Isolation of Methanobrevibacter smithii from human feces.

Fecal specimens from nine adults were examined for the presence of methanogenic bacteria. Enrichment cultures of five specimens produced methane in 5 days. Of these five specimens, three were tested and produced methane during a short-term incubation. Four specimens did not produce methane in either short-term incubation or in enrichment culture. Each methanogenic culture contained methanogens similar in morphology to organisms of the genus Methanobrevibacter and showed factor-420 fluorescence by fluorescence microscopy. Pure cultures were obtained from four of the five methanogenic enrichment cultures. Each isolate grew and formed methane from either H2-CO2 or formate, but growth obtained with formate was poor. None of the isolates used acetate, methanol, or trimethylamine. All isolates grew in the presence of bile salts. In immunological studies, each isolate was closely related to the type strain of Methanobrevibacter smithii, a finding consistent with the physiological and morphological similarities between the isolates and the type strain.     

Enumeration of Methanobrevibacter smithii in human feces

A platin 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 1–8×10¹¹/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 thay 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.     

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.     

Interactions between anaerobic cellulolytic bacteria and fungi in the presence of Methanobrevibacter smithii

A mixed inoculum of cellulolytic rumen bacteria depressed straw degradation by a mixed culture of cellulolytic fungi grown in the presence of Methanobrevibacter smithii. The inhibitory effect appeared to be caused by Ruminococcus albus strain JI and R. flavefaciens strain 007. Ruminococcus albus strain J1 also depressed straw degradation by the fungi, but R. albus strain SY3 and three strains of Bacteroides (Fibrobacter) succinogenes tested showed little or no inhibitory activity. It seems that some ruminococci show competitive or antagonistic activity towards certain rumen fungi.     

Structure of genes and an insertion element in the methane producing archaebacterium Methanobrevibacter smithii

Summary DNA fragments cloned from the methanogenic archaebacterium Methanobrevibacter smithii which complement mutations in the purE and proC genes of E. coli have been sequenced. Sequence analyses, transposon mutagenesis and expression in E. coli minicells indicate that purE and proC complementations result from the synthesis of M. smithii polypeptides with molecular weights of 36,697 and 27,836 respectively. The encoding genes appear to be located in operons. The M. smithii genome contains 69% A/T basepairs (bp) which is reflected in unusual codon usages and intergenic regions containing approximately 85% A/T bp. An insertion element, designated ISM1, was found within the cloned M. smithii DNA located adjacent to the proC complementing region. ISM1 is 1381 bp in length, has 29 bp terminal inverted repeat sequences and contains one major ORF encoded in 87% of the ISM1 sequence. ISM1 is mobile, present in approximately 10 copies per genome and integration duplicates 8 bp at the site of insertion. The duplicated sequences show homology with sequences within the 29bp terminal repeat sequence of ISM1. Comparison of our data with sequences from halophilic archaebacteria suggests that 5GAANTTTCA and 5TTTTAATATAAA may be consensus promoter sequences for archaebacteria. These sequences closely resemble the consensus sequences which precede Drosophila heatshock genes (Pelham 1982; Davidson et al. 1983). Methanogens appear to employ the eubacterial system of mRNA: 16SrRNA hybridization to ensure initiation of translation; the consensus ribosome binding sequence is 5AGGTGA.     

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.     

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.     

High Prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae Detected in the Human Gut Using an Improved DNA Detection Protocol

Background

The low and variable prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae DNA in human stool contrasts with the paramount role of these methanogenic Archaea in digestion processes. We hypothesized that this contrast is a consequence of the inefficiencies of current protocols for archaeon DNA extraction. We developed a new protocol for the extraction and PCR-based detection of M. smithii and M. stadtmanae DNA in human stool.

Methodology/Principal Findings

Stool specimens collected from 700 individuals were filtered, mechanically lysed twice, and incubated overnight with proteinase K prior to DNA extraction using a commercial DNA extraction kit. Total DNA was used as a template for quantitative real-time PCR targeting M. smithii and M. stadtmanae 16S rRNA and rpoB genes. Amplification of 16S rRNA and rpoB yielded positive detection of M. smithii in 95.7% and M. stadtmanae in 29.4% of specimens. Sequencing of 16S rRNA gene PCR products from 30 randomly selected specimens (15 for M. smithii and 15 for M. stadtmanae) yielded a sequence similarity of 99–100% using the reference M. smithii ATCC 35061 and M. stadtmanae DSM 3091 sequences.

Conclusions/Significance

In contrast to previous reports, these data indicate a high prevalence of the methanogens M. smithii and M. stadtmanae in the human gut, with the former being an almost ubiquitous inhabitant of the intestinal microbiome.     

Detection of the nifH gene of Methanobrevibacter smithii: a potential tool to identify sewage pollution in recreational waters

AIMS: The goal of this study was to develop and test the efficacy of a PCR assay for the environmental detection of the nifH gene of Methanobrevibacter smithii, a methanogen found in human faeces and sewage. METHODS AND RESULTS: PCR primers for the nifH gene of M. smithii were designed, tested and used to detect the presence or absence of this organism in faecal and environmental samples. Specificity analysis showed that the Mnif primers amplified products only in M. smithii pure culture strains (100%), human faeces (29%), human sewage samples (93%) and sewage-contaminated water samples (100%). No amplification was observed when primers were tested against 43 bacterial stock cultures, 204 animal faecal samples, 548 environmental bacterial isolates and water samples from a bovine waste lagoon and adjacent polluted creek. Sequencing of PCR products from sewers demonstrated that a 222-bp product was the nifH gene of M. smithii. The minimal amount of total DNA required for the detection of M. smithii was 10 ng for human faeces, 10 ng for faecally contaminated water and 5 ng for sewage. Recreational water seeded with M. smithii established a lower detection limit of 13 cells ml(-1). CONCLUSIONS: The Mnif assay developed during this investigation showed successful detection of M. smithii in individual human faecal samples, sewage and sewage-contaminated water but not in uncontaminated marine water or bovine-contaminated waters. The Mnif assay appears to be a potentially useful method to detect sewage-polluted coastal waters. SIGNIFICANCE AND IMPACT OF THE STUDY: This study was the first to utilize methanogens as an indicator of sewage pollution. Mnif PCR detection of M. smithii was shown to be a rapid, inexpensive and reliable test for determining the presence or absence of sewage pollution in coastal recreational waters.     

Stability of the microbial populations of the caecum of guinea-pigs and rabbits

The microbial population of the caecum of rabbits and guinea-pigs, respectively, normally exhibits distinctive features which can be tabulated precisely, and which persist when the animals are kept in the same cage and fed on the same diet. A pronounced resistance, therefore, opposes cross-infection and tends to secure the stability of the population whose characteristics afford useful criteria for the microbiological control of in vitro incubation experiments.     

Circadian analysis of large human populations: Inferences from the power grid

Few, if any studies have focused on the daily rhythmic nature of modern industrialized populations. The present study utilized real-time load data from the U.S. Pacific Northwest electrical power grid as a reflection of human operative household activity. This approach involved actigraphic analyses of continuously streaming internet data (provided in 5?min bins) from a human subject pool of approximately 43 million primarily residential users. Rhythm analyses reveal striking seasonal and intra-week differences in human activity patterns, largely devoid of manufacturing and automated load interference. Length of the diurnal activity period (alpha) is longer during the spring than the summer (16.64?h versus 15.98?h, respectively; p?<?0.01). As expected, significantly more activity occurs in the solar dark phase during the winter than during the summer (6.29?h versus 2.03?h, respectively; p?<?0.01). Interestingly, throughout the year a “weekend effect” is evident, where morning activity onset occurs approximately 1?h later than during the work week (5:54 am versus 6:52 am, respectively; p?<?0.01). This indicates a general phase-delaying response to the absence of job-related or other weekday morning arousal cues, substantiating a preference or need to sleep longer on weekends. Finally, a shift in onset time can be seen during the transition to Day Light Saving Time, but not the transition back to Standard Time. The use of grid power load as a means for human actimetry assessment thus offers new insights into the collective diurnal activity patterns of large human populations.     

A real-time qPCR assay for the detection of the nifH gene of Methanobrevibacter smithii, a potential indicator of sewage pollution

Aims: To degrade ether‐type polyurethane (ether‐PUR), ether‐PUR–degrading micro‐organism was isolated. Moreover, ether‐PUR–degrading mechanisms were analysed using model compounds of ether‐PUR. Methods and Results: A fungus designated as strain PURDK2, capable of changing the configuration of ether‐PUR, has been isolated. This isolated fungus was identified as Alternaria sp. Using a scanning electron microscope, the grid structure of ether‐PUR was shown to be melted and disrupted by the fungus. The degradation of ether‐PUR by the fungus was analysed, and the ether‐PUR was degraded by the fungus by about 27·5%. To analyse the urethane‐bond degradation by the fungus, a degraded product of ethylphenylcarbamate was analysed using GC/MS. Aniline and ethanol were detected by degradation with the supernatant, indicating that the fungus secreted urethane‐bond–degrading enzyme(s). PURDK2 also degraded urea bonds when diphenylmethane‐4,4′‐dibutylurea was used as a substrate. Conclusions: The enzyme(s) from PURDK2 degraded urethane and urea bonds to convert the high molecular weight structure of ether‐PUR to small molecules; and then the fungus seems to use the small molecules as an energy source. Significance and Impact of the Study: Ether‐PUR–degrading fungus, strain PURDK2, was isolated, and the urethane‐ and urea‐bonds–degrading enzymes from strain PURDK2 could contribute to the material recycling of ether‐PUR.     

Chromosomal banding patterns in human large bowel adenomas

Summary The human thyroglobulin gene was mapped by in situ hybridization whereby a ³H-labeled recombinant plasmid DNA containing a fragment of 2.3 kilobases of human thyroglobulin gene was hybridized to human chromosome preparations. A high proportion (25%) of hybridized metaphases exhibited silver grains at the distal portion of the long arm of chromosome 8. Analysis of the grain position at this site indicated that the chromosomal localization of the human thyroglobulin gene was 8q242-8q243.     

Undecompressed microbial populations from the deep sea.

Metabolic transformations of glutamate and Casamino Acids by natural microbial populations collected from deep waters (1,600 to 3,100 m) were studied in decompressed and undecompressed samples. Pressure-retaining sampling/incubation vessels and appropriate subsampling/incubation vessels and appropriate subsampling techniques permitted time course experiments. In all cases the metabolic activity in undecompressed samples was lower than it was when incubated at 1 atm. Surface water controls showed a reduced activity upon compression. The processes involving substrate incorporation into cell material were more pressure sensitive than was respiration. The low utilization of substrates, previously found by in situ incubations for up to 12 months, was confirmed and demonstrated to consist of an initial phase of activity, in the range of 5 to 60 times lower than the controls, followed by a stationary phase of virtually no substrate utilization. No barophilic growth response (higher rates at elevated pressure than at 1 atm) was recorded; all populations observed exhibition various degrees of barotolerance.     

Biofilms in the large bowel suggest an apparent function of the human vermiform appendix

The human vermiform ("worm-like") appendix is a 5-10cm long and 0.5-1cm wide pouch that extends from the cecum of the large bowel. The architecture of the human appendix is unique among mammals, and few mammals other than humans have an appendix at all. The function of the human appendix has long been a matter of debate, with the structure often considered to be a vestige of evolutionary development despite evidence to the contrary based on comparative primate anatomy. The appendix is thought to have some immune function based on its association with substantial lymphatic tissue, although the specific nature of that putative function is unknown. Based (a) on a recently acquired understanding of immune-mediated biofilm formation by commensal bacteria in the mammalian gut, (b) on biofilm distribution in the large bowel, (c) the association of lymphoid tissue with the appendix, (d) the potential for biofilms to protect and support colonization by commensal bacteria, and (e) on the architecture of the human bowel, we propose that the human appendix is well suited as a "safe house" for commensal bacteria, providing support for bacterial growth and potentially facilitating re-inoculation of the colon in the event that the contents of the intestinal tract are purged following exposure to a pathogen.     

First isolation of Desulfovibrio from the human vaginal flora

Four Desulfovibrio species, including 2 subtypes of 1 species, namely, Desulfovibrio piger, Desulfovibrio desulfuricans MB subtype and Essex 6 subtype, Desulfovibrio fairfieldensis, and Desulfovibrio vulgaris, have been isolated from the human oral and intestinal flora, but not previously from the vaginal flora. They are opportunistic pathogens and have been considered as possible environmental and etiologic agents involved in ulcerative colitis and chronic periodontitis. We isolated Desulfovibrio intestinalis from vaginal specimens of four Japanese women; a species which has not been previously isolated from humans. The vaginal isolates were highly resistant to cefoxitin, piperacillin, and piperacillin-tazobactam but were susceptible to the other antimicrobial agents tested. Our findings suggested that vaginal Desulfovibrio species may be involved in gynecological or obstetric pathology, and provides additional information of the medical relevance on human Desulfovibrio species.     

黄牛泌乳系统正常菌群的检测

采用稀释滴种法对30头健康黄牛泌乳系统的10种主要正常菌群分四个部位(乳头、乳池、输乳管和腺泡)进行定性、定量检测。结果有6种菌群被检测出,按数量由多至少依次为棒状杆菌、乳杆菌、葡萄球菌、芽胞杆菌、双歧杆菌和肠杆菌。乳头的优势菌群为棒状杆菌、芽胞杆菌和葡萄球菌;乳池、输乳管和腺泡的优势菌群均为棒状杆菌和乳杆菌。黄牛泌乳系统各正常菌群的数量均较少,远低于其他孔道系统。