Methanobrevibacter Smithii Research Articles

Review article: inhibition of methanogenic archaea by statins as a targeted management strategy for constipation and related disorders.

SummaryBackgroundObservational studies show a strong association between delayed intestinal transit and the production of methane. Experimental data suggest a direct inhibitory activity of methane on the colonic and ileal smooth muscle and a possible role for methane as a gasotransmitter. Archaea are the only confirmed biological sources of methane in nature and Methanobrevibacter smithii is the predominant methanogen in the human intestine.AimTo review the biosynthesis and composition of archaeal cell membranes, archaeal methanogenesis and the mechanism of action of statins in this context.MethodsNarrative review of the literature.ResultsStatins can inhibit archaeal cell membrane biosynthesis without affecting bacterial numbers as demonstrated in livestock and humans. This opens the possibility of a therapeutic intervention that targets a specific aetiological factor of constipation while protecting the intestinal microbiome. While it is generally believed that statins inhibit methane production via their effect on cell membrane biosynthesis, mediated by inhibition of the HMG‐CoA reductase, there is accumulating evidence for an alternative or additional mechanism of action where statins inhibit methanogenesis directly. It appears that this other mechanism may predominate when the lactone form of statins, particularly lovastatin lactone, is administered.ConclusionsClinical development appears promising. A phase 2 clinical trial is currently in progress that evaluates the effect of lovastatin lactone on methanogenesis and symptoms in patients with irritable bowel syndrome with constipation. The review concludes with an outlook for the future and subsequent work that needs to be done.

Correlation between body mass index and faecal microbiota from children

Childhood obesity is an increasing problem at the global level and considered as a risk factor for obesity development and the associated co-morbidities in adult life. In this study, the occurrence of Bacteroides fragilis group, Clostridium spp., Bifidobacterium spp. and Escherichia coli in 84 faecal samples from 30 obese, 24 overweight and 30 lean children was verified by culture technique and quantitative determination by quantitative PCR. In addition, Lactobacillus spp. and Methanobrevibacter smithii were also analysed. A correlation between the body mass index (BMI) and these bacteria was sought. Bacteroides vulgatus, Clostridium perfringens and Bifidobacterium adolescentis were most prevalent in all samples evaluated by culture-method. The B. fragilis group were found at high concentrations in obese and overweight children when compared with the lean ones (p 0.015). The obese and overweight children harboured higher numbers of Lactobacillus spp. than lean children (p 0.022). The faecal concentrations of the B. fragilis group (r = 0.24; p 0.026) and Lactobacillus spp. (r = 0.44; p 0.002) were positively correlated with BMI. Bifidobacterium spp. were found in higher numbers in the lean group than the overweight and obese ones (p 0.042). Furthermore, a negative correlation between BMI and Bifidobacterium spp. copy number (r = −0.22; p 0.039) was observed. Our findings show some difference in the intestinal microbial ecosystem of obese children compared with the lean ones and a significant association between number of Lactobacillus spp. and B. fragilis group and BMI.

Gut colonization with methanobrevibacter smithii is associated with childhood weight development

To prospectively investigate the presence and counts of archaea in feces of 472 children in association with weight development from 6 to 10years of age. Within the KOALA Birth Cohort Study, a single fecal sample from each child was analyzed by quantitative polymerase chain reaction to quantify archaea (Methanobrevibacter smithii, Methanosphera stadtmanae). Anthropometric outcomes (overweight [body mass index {BMI}≥85th percentile], age- and sex-standardized BMI, weight, and height z-scores) were repeatedly measured at ages (mean±SD) of 6.2±0.5, 6.8±0.5, 7.8±0.5, and 8.8±0.5years. Generalized estimating equation was used for statistical analysis while controlling for confounders. Methanobrevibacter smithii colonization was associated with an increased risk of overweight (adjusted odds ratio [OR]=2.69; 95% confidence interval [CI] 0.96-7.54) from 6 to 10years of age. Children with high levels (>7 log10 copies/g feces) of this archaeon were at highest risk for overweight (OR=3.27; 95% CI 1.09-9.83). Moreover, M. smithii colonization was associated with higher weight z-scores (adj. β 0.18; 95% CI 0.00-0.36), but not with height. For BMI z-scores, the interaction (P=0.008) between M. smithii and age was statistically significant, implying children colonized with M. smithii had increasing BMI z-scores with age. Presence and higher counts of M. smithii in the gut of children are associated with higher weight z-scores, higher BMI z-scores, and overweight.

High-throughput DNA sequencing of the moose rumen from different geographical locations reveals a core ruminal methanogenic archaeal diversity and a differential ciliate protozoal diversity.

Moose rumen samples from Vermont, Alaska and Norway were investigated for methanogenic archaeal and protozoal density using real-time PCR, and diversity using high-throughput sequencing of the 16S and 18S rRNA genes. Vermont moose showed the highest protozoal and methanogen densities. Alaskan samples had the highest percentages of Methanobrevibacter smithii, followed by the Norwegian samples. One Norwegian sample contained 43 % Methanobrevibacter thaueri, whilst all other samples contained < 10 %. Vermont samples had large percentages of Methanobrevibacter ruminantium, as did two Norwegian samples. Methanosphaera stadtmanae represented one-third of sequences in three samples. Samples were heterogeneous based on gender, geographical location and weight class using analysis of molecular variance (AMOVA). Two Alaskan moose contained >70 % Polyplastron multivesiculatum and one contained >75 % Entodinium spp. Protozoa from Norwegian moose belonged predominantly (>50 %) to the genus Entodinium, especially Entodinium caudatum. Norwegian moose contained a large proportion of sequences (25–97 %) which could not be classified beyond family. Protozoa from Vermont samples were predominantly Eudiplodinium rostratum (>75 %), with up to 7 % Diploplastron affine. Four of the eight Vermont samples also contained 5–12 % Entodinium spp. Samples were heterogeneous based on AMOVA, principal coordinate analysis and UniFrac. This study gives the first insight into the methanogenic archaeal diversity in the moose rumen. The high percentage of rumen archaeal species associated with high starch diets found in Alaskan moose corresponds well to previous data suggesting that they feed on plants high in starch. Similarly, the higher percentage of species related to forage diets in Vermont moose also relates well to their higher intake of fibre.

Lovastatin Lactone Inhibits Methane Production in Human Stool Homogenates

Introduction: Research has linked an altered gut microbiome to symptoms of irritable bowel syndrome (IBS). In constipation-predominant IBS (IBS-C), methane production appears important as methane levels on breath are proportional to the degree of constipation, and antibiotic elimination of methane significantly improves constipation. The predominant methane-producing organism in humans is Methanobrevibacter smithii. Recent data suggests certain HMG-CoA reductase inhibitors (statins) may reduce methane production by inhibiting methanogenesis. This study examined the effects of various statins on methane production in fresh human stool homogenates. Methods: Five female subjects recruited based on high breath methane production (average=69.6ppm) provided a total of 8 fresh stool samples each. Samples were homogenized in 1XPBS (2g stool/3ml PBS) in an anaerobic chamber at 37°C and divided into 5 stoppered flasks. To assess statin effects, head gas withdrawn through a stopcock was collected at baseline, then every 30min for a total of 270min, and analyzed on a Quintron Model SC gas chromatograph for methane levels. Nine statins were initially assessed for methane inhibition at a concentration of 5mg/g stool: lovastatin lactone and hydroxyacid, pravastatin lactone and hydroxyacid, simvastatin lactone, mevastatin lactone, rosuvastatin lactone, and atorvastatin lactone and hydroxyacid. Once the ideal statin was determined, the effects of concentrations of 0.04, 0.12, 0.48, 1, 5, and 10mg/g stool were tested by sampling head gas every 90min after statin addition, up to 720min.Further assessments were then performed comparing three forms of lovastatin (lactone, diol and hydroxyacid). Results: Lovastatin lactone was identified as the only effective methane inhibitor (Figure 1), significantly inhibiting methane levels by -65% of the control stool. Lovastatin lactone at 5mg/g produced the maximum inhibiting effect resulting in an average methane level of 3% of the control over time (Figure 2). In a final validation comparison of 3 forms of lovastatin (5mg/g), both the lactone and diol forms proved to be effective. In all assessments, hydroxyacid forms were least able to inhibit methane production in fresh stool samples.Figure 1Figure 2Conclusion: Lovastatin lactone and lovastatin diol at concentrations of 5mg/g stool significantly reduce methane production in human stool homogenates. Future studies are needed to assess the effect of this statin on methane production and IBS-C symptoms in humans.

Amino Acid and Peptide Utilization Profiles of the Fluoroacetate-Degrading Bacterium Synergistetes Strain MFA1 Under Varying Conditions.

Synergistetes strain MFA1 is an asaccharolytic ruminal bacterium isolated based on its ability to degrade fluoroacetate, a plant toxin. The amino acid and peptide requirements of the bacterium were investigated under different culturing conditions. The growth of strain MFA1 and its fluoroacetate degradation rate were enhanced by peptide-rich protein hydrolysates (tryptone and yeast extract) compared to casamino acid, an amino acid-rich protein hydrolysate. Complete utilization and preference for arginine, asparagine, glutamate, glycine, and histidine as free amino acids from yeast extract were observed, while the utilization of serine, threonine, and lysine in free form and peptide-bound glutamate was stimulated during growth on fluoroacetate. A predominant peptide in yeast extract preferentially utilized by strain MFA1 was partially characterized by high-liquid performance chromatography-mass spectrometry as a hepta-glutamate oligopeptide. Similar utilization profiles of amino acids were observed between the co-culture of strain MFA1 with Methanobrevibacter smithii without fluoroacetate and pure strain MFA1 culture with fluoroacetate. This suggests that growth of strain MFA1 could be enhanced by a reduction of hydrogen partial pressure as a result of hydrogen removal by a methanogen or reduction of fluoroacetate.

Methanogen communities in stools of humans of different age and health status and co-occurrence with bacteria.

Hydrogenotrophic methanogens live in a synthrophic relationship with the human gut microbiota as the terminal part of the anaerobic food chain. Methanobrevibacter smithii of the Methanobacteriales is the prevailing archaeal species. Recently, methylotrophic archaea of the novel order Methanomassiliicoccales were isolated from human stools. Few data exist on the prevalence, abundance, persistence and ecology of these methanogens in humans. This study investigated methanogen communities in 26 healthy and obese children (8-14 years) and 18 adults (28-78 years) using quantitative PCR. Samples were obtained from nine females before and after giving birth. Bacterial groups linked to the abundance of methanogens in adult females were identified using a 16S rRNA gene amplicon data set. A total of 89% and 65% of adults and children, respectively, carried Methanobacteriales. Methanomassiliicoccales were recovered from 50% of the adults and one child. Mean relative abundance of Methanomassiliicoccales in adults was lower than that of Methanobacteriales (0.10% versus 0.52%). Both Methanobacteriales and Methanomassiliicoccales formed stable communities in females before and after giving birth. Methanobacteriales co-occurred with bacterial taxonomic groups associated with the trophic chain from carbohydrate degradation to hydrogen and formate formation. Relative abundance was inversely correlated to Blautia. Negative correlation with little characterized groups within the Clostridiales indicated possible interactions of Methanomassiliicoccales with the bacterial community.

The camel faecal metagenome under different systems of management: Phylogenetic and gene-centric approach

The study investigated the composition of microorganisms in the camel (Camelus dromedarius) faecal samples, maintained under two groups of intensive (group 1) and extensive (group 2) systems of management at Jaisalmer district of Rajasthan, India by using culture-independent approach. The study included the comparison of camel faecal microbiota to other currently available gut/faecal metagenomes, in order to reveal potential differences in these gut environments. After shotgun sequencing of the camel fecal microbiome, we used the Metagenomics-Rapid Annotation Server Tool (MG-RAST) to identify the microbial diversity and metabolic potential of the camel gut. Of the sequenced data that passed quality control, 1.3% and 2.2% sequences contained ribosomal RNA genes in groups 1 and 2, respectively. The annotated protein features which were assigned to functional categories were 63.6% and 69.5%, in groups 1 and 2, respectively. The domain level breakdown of our samples revealed bacteria was the major domain in both systems of management. At the phylum level, Firmicutes (61.40% and 29.17%), Proteobacteria (11.10% and 47.85%), Bacteroidetes (8.04% and 3.83%) and Verrucomicrobia (7.48% and 10.35%) were predominant in the fecal microbial community of groups 1 and 2, respectively. The fecal metagenomes revealed Euryarchaeota phylum with Methanobrevibacter smithii as the major archaeal species in both these groups. Functional analysis using subsystems of MG-RAST revealed that sequences for protein metabolism were abundant in group 1 and those for metabolism of carbohydrates predominated in group 2. Glycoside hydrolases of carbohydrates functional category were seen in group 2. High taxonomic similarity of group 1 with cow rumen metagenomes, cattle faecal pool, canine gastro-intestine as well as group 2 camels with the termite gut was observed. Functional similarities of camel faecal metagenomes of groups 1 and 2 with cattle faecal pool and camel foregut were noticed. Altogether, these data suggest that agricultural and animal husbandry practices can impose significant selective pressures on the gut microbiota. The present study provided a baseline for understanding the complexity of camel gut microbial ecology while also highlighting striking similarities and differences when compared to other animal gastrointestinal environments and in future could help in developing strategies for improving the existing management conditions.

Effects of hydrogen in headspace and bicarbonate in media on rumen fermentation, methane production and methanogenic population using in vitro gas production techniques

An in vitro batch culture study was conducted to investigate (1) 13C-labeled methane (CH4) production efficiency (PE) which was produced from the reduction of 13C-labeled sodium bicarbonate (NaH13CO3) in media; and (2) the effects of NaHCO3 and hydrogen (H2) additions on ruminal fermentation, CH4 production and methanogenic populations in a 4×3 factorial design with lucerne hays as substrates. Factors were NaH13CO3 concentration in media (0, 11.9, 23.8 or 47.6μmol/ml) and H2 volume in headspace (0, 36 or 60ml). The 13CH4 PE produced from NaH13CO3 was linearly increased (P<0.01) with H2 addition, but quadratically decreased (P<0.01) in response to NaH13CO3 addition. Moreover, addition of H2 quadratically increased (P<0.01) total CH4 production, apparent dry matter degradability (ADMD), total volatile fatty acids (VFA), molar proportion of acetate and populations of Methanosarcina barkeri (M. barkeri), Methanosphaera stadtmanae (M. stadtmanae), Methanobrevibacter ruminantium M1 (M. ruminantium) and Methanobrevibacter smithii (M. smithii). Quadratic effects (P<0.041) were observed for total CH4 production, populations of M. barkeri, M. stadtmanae, M. ruminantium and M. smithii in response to NaHCO3 addition. Additionally, the addition of NaHCO3 linearly increased (P<0.01) the total VFA. The results of methanogenic populations during incubation showed that the copy number of 16S rRNA gene for M. barkeri was increased (P=0.023) by incubation time with the highest value being at 24h, but no differences were observed for the M. stadtamanae, M. ruminantium and M. smithii gene copy numbers among the incubation time points. These results demonstrate that both CO2 and H2 concentrations affect methanogenesis in the rumen. Furthermore, the present study suggests that headspace hydrogen and media bicarbonate play roles in regulating feed fermentation, CH4 production and methanogenic populations in vitro.

Archaeal microbiota population in piglet feces shifts in response to weaning: Methanobrevibacter smithii is replaced with Methanobrevibacter boviskoreani.

Methanogens commonly inhabit swine intestine. We analyzed the gut archaeal population by extracting DNA from the feces of nine piglets. We performed PCR to target the V6-V8 region of the 16S rRNA gene. Subsequent denaturing gradient gel electrophoresis (DGGE) revealed the presence of Methanobrevibacter boviskoreani, which has not previously been identified in pigs. We confirmed these data with a PCR-DGGE analysis of the mcrA gene, and subsequent sequencing. At 63 days old, the only band in fecal samples corresponded to M. boviskoreani. The DGGE analysis also showed that Methanobrevibacter smithii, which was abundant at 28 days, was dramatically reduced at 42 days, and it completely disappeared at 63 days. To confirm these data, we quantified M. smithii and the total archaeal population by quantitative PCR (qPCR); moreover, we designed a new set of species-specific primers based on the 16S rRNA gene of M. boviskoreani. The qPCR results confirmed the reduction in M. smithii over time and a simultaneous increase in M. boviskoreani. At 63 days, the total numbers of archaea and M. boviskoreani genomes were comparable, which suggested that M. boviskoreani represented the dominant archaea. This work showed that the archaeal population shifted during weaning, and M. boviskoreani replaced M. smithii.

The repertoire of archaea cultivated from severe periodontitis.

In previous studies, the abundance and diversity of methanogenic archaea in the dental microbiota have been analysed by the detection of specific DNA sequences by PCR-based investigations and metagenomic studies. Few data issued regarding methanogens actually living in dental plaque. We collected dental plaque specimens in 15 control individuals and 65 periodontitis patients. Dental plaque specimens were cultured in an anoxic liquid medium for methanogens in the presence of negative control tubes. Dental plaque methanogens were cultured from 1/15 (6.67%) control and 36/65 (55.38%) periodontitis patient samples (p<0.001). The cultures yielded Methanobrevibacter oralis in one control and thirty-one patients, Methanobrevibacter smithii in two patients and a potential new species named Methanobrevibacter sp. strain N13 in three patients with severe periodontitis. Our observations of living methanogens, strengthen previous observations made on DNA-based studies regarding the role of methanogens, in periodontitis.

Distribution of glycerol ether lipids in halophilic, methanogenic and hyperthermophilic archaea

Four representatives of methanogenic Euryarchaeota (Methanosarcina mazei strain Gö1, Methanosphaera stadtmanae, Methanobrevibacter smithii and Methanosaeta thermophila), the hyperthermophilic euryarchaeon Thermococcus kodakarensis and the halophilic euryarchaeon Haloferax volcanii were studied for their glycerol ether lipid composition. The predominant core membrane lipid in all of them was archaeol, which was accompanied by variable quantities of sn-2-hydroxyarchaeol in the methanogens M. mazei (Methanosarcinales) and M. stadtmanae (Methanobacteriales). All methanogenic and hyperthermophilic Euryarchaeota also contained comparatively high abundances of the glycerol dialkyl glycerol tetraether without a cyclopentane moiety (GDGT-0). The methanoarchaeon M. stadtmanae, in addition to GDGT-0, contained GDGT core lipid structures with 1–4 cyclopentane moieties (GDGTs 1–4). We also found minor amounts of a glycerol trialkyl glycerol tetraether (GTGT) and a glycerol dialkanol diether (GDD), both of which did not contain cyclopentane moieties, as well as methylated and dimethylated GDGT-0 in all the archaea with the exception of H. volcanii. Like its GDGT distribution, M. stadtmanae showed an extended range of GDD structures with up to two cyclopentane ring systems. Our results thus indicate that both methanogenic and hyperthermophilic Euryarchaeota may represent source organisms of GTGT-0, GDDs and methylated-GDGTs in natural environments. All the latter components have recently been reported to be ubiquitously distributed in marine sediments but their biological origin is largely unknown. Moreover, a suite of unsaturated GDGTs without a cyclopentane moiety and up to four double bonds in the hyperthermophile T. kodakarensis was tentatively assigned.

Diversity of human-associated Methanobrevibacter smithii isolates revealed by multispacer sequence typing.

Methanobrevibacter smithii is the main archaea in human, detoxifying molecular hydrogen resulting from anaerobic bacteria fermentations into gaseous methane. Its identification relies on gene sequencing, but no method is available to discriminate among genetic variants of M. smithii. Here, we developed a multispacer sequence typing (MST) for genotyping the genetic variants of M. smithii. Four intergenic spacers recovered from the M. smithii reference genome were PCR amplified and sequenced in three M. smithii reference strains and in a collection of 22 M. smithii isolates from the oral cavity in two individuals and the gut of 10 additional individuals. Sequencing yielded 216 genetic polymorphisms including 89 single nucleotide polymorphisms (41.2 %), 83 insertions (38.4 %), and 44 deletions (20.4 %). Combining these genetic polymorphisms yielded 15 genotypes with an index of discrimination of 0.942 (confidence interval 0.9-0.984; P < 0.05). Five M. smithii isolates made from the oral cavity yielded five different genotypes; seven gut isolates yielded nine different genotypes; genotypes MST5 and MST6 were found both in the oral cavity and the gut. Multiple genotypes were identified in some individuals at the same anatomical site. MST is a sequencing-based method which discriminates several genetic variants within M. smithii. Individuals may harbor several contemporary genetic variants of M. smithii in the oral cavity and gut. MST will allow studying population dynamics of M. smithii and tracing its circulation between individuals and their environment.

Archaea prevalence in inflamed pulp tissues.

Archaea have been detected in several ecological niches of the human body such as the large intestine, skin, vagina as well as the oral cavity. At present, archaea are recognized as nonpathogenic microorganisms. However, some data indicate that they may be involved in the etiopathogenesis of several diseases, including intestinal diseases as well as oral diseases: periodontitis, peri-implantitis and endodontitis. In this study, on the basis of 16S rRNA gene sequence analysis, we examined whether archaea might be present in inflamed pulp tissues and contribute to the development of endodontic infection. In comparison, we also determined selected bacterial species associated with endodontitis. We detected archaea in 85% of infected endodontic samples. In addition, Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola were present in inflamed pulp tissue samples and Treponema denticola occurred with the highest frequency (70%). Further analysis revealed the presence of methanogenic archaea in analyzed samples. Direct sequencing of archaeal 16S rRNA gene PCR products indicated the occurrence of methanogenic archaea in inflamed pulp tissues; phylogenetically most similar were Methanobrevibacter oralis and Methanobrevibacter smithii. Therefore, our results show that methanogenic archaea are present in inflamed pulp tissues and may participate in the development of endodontic infection.

Investigation of a new acetogen isolated from an enrichment of the tammar wallaby forestomach.

BackgroundForestomach fermentation in Australian marsupials such as wallabies and kangaroos, though analogous to rumen fermentation, results in lower methane emissions. Insights into hydrogenotrophy in these systems could help in devising strategies to reduce ruminal methanogenesis. Reductive acetogenesis may be a significant hydrogen sink in these systems and previous molecular analyses have revealed a novel diversity of putative acetogens in the tammar wallaby forestomach.ResultsMethanogen-inhibited enrichment cultures prepared from tammar wallaby forestomach contents consumed hydrogen and produced primarily acetate. Functional gene (formyltetrahydrofolate synthetase and acetyl-CoA synthase) analyses revealed a restricted diversity of Clostridiales species as the putative acetogens in the cultures. A new acetogen (growth on H2/CO2 with acetate as primary end product) designated isolate TWA4, was obtained from the cultures. Isolate TWA4 classified within the Lachnospiraceae and demonstrated >97% rrs identity to previously isolated kangaroo acetogens. Isolate TWA4 was a potent hydrogenotroph and demonstrated excellent mixotrophic growth (concomitant consumption of hydrogen during heterotrophic growth) with glycerol. Mixotrophic growth of isolate TWA4 on glycerol resulted in increased cell densities and acetate production compared to autotrophic growth. Co-cultures with an autotrophic methanogen Methanobrevibacter smithii revealed that isolate TWA4 performed reductive acetogenesis under high hydrogen concentration (>5 mM), but not at low concentrations. Under heterotrophic growth conditions, isolate TWA4 did not significantly stimulate methanogenesis in a co-culture with M. smithii contrary to the expectation for organisms growing fermentatively.ConclusionsThe unique properties of tammar wallaby acetogens might be contributing factors to reduced methanogen numbers and methane emissions from tammar wallaby forestomach fermentation, compared to ruminal fermentation. The macropod forestomach may be a useful source of acetogens for future strategies to reduce methane emissions from ruminants, particularly if these strategies also include some level of methane suppression and/or acetogen stimulation, for example by harnessing mixotrophic growth capabilitiesElectronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0314-3) contains supplementary material, which is available to authorized users.

Alternative fecal indicators and their empirical relationships with enteric viruses, Salmonella enterica, and Pseudomonas aeruginosa in surface waters of a tropical urban catchment.

The suitability of traditional microbial indicators (i.e., Escherichia coli and enterococci) has been challenged due to the lack of correlation with pathogens and evidence of possible regrowth in the natural environment. In this study, the relationships between alternative microbial indicators of potential human fecal contamination (Bacteroides thetaiotaomicron, Methanobrevibacter smithii, human polyomaviruses [HPyVs], and F+ and somatic coliphages) and pathogens (Salmonella spp., Pseudomonas aeruginosa, rotavirus, astrovirus, norovirus GI, norovirus GII, and adenovirus) were compared with those of traditional microbial indicators, as well as environmental parameters (temperature, conductivity, salinity, pH, dissolved oxygen, total organic carbon, total suspended solids, turbidity, total nitrogen, and total phosphorus). Water samples were collected from surface waters of urban catchments in Singapore. Salmonella and P. aeruginosa had significant positive correlations with most of the microbial indicators, especially E. coli and enterococci. Norovirus GII showed moderately strong positive correlations with most of the microbial indicators, except for HPyVs and coliphages. In general, high geometric means and significant correlations between human-specific markers and pathogens suggest the possibility of sewage contamination in some areas. The simultaneous detection of human-specific markers (i.e., B. thetaiotaomicron, M. smithii, and HPyVs) with E. coli and enterococcus supports the likelihood of recent fecal contamination, since the human-specific markers are unable to regrow in natural surface waters. Multiple-linear-regression results further confirm that the inclusion of M. smithii and HPyVs, together with traditional indicators, would better predict the occurrence of pathogens. Further study is needed to determine the applicability of such models to different geographical locations and environmental conditions.

Temporal analysis of the effect of extruded flaxseed on the swine gut microbiota.

Flaxseed is a rich source of α-linolenic acid, an essential ω-3 fatty acid reported to have beneficial health effects in humans. Feeding swine a diet supplemented with flaxseed has been found to enrich pork products with ω-3 fatty acids. However, the effect of flaxseed supplementation on the swine gut microbiota has not been assessed to date. The purpose of this study was to investigate if extruded flaxseed has any impact on the bacterial and archaeal microbiota in the feces of growing-finishing pigs over a 51-day period, using denaturing gradient gel electrophoresis (DGGE) and real-time PCR. Bacterial DGGE profile analysis revealed major temporal shifts in the bacterial microbiota with only minor ones related to diet. The archaeal microbiota was significantly less diverse than that of Bacteria. The majority of bacterial DGGE bands sequenced belonged to the Firmicutes phylum while the archaeal DGGE bands were found to consist of only 2 species, Methanobrevibacter smithii and Methanosphaera stadtmanae. The abundance of Bacteroidetes decreased significantly from day 0 to day 21 in all diet groups while the abundance of Firmicutes was relatively stable across all diet cohorts and sampling times. There was also no significant correlation between pig mass and the ratio of Firmicutes to Bacteroidetes. While the addition of extruded flaxseed to the feed of growing-finishing pigs was beneficial for improving ω-3 fatty acid content of pork, it had no detectable impact on the fecal bacterial and archaeal microbiota, suggesting that extruded flaxseed may be used to improve meat quality without adverse effect on the swine gut microbiota or animal performance.

Biofilm formation of mucosa-associated methanoarchaeal strains.

Although in nature most microorganisms are known to occur predominantly in consortia or biofilms, data on archaeal biofilm formation are in general scarce. Here, the ability of three methanoarchaeal strains, Methanobrevibacter smithii and Methanosphaera stadtmanae, which form part of the human gut microbiota, and the Methanosarcina mazei strain Gö1 to grow on different surfaces and form biofilms was investigated. All three strains adhered to the substrate mica and grew predominantly as bilayers on its surface as demonstrated by confocal laser scanning microscopy analyses, though the formation of multi-layered biofilms of Methanosphaera stadtmanae and Methanobrevibacter smithii was observed as well. Stable biofilm formation was further confirmed by scanning electron microscopy analysis. Methanosarcina mazei and Methanobrevibacter smithii also formed multi-layered biofilms in uncoated plastic μ-dishesTM, which were very similar in morphology and reached a height of up to 40 μm. In contrast, biofilms formed by Methanosphaera stadtmanae reached only a height of 2 μm. Staining with the two lectins ConA and IB4 indicated that all three strains produced relatively low amounts of extracellular polysaccharides most likely containing glucose, mannose, and galactose. Taken together, this study provides the first evidence that methanoarchaea can develop and form biofilms on different substrates and thus, will contribute to our knowledge on the appearance and physiological role of Methanobrevibacter smithii and Methanosphaera stadtmanae in the human intestine.

Development and Integration of Quantitative Real-Time PCR Methods for Detection of Mitochondrial DNA and Methanobrevibacter smithii nifH Gene as Novel Microbial Source Tracking Tools

A novel microbial source tracking (MST) method based on the detection of human and non-human markers was developed and applied to track the origin of fecal pollution in water systems. Mitochondrial DNA sequences were used to develop new quantitative real-time polymerase chain reaction (qPCR) assays for dog, poultry, and gull. The targets were included as part of a toolbox including human, cow, pig, and sheep assays. A primer and probe set for the detection of the human-specific nifH gene of Methanobrevibacter smithii was also designed as an indicator of human fecal contamination. The assays were tested for specificity and applied to fecal-spiked surface waters and environmental samples collected from two river catchments impacted by sources of human and non-human fecal contamination. The MST methods described were applicable to both spiked waters and environmental samples, and using the two approaches the origin of fecal pollution could be successfully determined in mixed source fecally polluted waters.

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

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