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Keystone species are thought to play a critical role in determining the structure and function of microbial communities. As they are important candidates for microbiome-targeted interventions, the identification and characterization of keystones is a pressing research goal. Both empirical as well as computational approaches to identify keystones have been proposed, and in particular correlation network analysis is frequently utilized to interrogate sequencing-based microbiome data. Here, we apply an established method for identifying putative keystone taxa in correlation networks. We develop a robust workflow for network construction and systematically evaluate the effects of taxonomic resolution on network properties and the identification of keystone taxa. We are able to identify correlation network keystone species and genera, but could not detect taxa with high keystone potential at lower taxonomic resolution. Based on the correlation patterns observed, we hypothesize that the identified putative keystone taxa have a stabilizing effect that is exerted on correlated taxa. Correlation network analysis further revealed subcommunities present in the dataset that are remarkably similar to previously described patterns. The interrogation of available metatranscriptomes also revealed distinct transcriptional states present in all putative keystone taxa. These results suggest that keystone taxa may have stabilizing properties in a subset of community members rather than global effects. The work presented here contributes to the understanding of correlation network keystone taxa and sheds light on their potential ecological significance.

Methanobrevibacter smithii (M. smithii), initially isolated from human feces, has been recognised as a distinct taxon within the Archaea domain following comprehensive phenotypic, genetic, and genomic analyses confirming its uniqueness among methanogens. Its diversity, encompassing 15 genotypes, mirrors that of biotic and host-associated ecosystems in which M. smithii plays a crucial role in detoxifying hydrogen from bacterial fermentations, converting it into mechanically expelled gaseous methane. In microbiota in contact with host epithelial mucosae, M. smithii centres metabolism-driven microbial networks with Bacteroides, Prevotella, Ruminococcus, Veillonella, Enterococcus, Escherichia, Enterobacter, Klebsiella, whereas symbiotic association with the nanoarchaea Candidatus Nanopusillus phoceensis determines small and large cell variants of M. smithii. The former translocate with bacteria to induce detectable inflammatory and serological responses and are co-cultured from blood, urine, and tissular abscesses with bacteria, prototyping M. smithii as a model organism for pathogenicity by association. The sources, mechanisms and dynamics of in utero and lifespan M. smithii acquisition, its diversity, and its susceptibility to molecules of environmental, veterinary, and medical interest still have to be deeply investigated, as only four strains of M. smithii are available in microbial collections, despite the pivotal role this neglected microorganism plays in microbiota physiology and pathologies.

Archaea are an understudied component of the human microbiome. In this study, the gut archaeome and bacteriome of 60 healthy adults from different region were analyzed by whole-genome shotgun sequencing. Archaea were ubiquitously found in a wide range of abundances, reaching up to 7.2 %. The dominant archaeal phylum was Methanobacteriota, specifically the family Methanobacteriaceae, encompassing more than 50 % of Archaea in 50 samples. The previously underestimated Thermoplasmatota, mostly composed of Methanomassiliicoccaceae, dominated in 10 subjects (>50 %) and was present in all others except one. Halobacteriota, the sole other archaeal phylum, occurred in negligible concentration, except for two samples (4.6–4.8 %). This finding confirmed that the human gut archaeome is primarily composed of methanogenic organisms and among the known methanogenic pathway: i) hydrogenotrophic reduction of CO2 is the predominant, being the genus Methanobrevibacter and the species Methanobrevibacter smithii the most abundant in the majority of the samples; ii) the second pathway, that involved Methanomassiliicoccales, was the hydrogenotrophic reduction of methyl-compounds; iii) dismutation of acetate or methyl-compounds seemed to be absent. Co-occurrence analysis allowed to unravel correlations between Archaea and Bacteria that shapes the overall structure of the microbial community, allowing to depict a clearer picture of the human gut archaeome.

Anorexia nervosa (AN) remains a challenging condition in psychiatric management and its pathogenesis is not yet fully understood. An imbalance in the gut microbiota composition may contribute to its pathophysiology. This review aims to explore the link between the human gut microbiota and AN (objective 1) or refeeding syndrome in AN (objective 2). The online databases MEDLINE and PsycINFO were searched for relevant studies. A total of 14 studies met the inclusion and exclusion criteria and only answered objective 1. A total of 476 AN patients, 554 healthy-weight (HC) controls, and 0 patients with other psychiatric disorders were included. Compared to HC, there were consistently reduced abundances of Faecalibacterium prausnitzii and Roseburia inulinivorans, and increased Methanobrevibacter smithii, in AN patients. Changes in alpha diversity were inconsistent, while beta diversity increased in four of six studies. Our model suggests that an imbalance in gut microbiota composition leads to reduced short-chain fatty acids, contributing to a proinflammatory state in AN, which is also common in other psychiatric comorbidities. Microbial changes may also contribute to the semistarvation state through endocrine changes and altered energy utilization.

Methane-producing Archaea can be found in a variety of habitats, including the gastrointestinal tract, where they are linked to various diseases. The majority of current monitoring methods can be slow and laborious. To facilitate gut methanogenic Archaea detection, we investigated flow cytometry for rapid quantification based on the autofluorescent F420 cofactor, an essential coenzyme in methanogenesis. The methanogenic population was distinguishable from the SYBR green (SG) and SYBR green/propidium iodide (SGPI) stained background microbiome based on elevated 452 nm emission in Methanobrevibacter smithii spiked controls. As a proof-of-concept, elevated F420-autofluorescence was used to detect and quantify methanogens in 10 faecal samples and 241 in vitro incubated faecal samples. The methanogenic population in faeces, determined through Archaea-specific 16S rRNA gene amplicon sequencing, consisted of Methanobrevibacter and Methanomassiliicoccus. F420-based methanogen quantification in SG and SGPI-stained faecal samples showed an accuracy of 90 and 100% against Archaea proportions determined with universal primers. When compared to methane and Archaea presence, methanogen categorisation in in vitro incubated faeces exhibited an accuracy of 71 and 75%, with a precision of 42 and 70%, respectively. To conclude, flow cytometry is a reproducible and fast method for the detection and quantification of gut methanogenic Archaea.

This Review aims to coalesce existing knowledge on the human archaeome, a less‐studied yet critical non‐bacterial component of the human microbiome, with a focus on its interaction with the immune system. Despite a largely bacteria‐centric focus in microbiome research, archaea present unique challenges and opportunities for understanding human health. We examine the archaeal distribution across different human body sites, such as the lower gastrointestinal tract (LGT), upper aerodigestive tract (UAT), urogenital tract (UGT), and skin. Variability in archaeal composition exists between sites; methanogens dominate the LGT, while Nitrososphaeria are prevalent on the skin and UAT. Archaea have yet to be classified as pathogens but show associations with conditions such as refractory sinusitis and vaginosis. In the LGT, methanogenic archaea play critical metabolic roles by converting bacterial end‐products into methane, correlating with various health conditions, including obesity and certain cancers. Finally, this work looks at the complex interactions between archaea and the human immune system at the molecular level. Recent research has illuminated the roles of specific archaeal molecules, such as RNA and glycerolipids, in stimulating immune responses via innate immune receptors like Toll‐like receptor 8 (TLR8) and ‘C‐type lectin domain family 4 member E’ (CLEC4E; also known as MINCLE). Additionally, metabolic by‐products of archaea, specifically methane, have demonstrated immunomodulatory effects through anti‐inflammatory and anti‐oxidative pathways. Despite these advancements, the mechanistic underpinnings of how archaea influence immune activity remain a fertile area for further investigation. [ABSTRACT FROM AUTHOR]

Methane-producing Archaea can be found in a variety of habitats, including the gastrointestinal tract, where they are linked to various diseases. The majority of current monitoring methods can be slow and laborious. To facilitate gut methanogenic Archaea detection, we investigated flow cytometry for rapid quantification based on the autofluorescent F420 cofactor, an essential coenzyme in methanogenesis. The methanogenic population was distinguishable from the SYBR green (SG) and SYBR green/propidium iodide (SGPI) stained background microbiome based on elevated 452 nm emission in Methanobrevibacter smithii spiked controls. As a proof-of-concept, elevated F420-autofluorescence was used to detect and quantify methanogens in 10 faecal samples and 241 in vitro incubated faecal samples. The methanogenic population in faeces, determined through Archaea-specific 16S rRNA gene amplicon sequencing, consisted of Methanobrevibacter and Methanomassiliicoccus. F420-based methanogen quantification in SG and SGPI-stained faecal samples showed an accuracy of 90 and 100% against Archaea proportions determined with universal primers. When compared to methane and Archaea presence, methanogen categorisation in in vitro incubated faeces exhibited an accuracy of 71 and 75%, with a precision of 42 and 70%, respectively. To conclude, flow cytometry is a reproducible and fast method for the detection and quantification of gut methanogenic Archaea. [ABSTRACT FROM AUTHOR]

Archaea are an understudied component of the human microbiome. In this study, the gut archaeome and bacteriome of 60 healthy adults from different region were analyzed by whole-genome shotgun sequencing. Archaea were ubiquitously found in a wide range of abundances, reaching up to 7.2 %. The dominant archaeal phylum was Methanobacteriota, specifically the family Methanobacteriaceae, encompassing more than 50 % of Archaea in 50 samples. The previously underestimated Thermoplasmatota, mostly composed of Methanomassiliicoccaceae, dominated in 10 subjects (>50 %) and was present in all others except one. Halobacteriota, the sole other archaeal phylum, occurred in negligible concentration, except for two samples (4.6-4.8 %). This finding confirmed that the human gut archaeome is primarily composed of methanogenic organisms and among the known methanogenic pathway: i) hydrogenotrophic reduction of CO2 is the predominant, being the genus Methanobrevibacter and the species Methanobrevibacter smithii the most abundant in the majority of the samples; ii) the second pathway, that involved Methanomassiliicoccales, was the hydrogenotrophic reduction of methyl-compounds; iii) dismutation of acetate or methyl-compounds seemed to be absent. Co-occurrence analysis allowed to unravel correlations between Archaea and Bacteria that shapes the overall structure of the microbial community, allowing to depict a clearer picture of the human gut archaeome. [ABSTRACT FROM AUTHOR]

Anorexia nervosa (AN) remains a challenging condition in psychiatric management and its pathogenesis is not yet fully understood. An imbalance in the gut microbiota composition may contribute to its pathophysiology. This review aims to explore the link between the human gut microbiota and AN (objective 1) or refeeding syndrome in AN (objective 2). The online databases MEDLINE and PsycINFO were searched for relevant studies. A total of 14 studies met the inclusion and exclusion criteria and only answered objective 1. A total of 476 AN patients, 554 healthy-weight (HC) controls, and 0 patients with other psychiatric disorders were included. Compared to HC, there were consistently reduced abundances of Faecalibacterium prausnitzii and Roseburia inulinivorans, and increased Methanobrevibacter smithii, in AN patients. Changes in alpha diversity were inconsistent, while beta diversity increased in four of six studies. Our model suggests that an imbalance in gut microbiota composition leads to reduced short-chain fatty acids, contributing to a proinflammatory state in AN, which is also common in other psychiatric comorbidities. Microbial changes may also contribute to the semistarvation state through endocrine changes and altered energy utilization. [ABSTRACT FROM AUTHOR]

Background: We recently demonstrated that diarrhea-predominant irritable bowel syndrome (IBS-D) subjects have higher relative abundance (RA) of hydrogen sulfide (H2S)-producing Fusobacterium and Desulfovibrio species, and constipation-predominant IBS (IBS-C) subjects have higher RA of methanogen Methanobrevibacter smithii. Aims: In this study, we investigate the effects of increased methanogens or H2S producers on stool phenotypes in rat models. Methods: Adult Sprague–Dawley rats were fed high-fat diet (HFD) for 60 days to increase M. smithii levels, then gavaged for 10 days with water (controls) or methanogenesis inhibitors. To increase H2S producers, rats were gavaged with F. varium or D. piger. Stool consistency (stool wet weight (SWW)) and gas production were measured. 16S rRNA gene sequencing was performed on stool samples. Results: In HFD diet-fed rats (N = 30), stool M. smithii levels were increased (P < 0.001) after 52 days, correlating with significantly decreased SWW (P < 0.0001) at 59 days (R = − 0.38, P = 0.037). Small bowel M. smithii levels decreased significantly in lovastatin lactone-treated rats (P < 0.0006), and SWW increased (normalized) in lovastatin hydroxyacid-treated rats (P = 0.0246), vs. controls (N = 10/group). SWW increased significantly in D. piger-gavaged rats (N = 16) on day 10 (P < 0.0001), and in F. varium-gavaged rats (N = 16) at all timepoints, vs. controls, with increased stool H2S production. 16S sequencing revealed stool microbiota alterations in rats gavaged with H2S producers, with higher relative abundance (RA) of other H2S producers, particularly Lachnospiraceae and Bilophila in F. varium-gavaged rats, and Sutterella in D. piger-gavaged rats. Conclusions: These findings suggest that increased M. smithii levels result in a constipation-like phenotype in a rat model that is partly reversible with methanogenesis inhibitors, whereas gavage with H2S producers D. piger or F. varium results in increased colonization with other H2S producers and diarrhea-like phenotypes. This supports roles for the increased RA of methanogens and H2S producers identified in IBS-C and IBS-D subjects, respectively, in contributing to stool phenotypes. [ABSTRACT FROM AUTHOR]

Orthopedic prosthesis infection must be medically managed after appropriate microbiological documentation. While bacteria and fungi are acknowledged to be causative opportunistic pathogens in this situation, the potential role of methanogens in orthopedic prosthesis infections is still unknown. In a retrospective study, a total of 100 joint and bone samples collected from 25 patients were screened by specific PCR assays for the detection of methanogens. PCR-positive samples were observed by autofluorescence, electron microscopy and tentatively cultured under specific culture conditions. Methanogens were detected by quantitative PCR in 4/100 samples, in the presence of negative controls. Sequencing identified Methanobrevibacter oralis in two cases, Methanobrevibacter smithii in one case and Methanobrevibacter wolinii in one case. Microscopic methods confirmed molecular findings and bacterial culture yielded two strains of Staphylococcus aureus, one strain of Staphylococcus epidermidis and one strain of Proteus mirabilis. These unprecedented data highlight the presence of methanogens in joint and bone samples of patients also diagnosed with bacterial orthopedic prosthesis infection, questioning the role of methanogens as additional opportunistic co-pathogens in this situation.

IntroductionType 1 diabetes (T1D) risk involves genetic susceptibility but also epigenetics, environment, and behaviors. Appropriate metabolic control, especially quickly after the diagnosis, is crucial for the patient quality of life.MethodsThis study aimed to produce a quantitative comparison of the behavior, nutrition habits, and gut microbiota composition between the onset and the 1-year follow-up in 35 children with T1D.Results and discussionAt follow-up, with the metabolic control, many parameters improved significantly, with respect to the onset, such as glycated hemoglobin (−19%), body mass index (BMI), and also nutritional behaviors, such as normal calorie intake (+6%), carbohydrate intake (−12%), extra portion request (−4%), and meals distribution during the day. Moreover, glycated hemoglobin decrement correlated with both total and rapid absorption carbohydrate intake (Spearman's rho = 0.288, 95% CI 0.066–0.510, p = 0.013), showing as the nutritional behavior supported the insulin therapy efficiency. The next-generation sequencing (NGS) analysis of microbiota revealed abundance differences for Ruminococcus bromii and Prevotella copri (higher at onset, p < 0.001) and the genera Succinivibrio and Faecalibacterium (lower at onset, p < 0.001), as a consequence of nutritional behavior, but it was not the only changing driver. The qRT-PCR analysis showed significant variations, in particular for Bacteroidetes and Bifidobacterium spp. (+1.56 log gene copies/g stool at follow-up, p < 0.001). During the year, in 11% of the patients, severe clinical episodes occurred (hypoglycemic or ketoacidosis). The likelihood of a severe hypoglycemic episode was modulated when the Methanobrevibacter smithii amount increased (odds ratio 3.7, 95% CI 1.2–11.4, p = 0.026). Integrated evaluation, including nutritional behavior and microbiota composition, could be considered predictive of the metabolic control management for children cohort with a recent diagnosis of T1D.

Methanobrevibacter smithii ( M. smithii ), initially isolated from human feces, has been recognised as a distinct taxon within the Archaea domain following comprehensive phenotypic, genetic, and genomic analyses confirming its uniqueness among methanogens. Its diversity, encompassing 15 genotypes, mirrors that of biotic and host-associated ecosystems in which M. smithii plays a crucial role in detoxifying hydrogen from bacterial fermentations, converting it into mechanically expelled gaseous methane. In microbiota in contact with host epithelial mucosae, M. smithii centres metabolism-driven microbial networks with Bacteroides , Prevotella , Ruminococcus , Veillonella , Enterococcus , Escherichia , Enterobacter , Klebsiella , whereas symbiotic association with the nanoarchaea Candidatus Nanopusillus phoceensis determines small and large cell variants of M. smithii . The former translocate with bacteria to induce detectable inflammatory and serological responses and are co-cultured from blood, urine, and tissular abscesses with bacteria, prototyping M. smithii as a model organism for pathogenicity by association. The sources, mechanisms and dynamics of in utero and lifespan M. smithii acquisition, its diversity, and its susceptibility to molecules of environmental, veterinary, and medical interest still have to be deeply investigated, as only four strains of M. smithii are available in microbial collections, despite the pivotal role this neglected microorganism plays in microbiota physiology and pathologies., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 Published by Elsevier Ltd.)

Background The spectrum of infections caused by methanogens remains to be described. We searched for methanogens in the blood of febrile patients using specific tools. Methods Blood culture samples routinely collected in patients with fever were prospectively screened by specific PCR assays for methanogens. Positive samples were observed by autofluorescence and electron microscopy, analyzed by metagenomics and cultured using previously developed methods. Blood culture bottles experimentally inoculated were used as controls. The presence of methanogens in vascular and cardiac tissues was assessed by indirect immunofluorescence, fluorescent in situ hybridization and PCR-based investigations. Results PCR detection attempted in 7,716 blood samples, was negative in all 1,312 aerobic bottles and 810 bacterial culture-negative anaerobic bottles. PCRs were positive in 27/5,594 (0.5%) bacterial culture-positive anaerobic bottles collected from 26 patients. Sequencing confirmed Methanobrevibacter smithii associated with staphylococci in 14 patients, Enterobacteriaceae in nine patients and streptococci in three patients. Metagenomics confirmed M. smithii in five samples, and M. smithii was isolated in broth from two samples; the genomes of these two isolates were sequenced. Blood cultures experimentally inoculated with Enterobacteriaceae, Staphylococcus epidermidis or Staphylococcus hominis yielded hydrogen, but no methane, authentifying observational data. Three patients diagnosed with infectious mitral endocarditis, were indisputably diagnosed by microscopy, PCR-based detections and culture: we showed M. smithii microscopically and by a specific PCR followed by sequencing method in two of three cardiovascular tissues. Conclusions Using appropriate laboratory methods, M. smithii is demonstrated as causing archaemia and endocarditis in febrile patients who are coinfected by bacteria. [ABSTRACT FROM AUTHOR]

To understand the dynamics of methanogens in the human intestinal microbiota, we investigated the presence of methanogens in meconium using a polyphasic approach including microscopy and PCR-sequencing in 33 meconium samples collected from 33 pre-term neonates, in accordance with current ethics regulation. In the presence of negative controls, 90.9% samples were real-time PCR-positive for methanogens and 69.7 % were PCR-sequencing positive, identified as Methanobrevibacter (M.) smithii. Further, auto-fluorescent analysis detected methanogens in the two meconium samples analyzed, with a morphology suggesting M. smithii. Multispacer Sequence Typing found M. smithii genotypes ST1 and ST2, previously described as intestinal microbiota inhabitants. C-section delivery and non-use of peripartum antibiotics significantly correlated with PCR-detection of methanogens in meconium. These data position M. smithii among the early inhabitants of the human gut, detectable immediately after birth and suggest the contribution of methanogens to the perinatal development of intestinal microbiota and physiology.

Untreated tonsillar phlegmon is a life-threatening condition commonly caused by Streptococcus pyogenes and Fusobacterium necrophorum, among other pathogens. Here, using specific laboratory tools, we detected Methanobrevibacter smithii in addition to S. pyogenes. This unprecedented observation questions the role of methanogens in phlegmon and the optimal treatment of this mixed infection.

Abstract Background The oral cavity of humans is inhabited by several hundreds of bacterial species and other microorganisms such as fungi and archaeal methanogens. Regarding methanogens, data have been obtained from oral cavity samples collected in Europe, America and Asia. There is no study published on the presence of methanogens in the oral cavity in persons living in Africa. The objective of our study was to bring new knowledge on the distribution of oral methanogens in persons living in Mali, Africa. Methods A total of 31 patients were included in the study during a 15-day collection period in September. Bacterial investigations consisted in culturing the bacteria in 5% sheep blood–enriched Columbia agar and PolyViteX agar plates. For archaeal research, we used various methods including culture, molecular biology and fluorescent in situ hybridization (FISH). Results Eight of 31 (26%) oral samples collected in eight patients consulting for stomatology diseases tested positive in polymerase chain-reaction (PCR)-based assays for methanogens including five cases of Methanobrevibacter oralis and one case each of Methanobrevibacter smithii, Methanobrevibacter massiliense and co-infection Methanobrevibacter oralis and Methanobrevibacter massiliense. Conclusions In this pilot study, we are reporting here the first characterization of methanogens in the oral cavity in eight patients in Mali. These methanogen species have already been documented in oral specimens collected from individuals in Europe, Asia, North America and Brazil.

Atractylodis macrocephalae Rhizoma is widely used as a functional food in Asia. It can enhance glucose and lipid metabolism in the obesity patients with adiposity. In this study, therapeutic effects and the underlying molecular mechanisms of its ethanol extract (AMK) were investigated in db/db mice. Oral treatment with AMK at a dose of 100 mg/kg for three weeks significantly decreased the blood glucose, serum triacylglycerol, total cholesterol, HOMA-IR, endotoxin and IL-1β. Besides, AMK increased the abundance of Bacteroides thetaiotaomicron and Methanobrevibacter smithii in gut microbiota and improved the histological abnormalities of liver, adipocytes, pancreas and small intestine in diabetic mice. Moreover, AMK upregulated the expressions of GLP-1R, PI3K and PDX-1, but downregulated FOXO1 and NF-κB p65 in liver and pancreas tissues of diabetic mice. These results suggested that AMK ameliorated glucose metabolism by regulating GLP-1R/PI3K/PDX-1 pathway and gut microbiota, and it could be developed as a medicinal herb for T2DM treatment.

The authors report the cases of 9 patients eventually diagnosed with methanogenic archaea refractory or recalcitrant chronic rhinosinusitis, a condition known to involve various anaerobic bacteria but in which the role of methanogenic archaea is unknown. The authors retrospectively searched these microorganisms by PCR in surgically-collected sinusal pus specimens from patients diagnosed with refractory sinusitis, defined by the persistance of sinus inflammation and related-symptoms for more than 12 weeks despite appropriate treatment. Of the 116 tested sinus surgical specimens, 12 (10.3%) from 9 patients (six females, three males; aged 20–71 years) were PCR-positive. These specimens were further investigated by fluorescence in-situ hybridization, PCR amplicon-sequencing and culture. Methanobrevibacter smithii was documented in four patients and Methanobrevibacter oralis in another four, one of whom was also culture-positive. They were associated with a mixed flora including Gram-positive and Gram-negative bacteria. In the latter patient, “Methanobrevibacter massiliense” was the sole microorganism detected. These results highlight methanogenic archaea as being part of a mixed anaerobic flora involved in refractory sinusitis, and suggest that the treatment of this condition should include an antibiotic active against methanogens, notably a nitroimidazole derivative.

Vaginosis is a dysbiotic condition of the vaginal cavity that has deleterious effects during pregnancy. The role of methanogens in this disease is unknown since current methods of investigation are not appropriate for the search of methanogens. We prospectively investigated the presence of methanogens in vaginal specimens collected from 33 women thereafter diagnosed with bacterial vaginosis and 92 women thereafter diagnosed without bacterial vaginosis (control group) by direct microscopic examination and fluorescent in situ hybridization, PCR-sequencing, and real-time PCR and isolation and culture. These investigations found only one methanogen, Methanobrevibacter smithii, exclusively in 97% bacterial vaginosis specimens and in two intermediate microbiota specimens. M. smithii was detected microscopically in 2/20 specimens analyzed, by PCR-based observations in 34/125 specimens with 99% sequence similarity with the reference 16S rRNA and mcrA gene sequences and was cultured in 9/40 specimens. These data suggest that the detection of M. smithii could be used as a biomarker for the laboratory diagnosis of bacterial vaginosis. [ABSTRACT FROM AUTHOR]

Methanogens, the sole microbes producing methane, are archaea commonly found in human anaerobic microbiota. Methanogens are emerging as opportunistic pathogens associated with dysbiosis and are also detected and cultured in anaerobic abscesses. Their presence in the respiratory tract is yet unknown. As a preliminary answer, prospective investigation of 908 respiratory tract samples using polyphasic approach combining PCR-sequencing, real-time PCR, fluorescent in situ hybridization (FISH), and methanogens culture was carried out. Methanobrevibacter smithii and Methanobrevibacter oralis DNA sequences, were detected in 21/527 (3.9%) sputum samples, 2/188 (1.06%) bronchoalveolar lavages, and none of 193 tracheo-bronchial aspirations. Further, fluorescence in situ hybridization detected methanogens in three sputum investigated specimens with stick morphology suggesting M. oralis and in another one bronchoalveolar lavage sample investigated, diplococal morphology suggesting M. smithii. These observations extend the known territory of methanogens to the respiratory tract and lay the foundations for further interpretation of their detection as pathogens in any future cases of isolation from bronchoalveolar lavages and the lungs.

It is hypothesized that direct and indirect homeostasis between gut microbiota plays a key role in different intestine disorders. Archaea methanogens, an ancient domain of single-celled organism, are major archaea in the digestive system. Recent evidence has shown that the variable prevalence of methanogens in different individuals could have certain effects on inflammatory bowel diseases (IBD). We aimed to assess the prevalence of Methanobrevibacter smithii between Iranian patients suffering from IBD and healthy control subjects. Stool DNA extracts from 47 healthy controls and 61 IBD patients were investigated. Quantitative real time PCR was performed for detecting Mbb. smithii load. We found a significantly decreased the Mbb. smithii load between IBD patients and healthy subjects. It is assumed that there is a reverse association between Mbb. smithii bacterial load and susceptibility to IBD, and this association could be extended to IBD patients in remission as we found that Mbb. smithii bacterial load is markedly higher among healthy subjects in comparison to IBD patients. [ABSTRACT FROM AUTHOR]

Erhualian and Landrace breeds are typical genetically obese and lean pigs, respectively. To compare the fecal methanogenic Archaeal community between these two pig breeds, fecal samples from different growth phase pigs were collected and used for PCR-denaturing gradient gel electrophoresis (DGGE) with two primer pairs (344fGC/519r and 519f/915rGC) and real-time PCR analysis. Results showed that a better separation and higher quality of bands pattern were obtained in DGGE profiles using primers 344fGC/519r as compared with primers 519f/915rGC. Sequencing of DGGE bands showed that the predominant methanogens in the feces of Erhualian and Landrace pigs belonged to Methanobrevibacter spp. and Methanosphaera spp. Real-time PCR analysis revealed that there was no significant difference in the numbers of fecal total methanogens between Erhualian and Landrace pigs; however, pig growth phase affected the numbers of 16S rRNA genes of total methanogens and Methanobrevibacter smithii. Dissociation curves of methyl coenzyme-M reductase subunit A (mcrA) gene fragments amplified with real-time PCR showed all samples possessed a single peak at 82°C, which might be associated with M. smithii. Samples from the same growth phase of each breed showed good replicative dissociation curves. The results suggest that the growth phase (including diet factor) other than genotype of pig may affect the fecal methanogenic Archaeal community of pigs.

Atractylodis macrocephalae Rhizoma is widely used as a functional food in Asia. It can enhance glucose and lipid metabolism in the obesity patients with adiposity. In this study, therapeutic effects and the underlying molecular mechanisms of its ethanol extract (AMK) were investigated in db/db mice. Oral treatment with AMK at a dose of 100 mg/kg for three weeks significantly decreased the blood glucose, serum triacylglycerol, total cholesterol, HOMA-IR, endotoxin and IL-1β. Besides, AMK increased the abundance of Bacteroides thetaiotaomicron and Methanobrevibacter smithii in gut microbiota and improved the histological abnormalities of liver, adipocytes, pancreas and small intestine in diabetic mice. Moreover, AMK upregulated the expressions of GLP-1R, PI3K and PDX-1, but downregulated FOXO1 and NF-κB p65 in liver and pancreas tissues of diabetic mice. These results suggested that AMK ameliorated glucose metabolism by regulating GLP-1R/PI3K/PDX-1 pathway and gut microbiota, and it could be developed as a medicinal herb for T2DM treatment. [ABSTRACT FROM AUTHOR]

Severe acute malnutrition is the world-leading cause of children under-five's death. Recentmetagenomics studies have established a link between gutmicrobiota and severe acute malnutrition, describing an immaturity with a striking depletion in oxygen-sensitive prokaryotes. Amoxicillin and therapeutic diet cure most of the children with severe acute malnutrition but an irreversible disruption of the gut microbiota is suspected in the refractory and most severe cases. In these cases, therapeutic diet may be unable to reverse the microbiota alteration leading to persistent impaired development or death. In addition, as enteric sepsis is a major cause of death in this context, identification of missing gut microbes to be tested as probiotics (live bacteria that confer a benefit to the host) to restore rapidly the healthy gut microbiota and prevent the gut pathogenic invasion is of foremost importance. In this study, stool samples of malnourished patients with kwashiorkor and healthy children were collected from Niger and Senegal and analyzed by culturomics and metagenomics. We found a globally decreased diversity, a decrease in the hitherto unknown diversity (new species isolation), a depletion in oxygen-sensitive prokaryotes including Methanobrevibacter smithii and an enrichment in potentially pathogenic Proteobacteria, Fusobacteria and Streptococcus gallolyticus. A complex of 12 species identified only in healthy children using culturomics and metagenomics were identified as probiotics candidates, providing a possible, defined, reproducible, safe, and convenient alternative to fecal transplantation to restore a healthy gut microbiota in malnourished children. Microbiotherapy based on selected strains has the potential to improve the current treatment of severe acute malnutrition and prevent relapse and death by reestablishing a healthy gut microbiota. [ABSTRACT FROM AUTHOR]

Methanobrevibacter smithii (M. smithii), the most prevalent and abundant gut methanogen, detoxifies hydrogen into methane and is, therefore, of paramount importance for the equilibrium of the gut microbiota. The isolation by culture of M. smithii has routinely relied upon hydrogen‑carbon dioxide-enriched, oxygen-deprived atmospheres. In this study, we developed a medium referred to as "GG", which allowed for M. smithii growth and isolation by culture in an oxygen-deprived atmosphere, with no supply of either hydrogen or carbon dioxide, making it easier to detect M. smithii by culture in clinical microbiology laboratories. • Methanobrevibacter smithii can grow without hydrogen using formate and probably acetate. • GG medium eases Methanobrevibacter smithii routine culture, avoiding the explosive hydrogen. • Methanobrevibacter smithii can be associated with bacteria that do not produce hydrogen such as Staphylococcus and Streptococcus genus. [ABSTRACT FROM AUTHOR]

The identification of fecal pollution in aquatic ecosystems is one of the requirements to assess the possible risks to human health. In this report, physicochemical parameters, Escherichia coli enumeration and Methanobrevibacter smithii nifH gene quantification were conducted at 13 marine waters in the coastal beaches of Rio de Janeiro, Brazil. The pH, turbidity, dissolved oxygen, temperature, and conductivity, carried out by mobile equipment, revealed varied levels due to specific conditions of the beaches. The bioindicators' enumerations were done by defined substrate method, conventional, and real-time PCR. Six marine beach sites (46 %) presenting E. coli levels in compliance with Brazilian water quality guidelines (<2500 MPN/100 mL) showed nifH gene between 5.7 × 10 to 9.5 × 10 copies. L revealing poor correlation between the two approaches. To our knowledge, this is the first inquiry in qPCR using nifH gene as a biomarker of human-specific sources of sewage pollution in marine waters in Brazil. In addition, our data suggests that alternative indicator nifH gene could be used, in combination with other markers, for source tracking studies to measure the quality of marine ecosystems thereby contributing to improved microbial risk assessment. [ABSTRACT FROM AUTHOR]

NADH oxidases (NOXs) catalysing the oxidation of NADH to yield NAD+ and H2O, H2O2, or both play an important role in protecting organisms from oxidative stress and maintaining the balance of NAD+ /NADH. A gene encoding NOX was identified from Methanobrevibacter smithii (NOX-ms), the predominant archaeon in the human gut ecosystem. Subsequent analyses showed that it is an FAD-containing protein with a subunit molecular mass of 48 kDa. NOX-ms was purified to homogeneity after expression in Escherichia coli. NOX-ms catalysed the oxidization of NADH and converted O2 to H2O with an optimal pH of 7.5 and a temperature optimum of approximately 37 °C. The Vmax and Km values were 42.6-44.1 unit/mg and 47.8-54.6 μM for NADH. The apparent Vmax and Km for oxygen were 189.5-196.1 unit/mg and 14.6-16.8 μM. The mutation analysis suggests that Cys42 in NOX-ms plays a key role in the four-electron reduction of O2 to H2O. Quantitative reverse transcription-PCR (RT-qPCR) revealed that transcription of NOX-ms was also up-regulated after exposing the cells to oxidative stress and glucose. Finally, the potential of NOX-ms as a target to control colonization of M. smithii and its possible applications are discussed. [ABSTRACT FROM AUTHOR]

Obesity constitutes a significant and rapidly increasing public health challenge and is associated with significant co-morbidities and healthcare costs. Although undoubtedly multifactorial, research over the last decade has demonstrated that the microbes that colonize the human gut may contribute to the development of obesity through roles in polysaccharide breakdown, nutrient absorption, inflammatory responses and gut permeability. Studies have consistently shown that the Firmicutes to Bacteroidetes ratio, in particular, is increased in obesity and reduces with weight loss. In addition, we and others have shown that the methanogenic Archaea may also contribute to altered metabolism and weight gain in the host. However, much remains to be learned about the roles of different gut microbial populations in weight gain and obesity and the underlying mechanisms before we can begin to approach targeted treatments. [ABSTRACT FROM AUTHOR]