UT were congruent with 16S rRNA gene phylogenies while exhibiting longer branch lengths, improving resolution of closely related taxa. ‘Universal’ thermosome primers were applied to profiling the archaeal community of dairy cow rumen and results compared with profiles based on the 16S rRNA gene and methyl co-enzyme M reductase (methanogen-specific) gene. Clone libraries generated from each target gene, as well as a pyrosequencing profile of one thermosome rumen library, revealed that all three targets consistently detected Methanobrevibacter smithii, Methanobrevibacter ruminantium and Methanosphaera stadtmanae as the dominant constituents; however, thermosome gene sequences were more diverse than either of the other targets providing a higher resolution description of the archaeal community. These findings demonstrate that a ‘universal’ thermosome PCR protocol is a powerful metagenomic tool for detecting and characterizing Archaea and archaeal communities.

The etiology of bioaerosol-related pulmonary diseases remains poorly understood. Recently, archaea emerged as prominent airborne components of agricultural environments, but the consequences of airway exposure to archaea remain unknown. Since subcomponents of archaea can be immunogenic, we used a murine model to study the pulmonary immune responses to two archaeal species found in agricultural facilities: Methanobrevibacter smithii (MBS) and Methanosphaera stadtmanae (MSS). Mice were administered intranasally with 6.25, 25 or 100 µg of MBS or MSS, once daily, 3 days a week, for 3 weeks. MSS induced more severe histopathological alterations than MBS with perivascular accumulation of granulocytes, pronounced thickening of the alveolar septa, alveolar macrophages accumulation and increased perivascular mononucleated cell accumulation. Analyses of bronchoalveolar lavage fluids revealed up to 3 times greater leukocyte accumulation with MSS compared to MBS. Instillation of 100 µg of MBS or MSS caused predominant accumulation of monocyte/macrophages (4.5×10(5) and 4.8×10(5) cells/ml respectively) followed by CD4(+) T cells (1.38×10(5) and 1.94×10(5) cells/ml respectively), B cells (0.73×10(5) and 1.28×10(5) cells/ml respectively), and CD8(+) T cells (0.20×10(5) and 0.31×10(5) cells/ml respectively) in the airways. Both archaeal species induced similar titers of antigen-specific IgGs in plasma. MSS but not MBS caused an accumulation of eosinophils and neutrophils in the lungs, which surprisingly, correlated inversely with the size of the inoculum. Stronger immunogenicity of MSS was confirmed by a 3 fold higher accumulation of myeloid dendritic cells in the airways, compared to MBS. Thus, the dose and species of archaea determine the magnitude and nature of the pulmonary immune response. This is the first report of an immunomodulatory role of archaeal species found in bioaerosols.

The human gut microbiota harbors three main groups of H 2 -consuming microbes: methanogens including the dominant archaeon, Methanobrevibacter smithii , a polyphyletic group of acetogens, and sulfate-reducing bacteria. Defining their roles in the gut is important for understanding how hydrogen metabolism affects the efficiency of fermentation of dietary components. We quantified methanogens in fecal samples from 40 healthy adult female monozygotic (MZ) and 28 dizygotic (DZ) twin pairs, analyzed bacterial 16S rRNA datasets generated from their fecal samples to identify taxa that co-occur with methanogens, sequenced the genomes of 20 M. smithii strains isolated from families of MZ and DZ twins, and performed RNA-Seq of a subset of strains to identify their responses to varied formate concentrations. The concordance rate for methanogen carriage was significantly higher for MZ versus DZ twin pairs. Co-occurrence analysis revealed 22 bacterial species-level taxa positively correlated with methanogens: all but two were members of the Clostridiales, with several being, or related to, known hydrogen-producing and -consuming bacteria. The M. smithii pan-genome contains 987 genes conserved in all strains, and 1,860 variably represented genes. Strains from MZ and DZ twin pairs had a similar degree of shared genes and SNPs, and were significantly more similar than strains isolated from mothers or members of other families. The 101 adhesin-like proteins (ALPs) in the pan-genome (45 ± 6 per strain) exhibit strain-specific differences in expression and responsiveness to formate. We hypothesize that M. smithii strains use their different repertoires of ALPs to create diversity in their metabolic niches, by allowing them to establish syntrophic relationships with bacterial partners with differing metabolic capabilities and patterns of co-occurrence.

The F420-dependent NADP oxidoreductase enzyme from Methanobrevibacter smithii catalyzes the important electron transfer step during methanogenesis. Therefore, it may act as potential target for blocking the process of methane formation. Its protein sequence is available in GenBank (accession number: ABQ86254.1) however no report has been found about its 3D protein structure. In this work, we first time claim 3D model structure of F420-dependent NADP oxidoreductase enzyme from Methanobrevibacter smithii by comparative homology modeling method. Swiss model and ESyPred3d (via Modeller 6v2) software’s were generated the 3D model by detecting 1JAX (A) as template along with sequence identities of 34.272% and 35.40%. Furthermore, PROCHECK with Ramachandran plot and ProSA analysis revealed that swiss model produced better model than Modeller6v2 with 98.90% of residues in favored and additional allowed regions (RM plot) as well as with ProSA Z score of -7.26. In addition, we investigated that the substrate F420 bound at the cavity of the model. Subsequently, inhibitor prediction study revealed that Lovastatin (-22.07 Kcal/mol) and Compactin (Mevastatin) (-21.91 Kcal/mol) produced more affinity for model structure of NADP oxidoreducatse as compared to F420 (-14.40 Kcal/mol). It indicates that the Lovastatin and Compactin (Mevastatin) compounds (Negative regulator) may act as potential inhibitor of F420 dependent NADP oxidoreducatse protein.

The effects of metronidazole, CO, methanogens, and CO 2 on the fermentation of glucose by the anaerobic fungus Neocallimastix sp. strain L2 were investigated. Both metronidazole and CO caused a shift in the fermentation products from predominantly H 2 , acetate, and formate to lactate as the major product and caused a lower glucose consumption rate and cell protein yield. An increased lactate dehydrogenase activity and a decreased hydrogenase activity were observed in cells grown under both culture conditions. In metronidazole-grown cells, the amount of hydrogenase protein was decreased compared with the amount in cells grown in the absence of metronidazole. When Neocallimastix sp. strain L2 was cocultured with the methanogenic bacterium Methanobrevibacter smithii , the fermentation pattern changed in the opposite direction: H 2 and acetate production increased at the expense of the electron sink products lactate, succinate, and ethanol. A concomitant decrease in the enzyme activities leading to these electron sink products was observed, as well as an increase in the glucose consumption rate and cell protein yield, compared with those of pure cultures of the fungus. Low levels of CO 2 in the gas phase resulted in increased H 2 and lactate formation and decreased production of formate, acetate, succinate, and ethanol, a decreased glucose consumption rate and cell protein yield, and a decrease in most of the hydrogenosomal enzyme activities. None of the tested culture conditions resulted in changed quantities of hydrogenosomal proteins. The results indicate that manipulation of the pattern of fermentation in Neocallimastix sp. strain L2 results in changes in enzyme activities but not in the proliferation or disappearance of hydrogenosomes.

Microbiological contamination from runoff is a human health concern in urbanized coastal environments, but the contamination sources are often unknown. This study quantified fecal indicator bacteria and compared the distributions of human-specific genetic markers and bacterial community composition during dry and wet weather in urban creeks draining two neighboring watersheds in Santa Barbara, CA. In a prior study conducted during exclusively dry weather, the creeks were contaminated with human waste as indicated by elevated numbers of the human-specific Bacteroidales marker HF183 (Sercu et al. in Environ Sci Technol 43:293–298, 2009). During the storm, fecal indicator bacterial numbers and loads increased orders of magnitude above dry weather conditions. Moreover, bacterial community composition drastically changed during rainfall and differed from dry weather flow by (1) increased bacterial diversity, (2) reduced spatial heterogeneity within and between watersheds, and (3) clone library sequences more related to terrestrial than freshwater taxa. Finally, the spatial patterns of human-associated genetic markers (HF183 and Methanobrevibacter smithii nifH gene) changed during wet weather, and the contribution of surface soils to M. smithii nifH gene detection was suspected. The increased fecal indicator bacteria numbers during wet weather were likely associated with terrestrial sources, instead of human waste sources that dominated during dry weather flow.

The gut microbiota has evolved intimate symbiotic relationships with the human host and is considered as an internal “microbial organ.” It has also been shown to exhibit an immensely diverse, complex composition and significant involvement in human health and disease by use of various high-throughput “omics” techniques. However, the molecular basis of these host–microbe interactions and the role of individual bacterial species remain unclear. In this Chapter, we discuss strategies and techniques for understanding the host–microbiome symbiosis, the modulation of human metabolic phenotype especially through the gut–liver axis, and potential therapies for the gut microbiota.

Ciliated protozoa are the principal component of the rumen microbiota. They contribute significantly to the digestion of ruminants. As anaerobic fermentative micro organisms, rumen ciliated protozoa produce a significant amount of hydrogen and formate. Methanogenic archaea therefore associate closely with rumen ciliated protozoa. The presence of episymbiotic methanogens in rumen ciliated protozoa has been demonstrated as early as 1980s by microscopy. The number of ciliate-associated methanogens increases from the 100-level to 104-level per cell of ciliate after feeding. Enhancement of hydrogen and/or formate production from the ciliates by feeding attracts free-living methanogens. There are a couple of studies about the phylogeny of the ciliate-associated methanogens based on a molecular ecological approach. A range of methanogenic archaeal 16S rDNA, representing Methanobacteriales, Methanomicrobiales and Methanosarcinales, has been detected as ciliate-associated methanogens. However, it is still difficult to draw a conclusion about a potentially specific interaction between a particular ciliate species and a species of methanogenic archaea from these limited studies.

The study of cell walls has progressed considerably in recent years and has provided directions for future research that will soon result in practical applications. Accumulation of a variety of residues, which are toxic or at least space filling, along with threats of increased oil prices and the deleterious effects on the biosphere caused by the use of traditional fuels have stimulated the development of alternative resources of energy tied to technologies for waste elimination–reutilization and fuel production, using microbes, especially methanogenic archaea (in short, methanogens). Progress in the study of the microflora of man and animals has demonstrated the critical role of the resident microbes, including methanogens, in health and disease. These advances have boosted the study of methanogens in natural and manufactured ecosystems, including those in the gastrointestinal and urinary tracts, particularly concerning intermicrobial and microbial–host interactions that involve the cell wall. Consequently, the development of immunochemical methods for analysis, modification, manipulation, and use of methanogens in animal and human medicines and in industry has become a priority, promising significant results. The data in this Chapter on the immunochemical and molecular characteristics of the cell walls of methanogens, encompassing a range of evolutionary branches, will be useful to develop tools with direct applications in environmental, biotechnological, and medical research and practical endeavors. For example, the identification of distinct immunotypes in Methanobrevibacter smithii, which is by far the most frequent methanogen inhabiting the human body and plays a role in pathology by enhancing the pathogenesis of associated microbes, is the first step for determining which are the immunotypes most relevant for disease development. The same principle is valid for research directed toward the elucidation of which methanogenic immunotype is the most efficient for bioconversion of wastes with the highest yield of useful byproducts in waste-treatment plants.

Archaea : an international microbiological journal 2010, 967271 (2010). doi:10.1155/2010/967271, Published by Heron Publ., Victoria, British Columbia

In order to determine the interactions between anaerobic bacteria and the performance of the upflow anaerobic sludge blanket, UASB, chemical oxygen demand, COD, volatile fatty acid, VFA, and methane gas productions were determined. The variations of anaerobic microorganisms in the anaerobic granular sludge were counted under different operational conditions. The study was composed of two parts. In the first part, the numbers of methanogens and acedogens in the anaerobic granular sludge were counted at different COD removal efficiencies. The relationships between the numbers of methanogens, the methane gas production and VFA production were investigated. Most probable number (MPN) results showed that the mean number of total methanogens were between 15×104 and 29×1049 MPN/g at COD removal efficiencies of 33% and 82% in an UASB reactor treating 2,4 DCP at loading rates of 0.024–0.116 g/l.day, respectively. Decreases in methane gas percentage could be attributed to lowering in number of methanogens. The results showed that the COD removal efficiencies elevated from 33% to 82% while the number of total acedogens decreased from 120 cfu/ml to 8 cfu/ml as the 2,4 DCP loading rates lowered from 0.116 g/l.day to 0.024 g/l.day. There is a strong correlation between acedogen, methanogen bacteria numbers and reactor performances. In the second part, Methanobacterium bryantii, Methanobacterium formicicum, Methanobrevibacter smithii, Methanococcus voltae, Methanosarcina mazei, Methanosarcina acetivorans, Methanogenium bourgense, and Methanospirillum hungatei were identified in the reactor treating 2,4 DCP.

Introduction: Increasing evidence suggests a role for methanogenic archaea (methanogens) in human health and disease via syntrophic interactions with bacteria. Here we assessed the prevalence and distribution of methanogens and possible associations with bacteria in oral biofilms. Methods: Forty-four periodontal and 32 endodontic samples from necrotic teeth with radiographic evidence of apical periodontitis were analysed. Terminal restriction fragment length polymorphism analysis based on the mcrA gene, specific to methanogens, was applied. The prevalence and amounts of methanogens in endodontic samples were compared with those of Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Treponema spp. and Synergistes spp. based on real-time quantitative polymerase chain reactions. Results: Besides dominance of the mcrA gene corresponding to Methanobrevibacter oralis, one mcrA gene type, for which no cultivated member has been reported previously, was identified in five periodontal samples and one endodontic sample. Rates of non-synonymous vs. synonymous nucleotide substitutions suggest that this mcrA gene type codes for a functionally active methyl-coenzyme M reductase. Methanobrevibacter smithii, the prominent methanogen in the human gut system, was not detected. Mean proportions of methanogens were comparable to Synergistes spp. ranging from 0.5 to 1.0% of the total microbial community. Treponema spp. dominated with a mean proportion of 10%, while the mean proportions of the other endodontic pathogens were below 0.1%. A positive association between methanogens and Synergistes spp. was found. Conclusion: Our data provide evidence of a novel, as yet uncultured methanogenic phylotype in association with oral infections, and indicate possible interactions between methanogens and Synergistes spp., the nature of which deserves further investigation.

Water quality is frequently impacted by microbial pollution from human and animal feces. Microbial source tracking (MST) can identify dominant pollution sources and improve assessment of health risk compared to indicator bacteria alone. This study aims to standardize and validate MST methods across laboratories in coastal Gulf of Mexico states. Three laboratories evaluated library-independent MST methods for human sewage detection via conventional PCR: (1) human-associated Bacteroidales, (2) human polyomaviruses (HPyVs), and (3) Methanobrevibacter smithii. All methods detected targets in human sewage seeded into buffer, freshwater or marine water (100% sensitivity). The limit of detection (LOD) for human sewage was lowest for the Bacteroidales assay (10(-5)-10(-6) dilution). LODs for HPyVs and M. smithii assays were similar to each other (10(-3)-10(-4)), but were higher than Bacteroidales. The HPyVs assay was 100% specific, showing no cross-reactivity to dog, cow, cat, bird, or wild animal feces among >300 samples from three Gulf Coast regions. The human Bacteroidales assay was 96% specific, but cross-reacted with 10% of dog and some chicken samples. The M. smithii assay was 98% specific with limited cross-reactivity with cow, dog and seagull samples. An experts' workshop concluded that all methods showed sufficient accuracy and reliability to move forward. SOPs will be distributed to collaborating laboratories for further inter-laboratory comparison, and field validation will occur in year 2.

The gastrointestinal (GI) tract is a dynamic environment and therefore the stability of the commensal community, or microbiota, is under constant challenge. Microscopic observations have revealed that the majority of bacteria present in the GI tract are not detected using standard culturing techniques, however with the application of culture-independent techniques it has been estimated that between 500 to 1000 bacterial species inhabit the human GI tract. Numerically predominant organisms in the microbiota belong to two eubacterial divisions, the Cytophaga-Flavobacterium-Bacteroides (CFB) and the Firmicutes, and fall into three main groups; Clostridium rRNA subcluster XIVa, Clostridium rRNA subcluster IV and Bacteroides. The prevalence and diversity of bacteria in different areas of the GI tract is influenced by the different conditions at these sites and thus the microbiota of the stomach and jejunum varies with that of the large intestine. Additionally, host genotype, age and diet have all been shown to affect microbial diversity in the GI tract. The distal intestine harbours the highest bacterial cell densities for any known ecosystem. Characterizing the species composition of the healthy microbiota may be a key step in identifying bacterial or associated physiological conditions that are present or absent in an unhealthy microbiota.

Recent evidence suggests that the microbial community in the human intestine may play an important role in the pathogenesis of obesity. We examined 184,094 sequences of microbial 16S rRNA genes from PCR amplicons by using the 454 pyrosequencing technology to compare the microbial community structures of 9 individuals, 3 in each of the categories of normal weight, morbidly obese, and post-gastric-bypass surgery. Phylogenetic analysis demonstrated that although the Bacteria in the human intestinal community were highly diverse, they fell mainly into 6 bacterial divisions that had distinct differences in the 3 study groups. Specifically, Firmicutes were dominant in normal-weight and obese individuals but significantly decreased in post-gastric-bypass individuals, who had a proportional increase of Gammaproteobacteria . Numbers of the H 2 -producing Prevotellaceae were highly enriched in the obese individuals. Unlike the highly diverse Bacteria , the Archaea comprised mainly members of the order Methanobacteriales , which are H 2 -oxidizing methanogens. Using real-time PCR, we detected significantly higher numbers of H 2 -utilizing methanogenic Archaea in obese individuals than in normal-weight or post-gastric-bypass individuals. The coexistence of H 2 -producing bacteria with relatively high numbers of H 2 -utilizing methanogenic Archaea in the gastrointestinal tract of obese individuals leads to the hypothesis that interspecies H 2 transfer between bacterial and archaeal species is an important mechanism for increasing energy uptake by the human large intestine in obese persons. The large bacterial population shift seen in the post-gastric-bypass individuals may reflect the double impact of the gut alteration caused by the surgical procedure and the consequent changes in food ingestion and digestion.

The effect of propionate toxicity at different pH values (6.5, 7.0, and 8.0) on methanogen-enriched sludge. Methanobrevibacter smithii, and Methanospirillum hungatii was studied. Organisms were grown in Balch medium 3 in Hungate tubes, and toxicity was characterized by a decrease in production of methane and in bacterial numbers. Propionate inhibited bacterial growth and cumulative methane production at concentrations as low as 20 mM. In the absence of propionate, the methanogen-enriched sludge and M. smithii showed better cumulative methane production at pH 6.5 and 7.0 than at pH 8.0. However, in the presence of propionate, these organisms showed better cumulative methane production at pH 8.0. M. hungatii differed in its behavior; the best values of cumulative methane production for this organism occurred at pH 7.0. Bacterial numbers reflected the microbial response to the presence of propionate. The highest counts of methanogenic bacteria were observed at pH 6.5 and 8.0. The numbers of methanogens were affected by the presence of propionate even at concentrations as low as 20 or 30 mM; at propionate concentrations above 80 mM, the methanogen count was affected by at least 2 orders of magnitude. Upon comparison of the responses of the pure cultures and the methanogen-enriched sludge to increasing propionate concentrations, it was found that the sensitivity of the pure cultures was similar to that of the methanogens in the sludge.

Pentoses and hexoses were removed simultaneously from lucerne (Medicago sativa) stem by Piromonas communis and Neocallimastix frontalis growing axenically or in coculture with Methanobrevibacter smithii. In monocultures, more arabinose (68–71%) than glucose (52–53%) or xylose (25–30%) was removed. In cocultures, xylose solubilization from cell walls increased for P. communis whereas glucose solubilization increased for both fungi. The rates of sugar removal were monosaccharide-dependent. For both fungi, the specific activities of extracellular hydrolytic enzymes were markedly elevated in methanogenic cocultures.

The diversity of methanogenic Archaea from the gut of 6 humans was investigated by targeting mcrA, a molecular metabolic marker of methanogenesis. Three operational taxonomic units (OTUs) were recovered from about 400 clones analyzed, two of which were attributed to the expected Methanobacteriales Methanobrevibacter smithii (4 volunteers) and Methanosphaera stadtmanae (1 volunteer). The third OTU (1 volunteer) corresponded to a distant phylotype that does not cluster with any of the five methanogenic orders. This result, also supported by 16S archaeal sequences retrieved from the same volunteer, strongly suggests there may be a sixth order and hence potential underestimation of the role of methanogens in gut physiology.