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Incidence of methanogenic bacteria in a sigmoidoscopy population: an association of methanogenic bacteria and diverticulosis.

This study determined the incidence and concentration of methane-producing bacteria in tap water enema samples of 130 individuals taken before sigmoidoscopy. The number of subjects classified in five major colonic groups were as follows: normal colon 36, diverticulosis 57, inflammatory bowel disease 11, colon polyps 34, and colon cancer 11. Some patients were placed in more than one category. Ninety four of the subjects or 72% had methanogenic bacteria ranging in concentration from 6 to about 3 X 10(10)/g dry weight of faeces. The predominant methanogen in all groups was Methanobrevibacter smithii. Chi-square analysis showed that the incidence of methanogens in concentrations of 10(7)/g dry weight of faeces or greater in patients with diverticulosis (58%) was significantly greater than in normal patients (25%). High methanogen concentrations are associated with excretion of methane in the breath.

Adult↗

Decompression sickness risk in rats by microbial removal of dissolved gas.

We present a method for reducing the risk of decompression sickness (DCS) in rats exposed to high pressures of H2. Suspensions of the human colonic microbe Methanobrevibacter smithii were introduced via a colonic cannula into the large intestines of the rats. While the rats breathed H2 in a hyperbaric chamber, the microbe metabolized some of the H2 diffusing into the intestine, converting H2 and CO2 to methane and water. Rate of release of methane from the rats, which was monitored by gas chromatography, varied with chamber H2 pressure. This rate was higher during decompression than during compression, suggesting that during decompression the microbe was metabolizing H2 stored in the rats' tissues. Rats treated with M. smithii had a 25% (5 of 20) incidence of DCS, which was significantly lower (P < 0.01) than the 56% (28 of 50) incidence of untreated controls, brought on by a standardized compression and decompression sequence. Thus using a microbe in the intestine to remove an estimated 5% of the body burden of H2 reduced DCS risk by more than one-half. This method of biochemical decompression may potentially facilitate human diving.

Animals↗

Increasing activity of H(2)-metabolizing microbes lowers decompression sickness risk in pigs during H(2) dives.

The risk of decompression sickness (DCS) was modulated by varying the biochemical activity used to eliminate some of the hydrogen (H(2)) stored in the tissues of pigs (19.4 +/- 0.2 kg) during hyperbaric exposures to H(2). Treated pigs (n = 16) received intestinal injections of Methanobrevibacter smithii, a microbe that metabolizes H(2) to water and CH(4). 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 H(2)). 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.

Animals↗

Modulation of decompression sickness risk in pigs with caffeine during H(2) biochemical decompression.

In H(2) biochemical decompression, H(2)-metabolizing intestinal microbes remove gas stored in tissues of animals breathing hyperbaric H(2), thereby reducing decompression sickness (DCS) risk. We hypothesized that increasing intestinal perfusion in pigs would increase the activity of intestinal Methanobrevibacter smithii, lowering DCS incidence further. Pigs (Sus scrofa, 17-23 kg, n = 20) that ingested caffeine (5 mg/kg) increased O(2) consumption rate in 1 atm air by ~20% for at least 3 h. Pigs were given caffeine alone or caffeine plus injections of M. smithii. Animals were compressed to 24 atm (20.5-23.1 atm H(2), 0.3-0.5 atm O(2)) for 3 h, then decompressed and observed for signs of DCS. In previous studies, DCS incidence in animals without caffeine treatment was significantly (P < 0.05) lower with M. smithii injections (7/16) than in controls (9/10). However, contrary to our hypothesis, DCS incidence was marginally higher (P = 0.057) in animals that received caffeine and M. smithii (9/10) than in animals that received caffeine but no M. smithii (4/10). More information on gas kinetics is needed before extending H(2) biochemical decompression to humans.

Animals↗

Archaeosomes varying in lipid composition differ in receptor-mediated endocytosis and differentially adjuvant immune responses to entrapped antigen.

Archaeosomes prepared from total polar lipids extracted from six archaeal species with divergent lipid compositions had the capacity to deliver antigen for presentation via both MHC class I and class II pathways. Lipid extracts from Halobacterium halobium and from Halococcus morrhuae strains 14039 and 16008 contained archaetidylglycerol methylphosphate and sulfated glycolipids rich in mannose residues, and lacked archaetidylserine, whereas the opposite was found in Methanobrevibacter smithii, Methanosarcina mazei and Methanococcus jannaschii. Annexin V labeling revealed a surface orientation of phosphoserine head groups in M. smithii, M. mazei and M. jannaschii archaeosomes. Uptake of rhodamine-labeled M. smithii or M. jannaschii archaeosomes by murine peritoneal macrophages was inhibited by unlabeled liposomes containing phosphatidylserine, by the sulfhydryl inhibitor N-ethylmaleimide, and by ATP depletion using azide plus fluoride, but not by H. halobium archaeosomes. In contrast, N-ethylmaleimide failed to inhibit uptake of the four other rhodamine-labeled archaeosome types, and azide plus fluoride did not inhibit uptake of H. halobium or H. morrhuae archaeosomes. These results suggest endocytosis of archaeosomes rich in surface-exposed phosphoserine head groups via a phosphatidylserine receptor, and energy-independent surface adsorption of certain other archaeosome composition classes. Lipid composition affected not only the endocytic mechanism, but also served to differentially modulate the activation of dendritic cells. The induction of IL-12 secretion from dendritic cells exposed to H. morrhuae 14039 archaeosomes was striking compared with cells exposed to archaeosomes from 16008. Thus, archaeosome types uniquely modulate antigen delivery and dendritic cell activation.

Adjuvants, Immunologic↗

Phylogenetic analysis of methanogens from the bovine rumen.

BACKGROUND: Interest in methanogens from ruminants has resulted from the role of methane in global warming and from the fact that cattle typically lose 6 % of ingested energy as methane. Several species of methanogens have been isolated from ruminants. However they are difficult to culture, few have been consistently found in high numbers, and it is likely that major species of rumen methanogens are yet to be identified. RESULTS: Total DNA from clarified bovine rumen fluid was amplified using primers specific for Archaeal 16S rRNA gene sequences (rDNA). Phylogenetic analysis of 41 rDNA sequences identified three clusters of methanogens. The largest cluster contained two distinct subclusters with rDNA sequences similar to Methanobrevibacter ruminantium 16S rDNA. A second cluster contained sequences related to 16S rDNA from Methanosphaera stadtmanae, an organism not previously described in the rumen. The third cluster contained rDNA sequences that may form a novel group of rumen methanogens. CONCLUSIONS: The current set of 16S rRNA hybridization probes targeting methanogenic Archaea does not cover the phylogenetic diversity present in the rumen and possibly other gastro-intestinal tract environments. New probes and quantitative PCR assays are needed to determine the distribution of the newly identified methanogen clusters in rumen microbial communities.

Animals↗

Characterization of a 4-methylbenzoate-degrading methanogenic consortium as determined by small-subunit rDNA sequence analysis.

A methanogenic consortium that degrades 4-methylbenzoate (MBA) as the sole carbon and energy source was successfully enriched in an upflow anaerobic sludge bed bioreactor and studied. Electron microscopic observation showed that long rods with a distinct collar feature resembling Desulfomonile tiedjei rods were the predominant population, and that these rods formed a close spatial orientation with Methanobrevibacter-like bacteria. In addition, thin filaments and bamboo-shaped filaments that highly resembled the acetoclastic Methanosaeta were also frequently observed. A 16S rDNA clone library was constructed for the domain Bacteria, and 20 sequence types or operational taxonomic units (OTUs) were found out of 139 clones screened. Phylogenetic analysis classified these 20 nearly full-length OTUs into the delta (50.3% of total clones) and gamma (4.3%) subdivisions of the division Proteobacteria, the green non-sulfur bacteria subdivision I (7.2%), Cytophagales (7.2%), Planctomycetes (5.7%), gram-positive low G + C group (8.6%), candidate divisions OP8, OP10 and OP11 (9.3%), and a novel candidate division MBA1 (7.2%) that had an interdivisional sequence similarity less than 75%. However, only 3 OTUs had a sequence similarity higher than 90% to known isolates or environmental 16S rDNA clones, suggesting that the microbial community was diversified and largely unidentified. In particular, those 8 OTUs found in the delta-Proteobacteria were either clustered into novel groups or showed a low sequence similarity to closely related bacteria. It is highly possible that the delta-Proteobacteria were the long rods with a distinct collar feature observed microscopically, and together with the methanogens were mainly responsible for the syntrophic degradation of MBA. The unique and novel microbial populations identified explained the requirement of a long start-up period of up to 426 d for the MBA-degrading consortium.

Journal Article↗

Phylogenetic analysis of methanogens in sheep rumen ecosystem and detection of Methanomicrobium mobile By fluorescence in situ hybridization.

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.

Animals↗

Detection of methanogens and proteobacteria from a single cell of rumen ciliate protozoa.

Rumen ciliate-associated bacteria and methanogenic archaea were analyzed by a 16S rRNA gene retrieved from a single cell of Polyplastron multivesiculatum, Isotricha intestinalis, and Ophryoscolex purkynjei. Rumen fluid was taken from a ruminally fistulated goat to prepare a ciliate fraction. Ciliate mixtures were incubated under mixtures of antibiotics for 48 h to eliminate extracellular bacteria. Individual cells of rumen ciliates were selected under microscopic observation after fixation with ethanol. Bacterial and archaeal 16S rRNA gene sequences were retrieved from each cell of three genera of ciliate. Two archaeal sequences related to Methanobrevibacter smithii were distributed to nearly all ciliate cells tested. These two methanogenic archaea were likely to be endosymbiotic methanogens commonly carried by the rumen ciliate, although some other sequences similar to the other genera were detected. A range of proteobacteria was retrieved from cells of P. multivesiculatum. Some sequences showed similarities to the previously known endosymbiotic proteobacteria. However, there were no proteobacteria that were carried by all the ciliate cells tested.

Animals↗

In vitro bacterial growth and in vivo ruminal microbiota populations associated with bloat in steers grazing wheat forage.

The role of ruminal bacteria in the frothy bloat complex common to cattle grazing winter wheat has not been previously determined. Two experiments, one in vitro and another in vivo, were designed to elucidate the effects of fresh wheat forage on bacterial growth, biofilm complexes, rumen fermentation end products, rumen bacterial diversity, and bloat potential. In Exp. 1, 6 strains of ruminal bacteria (Streptococcus bovis strain 26, Prevotella ruminicola strain 23, Eubacterium ruminantium B1C23, Ruminococcus albus SY3, Fibrobacter succinogenes ssp. S85, and Ruminococcus flavefaciens C94) were used in vitro to determine the effect of soluble plant protein from winter wheat forage on specific bacterial growth rate, biofilm complexes, VFA, and ruminal H2 and CH4 in mono or coculture with Methanobrevibacter smithii. The specific growth rate in plant protein medium containing soluble plant protein (3.27% nitrogen) was measured during a 24-h incubation at 39 degrees C in Hungate tubes under a CO2 gas phase. A monoculture of M. smithii was grown similarly, except under H2:CO2 (1:1), in a basal methanogen growth medium supplemented likewise with soluble plant protein. In Exp. 2, 6 ruminally cannulated steers grazing wheat forage were used to evaluate the influence of bloat on the production of biofilm complexes, ruminal microbial biodiversity patterns, and ruminal fluid protein fractions. In Exp. 1, cultures of R. albus (P < 0.01) and R. flavefaciens (P < 0.05) produced the most H2 among strains and resulted in greater (P < 0.01) CH4 production when cocultured with M. smithii than other coculture combinations. Cultures of S. bovis and E. ruminantium + M. smithii produced the most biofilm mass among strains. In Exp. 2, when diets changed from bermudagrass hay to wheat forage, biofilm production increased (P < 0.01). Biofilm production, concentrations of whole ruminal content (P < 0.01), and cheesecloth filtrate protein fractions (P < 0.05) in the ruminal fluid were greater on d 50 for bloated than for nonbloated steers when grazing wheat forage. The molecular analysis of the 16S rDNA showed that 2 different ruminal microbiota populations developed between bloated and nonbloated animals grazing wheat forage. Bloat in cattle grazing wheat pastures may be caused by increased production of biofilm, resulting from a diet-influenced switch in the rumen bacterial population.

Acetates↗

Phosphatidylserine receptor-mediated recognition of archaeosome adjuvant promotes endocytosis and MHC class I cross-presentation of the entrapped antigen by phagosome-to-cytosol transport and classical processing.

Archaeal isopranoid glycerolipid vesicles (archaeosomes) serve as strong adjuvants for cell-mediated responses to entrapped Ag. We analyzed the processing pathway of OVA entrapped in archaeosomes composed of Methanobrevibacter smithii lipids, high in archaetidylserine (OVA-archaeosomes). In vitro, OVA-archaeosomes stimulated spleen cells from OVA-TCR-transgenic mice, D011.10 (CD4(+) cells expressing OVA(323-339) TCR) or OT1 (>90% CD8(+) OVA(257-264) cells), indicating both MHC class I and II presentations. In vivo, when naive (Thy1.2(+)) CFSE-labeled OT1 cells were transferred into OVA-archaeosome-immunized Thy 1.1(+) recipient mice, there was profound accumulation and cycling of donor-specific cells, and differentiation of H-2K(b)Ova(257-264) CD8(+) T cells into CD44(high)CD62L(low) effectors. Both macrophages and dendritic cells (DCs) efficiently cross-presented OVA-archaeosomes on MHC class I. Blocking phagocytosis by phosphatidylserine-specific receptor agonists strongly inhibited MHC class I presentation of OVA-archaeosomes, whereas blocking mannose receptors or FcRs lacked effect, indicating specific recognition of the archaetidylserine head group of M. smithii lipids by APCs. In addition, inhibitors of endosomal acidification blocked MHC class I processing of OVA-archaeosomes, whereas endosomal protease inhibitors lacked effect, suggesting acidification-dependent phagosome-to-cytosol diversion. Proteasomal inhibitors blocked OVA-archaeosome MHC class I presentation, confirming cytosolic processing. Both in vitro and in vivo, OVA-archaeosome MHC class I presentation required TAP. Ag-free archaeosomes also activated DC costimulation and cytokine production, without overt inflammation. Phosphatidylserine-specific receptor-mediated endocytosis is a mechanism of apoptotic cell clearance and DCs cross-present Ags sampled from apoptotic cells. Our results reveal the novel ability of archaeosomes to exploit this mechanism for cytosolic MHC class I Ag processing, and provide an effective particulate vaccination strategy.

Adjuvants, Immunologic↗

[Catalase and superoxide dismutase in the cells of strictly anaerobic microorganisms].

Strictly anaerobic microorganisms relating to various physiological groups were screened for catalase and superoxide dismutase (SOD) activity. All of the investigated anaerobes possessed the SOD activity, necessary for protection against toxic products of oxygen reduction. High specific activities of SOD were found in Acetobacterium woodii and Acetobacterium wieringae. Most of the investigated clostridia and acetogens were catalase-negative. A significant activity of catalase was found in Thermohydrogenium kirishiense, in representatives of the genus Desulfotomaculum, and in several methanogens. Methanobrevibacter arboriphilus had an exceptionally high catalase activity after growth in medium supplemented with hemin. Hemin also produced a strong positive effect on the catalase activity in many other anaerobic microorganisms. In methanogens, the activities of the enzymes of antioxidant defense varied in wide ranges depending on the stage of growth and the energy source.

Bacteria, Anaerobic↗

Archaeosomes induce enhanced cytotoxic T lymphocyte responses to entrapped soluble protein in the absence of interleukin 12 and protect against tumor challenge.

Archaeosome adjuvants formulated as archaeal ether glycerolipid vesicles induce strong CD4(+) as well as CD8(+) CTL responses to entrapped soluble antigens. Immunization of mice with ovalbumin (OVA) entrapped in archaeosomes composed of the total polar lipids of Methanobrevibacter smithii resulted in a potent OVA-specific CD8(+) T-cell response, and subsequently, the mice dramatically resisted solid tumor growth of OVA-expressing EG.7 cells and lung metastasis of B16OVA melanoma cells. Prophylactic protection was antigen-specific because tumor curtailment was not seen in mice injected with antigen-free archaeosomes. Similarly, there was no protection against B16 melanoma cells lacking OVA expression. Furthermore, in vivo depletion of CD8(+) T cells abrogated the protective response, indicating that the antitumor immunity was mediated by CTLs. Depletion of CD4(+) T cells also resulted in partial loss of tumor protection, suggesting a beneficial role for T-helper cells. Interestingly OVA-archaeosomes induced enhanced CTL response in the absence of interleukin 12 and IFN-gamma. Furthermore, interleukin 12-deficient mice mounted strong tumor protection. However, IFN-gamma-deficient mice, despite the strong CTL response, were only transiently protected, revealing a need for IFN-gamma response in tumor protection. Archaeosomes also facilitated therapeutic protection when injected into mice concurrent with tumor cells. Interestingly, even archaeosomes lacking entrapped antigen mediated therapeutic protection, and this correlated to the activation of innate immunity as evident by the increased tumor-infiltrating natural killer and dendritic cells. Thus, archaeosomes represent effective tumor antigen delivery vehicles that can mediate protection by activating both innate as well as acquired immunity.

Adjuvants, Immunologic↗

[Susceptibility of archaea to the antibiotic effect of the parasporal inclusion proteins from Bacillus thuringiensis subspecies].

The proteins of parasporal inclusions from three Bacillus thuringiensis subspecies (kurstaki, amagiensis, and monterrey) inhibited growth of methanogenic archaea of two species belonging to two genera, Methanobrevibacter arboriphilus and Methanosarcina barkeri. The minimal inhibitory concentrations of these proteins were 20 to 50 micrograms/ml. Lysozyme exhibited similar bactericidal effect on archaea. The perspective of comparative studies on the effect of polyfunctional proteins on bacteria and archaea is discussed.

Archaea↗

[Diversity and function of symbiotic microbes in the gut of lower termites].

The gut of lower termites harbor a complex microbial community, including eukaryotic flagellates and prokaryotic bacteria and archaea, which play important roles in the wood-cellulose digestion of these termites. The hindguts of lower termites are characterized with an enlarged paunch with steep oxygen and hydrogen gradient and not randomly distributed abundant microorganisms. The symbiotic flagellates in the gut of lower termites, which are normally associated with epibionts or endobionts, are phylogenetically affiliated with Trichomonadida, Hypermastigida and Oxymonadida. They endocytose wood particles and ferment the polysaccharide components to acetate, CO2 and H2, which are further metabolized by symbiotic prokaryotes as energy and nutrition sources in the termites. Cellulose genes such as glycosyl hydrolase family 45 have been identified in gut protists with molecular approach. Phylogenetic analysis have revealed that in the gut of lower termites, such as several Reticulitermites species, most of the dominant bacteria belong to phyla like "Termite group 1", Spirochaetes, Firmicutes, Bacteroidetes, and Proteobacteria. These bacteria clones normally form distinct termite clusters in phylogenetic trees, which show the existence of specific microbial diversity in the termite guts. Most of the symbiotic archaea in the gut of lower termites are methanogens and affiliated with the genus Methanobrevibacter by phylogenetic analysis and pure culture. The symbiotic bacteria and archaea may involve in the reduction of CO2 and the metabolism of N2 in these termite guts. However, the functions and metabolic mechanisms of the symbiotic flagellates and prokaryotes in the gut of lower termites are still remaining to be further elucidated.

Animals↗

[Oral bacterial flora and its pathogenic potential].

The oral flora is a complex body formed by at least a hundred bacterial species which can be more or less regular residents in the oral cavity. Thanks to a developing taxonomy related to progress made in bacterial identification, these oral species can be separated into five groups based Gram staining and morphology: the Gram positive cocci group (Streptococcus, Staphylococcus and related geni), the Gram negative cocci group (Neisseria, Veillonella and related geni), the Gram positive bacilli group (Corynebacterium, Actinomyces, Lactobacillus, Methanobrevibacter and related geni), the Gram negative bacilli group (Haemophilus, Campylobacter, Bacteroides and related geni) and finally the spirochete group (Treponema).

Bacteria↗

Cloning and characterization of a 12-gene cluster from Bacillus subtilis encoding nine enzymes for de novo purine nucleotide synthesis.

An approximately 16-kilobase pair region of the Bacillus subtilis chromosome at 55 degrees containing genes for de novo purine nucleotide synthesis (Piggot, P. J., and Hoch, J. A. (1985) Microbiol. Rev. 49, 158-179) was cloned. The nucleotide sequence of over 13 kilobase pairs indicates that this region contains a cluster of 12 genes, 11 of which encode enzymes that catalyze the 10 reactions for de novo purine nucleotide synthesis from 5-phosphoribosyl 1-pyrophosphate to IMP. The genes were identified by complementation of Escherichia coli pur mutants and by sequence comparisons with homologous enzymes. The cluster is likely an operon and is organized into three groups of overlapping genes followed by the last gene: purEKB-purC(orf)QLF-purMNH(J)-purD. Sequence comparisons provide evidence for homology of monofunctional purine nucleotide biosynthetic enzymes from B. subtilis with the corresponding multifunctional enzymes from yeast and Drosophila. Sequence alignment of the phosphoribosylaminoimidazole carboxylase heterodimer from B. subtilis with the monomeric enzyme from Methanobrevibacter smithii indicates an evolutionary relationship between these two enzymes. S1 nuclease analysis was used to map the mRNA 5' and 3' ends and to estimate levels of mRNA. These experiments indicate that synthesis of purine nucleotides is regulated independently by adenine and guanine nucleotides. Adenine nucleotides regulate transcription initiation. Guanine nucleotides regulate transcription by a termination-antitermination mechanism in a 242-nucleotide 5' untranslated mRNA leader region. Groups of overlapping genes, regulated at least in part by transcription termination-antitermination is likely to be a common theme for genetic organization and regulation of biosynthetic genes in this Gram-positive organism.

Amino Acid Sequence↗

7-methylpterin derivatives in extracts of methanogens characterized by a relatively low methanopterin content.

Cofactor extracts of five hydrogenotrophic methanogenic bacteria which contain relatively low amounts of methanopterin were screened for the presence of 7-methylpterin derivatives. Extracts of Methanospirillum hungatei, Methanobrevibacter smithii and Methanoplanus endosymbiosus were found to contain 7-methylpterin and methanopterin. These compounds were absent in extracts of Methanogenium thermophilicum and Methanogenium tatii. An unidentified methanopterin-like compound was detected in extracts of these two species and of Msp. hungatei, while a 7-methylpterin-like compound was found in extracts of Mp. endosymbiosus. A possible physiological role of the latter two compounds is discussed.

Biological Factors↗