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PubMed1982

Enumeration of Methanobrevibacter smithii in human feces.

A plating medium containing cephalothin and clindamycin was developed for enumeration and isolation of methanogens in human feces. Specimens from nine CH4-producing subjects had total anaerobe counts of 1-8 x 1011/g dry weight. Methanogen counts on the antibiotic medium ranged from 0.001 - 12.6% of the total anaerobe count. There was no correlation between age, sex or percent dry fecal weight and the ratio of methanogens to total counts. Specimens from eight non-CH4-producing individuals contained bacteria that yielded nonmethanogenic colonies on the antibiotic medium. The means +/- SD of the logarithm of the total counts per gram dry weight were 11.4 /+- 0.29 and 11.38 +/- 0.44 for the positive and negative groups respectively. Values for the antibiotic-resistant flora were 8.8 /+- 1.13 and 7.78 /+- 1.08 respectively. Methanogens were isolated from the most dilute inoculum of each specimen from CH4-producing subjects. All isolates were morphologically, physiologically, and immunologically identical to Methanobrevibacter smithii. Growth of methanogens in media that were essentially extracts of CH4-negative feces suggested that no nutrients were lacking or inhibitors present in intestinal contents that prevent the growth of methanogens in these individuals.

Adult↗
PubMed1998

Methanobrevibacter filiformis sp. nov., A filamentous methanogen from termite hindguts.

A morphologically distinct, filamentous methanogen was isolated from hindguts of the subterranean termite, Reticulitermes flavipes (Kollar) (Rhinotermitidae), wherein it was part of the microbiota colonizing the hindgut wall. Individual filaments of strain RFM-3 were 0.23-0.28 micron in diameter and usually > 50 micron in length and aggregated into flocs that were often >/= 0.1 mm in diameter. Optimal growth of strain RFM-3 was obtained at pH 7.0-7.2 and 30 degrees C with a yeast-extract-supplemented, dithiothreitol-reduced medium in which cells produced stoichiometric amounts of methane from H2 + CO2. The morphology and gram-positive staining reaction of strain RFM-3, as well as its resistance to cell lysis by various chemical agents and its restriction to H2 + CO2 as an energy source, suggested that it was a member of the Methanobacteriaceae. The nucleotide sequence of the SSU-rRNA-encoding gene of strain RFM-3 confirmed this affiliation and also supported its recognition as a new species of Methanobrevibacter, for which the epithet filiformis is herewith proposed. Although M. filiformis was one of the dominant methanogens in R. flavipes collected from Woods Hole (Mass., USA), cells of similar morphology were not consistently observed in R. flavipes collected from different geographical locations.

Animals↗
PubMed2000

Anaerobic bioconversion of cellulose by Ruminococcus albus, Methanobrevibacter smithii, and Methanosarcina barkeri.

A system is described that combines the fermentation of cellulose to acetate, CH4, and CO2 by Ruminococcus albus and Methanobrevibacter smithii with the fermentation of acetate to CH4 and CO2 by Methanosarcina barkeri to convert cellulose to CH4 and CO2. A cellulose-containing medium was pumped into a co-culture of the cellulolytic R. albus and the H2-using methanogen, Mb. smithii. The effluent was fed into a holding reservoir, adjusted to pH 4.5, and then pumped into a culture of Ms. barkeri maintained at constant volume by pumping out culture contents. Fermentation of 1% cellulose to CH4 and CO2 was accomplished during 132 days of operation with retention times (RTs) of the Ms. barkeri culture of 7.5-3.8 days. Rates of acetate utilization were 9.5-17.3 mmol l(-1) day(-1) and increased with decreasing RT. The Ks for acetate utilization was 6-8 mM. The two-stage system can be used as a model system for studying biological and physical parameters that influence the bioconversion process. Our results suggest that manipulating the different phases of cellulose fermentation separately can effectively balance the pH and ionic requirements of the acid-producing phase with the acid-using phase of the overall fermentation.

Acetic Acid↗
PubMed2000

The effect of a methanogen, Methanobrevibacter smithii, on the growth rate, organic acid production, and specific ATP activity of three predominant ruminal cellulolytic bacteria.

Three predominant ruminal cellulolytic organisms, Fibrobacter succinogenes S85, Ruminococcus albus 8, and R. flavefaciens FD-1, were cultured with a methanogen, Methanobrevibacter smithii. Growth rates, bacterial protein, organic acids, and methane production were measured. When grown in diculture with the methanogen, a fermentative advantage was observed with F. succinogenes S85 as seen by an increase in specific rate of ATP production and organic acid concentration. The introduction of the methanogen did not improve the growth rate, organic acid yield, or specific rate of ATP production for R. albus 8. The growth rate and amount of organic acid end products increased when R. flavefaciens FD-1 was cultured with the methanogen; however, the specific activity of ATP production did not increase.

Adenosine Triphosphate↗
PubMed1999

A structural comparison of the total polar lipids from the human archaea Methanobrevibacter smithii and Methanosphaera stadtmanae and its relevance to the adjuvant activities of their liposomes.

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 beta-Glcp-(1,6)-beta-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.

Adjuvants, Immunologic↗
PubMed1985

Is coenzyme M bound to factor F430 in methanogenic bacteria? Experiments with Methanobrevibacter ruminantium.

Coenzyme M (2-mercaptoethane sulfonic acid) and factor F430 (a nickel porphinoid) are coenzymes found in methanogenic bacteria. Recently it has been proposed that in these bacteria a coenzyme MF430 also exists which plays a key role in methane formation and in which coenzyme M and F430 are bound to each other. To test this hypothesis Methanobrevibacter ruminantium, which requires coenzyme M as a vitamin, was grown in the presence of [2-14C]CoMSH. F430 and 'CoM' (mixture of CoMSH and its disulfides) were quantitatively extracted from these cells and from partially purified methyl-CoM reductase using various methods. The extracts were chromatographed on cellulose or Sephadex G-10. Under all conditions factor F430 and 'CoM' were completely (greater than 99%) separated. There was no indication for the existence of a protein-free F430 species containing covalently bound coenzyme M in Mb. ruminantium. The results support the structure previously assigned to coenzyme F430.

Binding Sites↗
PubMed1990

Effect of coumarin on glucose uptake by anaerobic rumen fungi in the presence and absence of Methanobrevibacter smithii.

The effect of coumarin (1,2 benzopyrone) on glucose utilisation by the anaerobic rumen fungi Neocallimastix frontalis and N. patriciarum has been compared with the effect of p-coumaric acid. Both compounds largely inhibited glucose utilisation by N. patriciarum strain Cx when present in the medium at a concentration of 2.5 mM, and had a similar effect on N. frontalis strain RE1 at 5 mM. Although in earlier studies co-culturing rumen fungi with Methanobrevibacter smithii enhanced resistance to ionophores, no comparable protective effect of M. smithii was found in the present study.

Animals↗
PubMed1983

Stability of Methanobrevibacter smithii populations in the microbial flora excreted from the human large bowel.

Total anaerobic bacteria and Methanobrevibacter smithii populations were enumerated in fecal specimens from two individuals over 10- and 13-month periods. The ratio of M. smithii to total anaerobic count varied between the individuals, but it was a relatively constant proportion of the large-bowel microbial flora within each individual. Neither a barium enema examination of one subject nor a radical change in the diet of the other had any long-term effect on the methanogen populations.

Adult↗
PubMed1990

Effect of Methanobrevibacter smithii on Xylanolytic Activity of Anaerobic Ruminal Fungi.

Three different ruminal anaerobic fungi, Neocallimastix frontalis PNK2, Sphaeromonas communis B7, and Piromonas communis B19, were grown axenically or in coculture with Methanobrevibacter smithii on xylan. N. frontalis and S. communis in monoculture and coculture accumulated xylobiose, xylose, and arabinose in the growth medium; arabinose was not metabolized, but xylobiose and xylose were subsequently used. The transient accumulation of xylose was much less evident in cocultures. Both the rate and extent of xylan utilization were increased by coculturing, and metabolite profiles became acetogenic as a result of interspecies hydrogen transfer; more acetate and less lactate were formed, while formate and hydrogen did not accumulate. For each of the three fungi, there were marked increases in the specific activities of extracellular xylanase (up to fivefold), alpha-l-arabinofuranosidase (up to fivefold), and beta-d-xylosidase (up to sevenfold) upon coculturing. The stimulating effect on fungal enzymes from coculturing with M. smithii was independent of the growth substrate, and the magnitude of the stimulation varied according to the enzymes and the incubation time. For an N. frontalis-M. smithii coculture, the positive stimulation was maintained during an extended (18-day) incubation period, and this affected not only hemicellulolytic enzymes but also polysaccharidase and glycoside hydrolase enzymes that were not involved in xylan breakdown. The specific activity of cell-bound endopeptidase was not increased under the coculture conditions used in this study. The higher enzyme activities in cocultures are discussed in relation to catabolite repression.

Journal Article↗
PubMed1991

Effect of propionate toxicity on methanogen-enriched sludge, Methanobrevibacter smithii, and Methanospirillum hungatii at different pH values.

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.

Colony Count, Microbial↗
PubMed1982

Antigenic analysis of Methanomicrobiales and Methanobrevibacter arboriphilus.

Preparation of new antisera has permitted more comprehensive immunological analyses of the two families of Methanomicrobiales. Methanomicrobiaceae and Methanosarcinaceae, and the species Methanobrevibacter arboriphilus. Immunological analysis was carried out with antibody probes against 23 strains, including almost all genera and species of methanogens. The absence of cross-reactions between families of methanogens was confirmed. Methanomicrobium and Methanogenium were found to be immunologically related. Extensive cross-reactions occurred among six strains of Methanosarcinaceae, but none occurred among three strains of M. arboriphilus when tested with the S probe, i.e., the last antiserum dilution of the titration curve's plateau.

Antigen-Antibody Complex↗
PubMed2000

Effects of the Fusarium spp. mycotoxins fusaric acid and deoxynivalenol on the growth of Ruminococcus albus and Methanobrevibacter ruminantium.

The Fusarium spp. mycotoxins fusaric acid and deoxynivalenol (DON) were tested for antimicrobial activity against Ruminococcus albus and Methanobrevibacter ruminantium. The growth of both organisms was inhibited by fusaric acid as low as 15 micrograms/mL (84 microM) but not by DON, at levels as high as 100 micrograms/mL (338 microM). No synergistic inhibitory effect was observed with DON plus fusaric acid. Neither organism was able to adapt to the fusaric acid and responses of each organism to the compound were different. The optical density (OD) maximum for R. albus, but not for M. ruminantium, was diminished after 28 days incubation at concentrations of fusaric acid below 240 micrograms/mL. Inhibition of R. albus started before significant growth had occurred, while M. ruminantium doubled twice before the onset of inhibition. Responses to picolinic acid, an analog of fusaric acid, were also dramatically different between the two microorganisms with M. ruminantium exhibiting a severe lag followed by a complete recovery of growth, while R. albus was only slightly inhibited with no lag. These results suggest that the mechanism of fusaric acid inhibition is specific to each microorganism. This is the first demonstration of the common mycotoxin fusaric acid inhibiting the growth of rumen bacteria.

Animals↗
PubMedDate not supplied

Isolation and antimicrobial susceptibility testing of fecal strains of the archaeon Methanobrevibacter smithii.

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.

Anti-Bacterial Agents↗
PubMed1986

A diphytanyl ether analog of phosphatidylserine from a methanogenic bacterium, Methanobrevibacter arboriphilus.

Several ninhydrin-positive lipids were found in methanogenic bacteria and the structure of one of them, designated as PNL2 from Methanobrevibacter arboriphilus, was identified as a diphytanyl ether analog of phosphatidylserine. The chromatographic behavior of the lipid on thin-layer plates and on a DEAE-cellulose column was identical to the ester form of phosphatidylserine. The infrared spectra showed the presence of amino, carboxyl, ether, and phosphate groups, and the absence of an ester linkage. The hydrophobic portion of the lipid was identified as diphytanyl glycerol diether on the basis of the mass spectrum of the acetolysis product and gas-liquid chromatography of the iodinated alkyl chain prepared by hydroiodic acid cleavage of PNL2. The fast atom bombardment-ionization and field desorption mass spectrum provided a molecular weight of 819 and several fragment ions consistent with the proposed structure. Hydrofluoric acid hydrolysis resulted in water-soluble products including serine, phosphoserine, and ammonia, which accounted for 95% of hydrolyzed PNL2. The lipid product of the hydrolysis was mainly the diether form of phosphatidic acid. This is the first report on the structural characterization of an amino-containing phospholipid in archaebacteria. Amino lipids have been found in many other methanogenic bacteria.

Chromatography, Gas↗
PubMed2004

16S ribosomal DNA-directed PCR primers for ruminal methanogens and identification of methanogens colonising young lambs.

The population densities and identities of methanogens colonising new-born lambs in a grazing flock were determined from rumen samples collected at regular intervals after birth. Methanogen colonisation was found at the first sampling (1-3 days after birth) and population densities reached around 10(4) methanogens per gram at 1 week of age. Population densities increased in an exponential manner to a maximum of 10(8)-10(9) per gram at 3 weeks of age. To identify methanogens, PCR primers specific for each of the Archaea; a grouping of the orders Methanomicrobiales, Methanosarcinales and Methanococcales; the order Methanobacteriales; the order Methanococcales; the order Methanosarcinales; the genus Methanobacterium; and the genus Methanobrevibacter were designed. Primer-pair specificities were confirmed in tests with target and non-target micro-organisms. PCR analysis of DNA extracts revealed that all the detectable ruminal methanogens belonged to the order Methanobacteriales, with no methanogens belonging to the Methanomicrobiales, the Methanosarcinales, or the Methanococcales being detected. In 3 lambs, the initial colonising methanogens were Methanobrevibacter spp. and in 2 lambs were a mixture of Methanobrevibacter and Methanobacterium spp. In the latter case, the initial colonising Methanobacterium spp. subsequently disappeared and were not detectable 12-19 days after birth. Seven weeks after birth, lambs contained only Methanobrevibacter spp. This study, the first to provide information on the identities of methanogens colonising pre-ruminants, suggests that the predominant methanogens found in the mature rumen establish very soon after birth and well before a functioning rumen develops.

Journal Article↗