Targeted isolation of Methanobrevibacter strains from fecal samples expands the cultivated human archaeome.

Archaea are vital components of the human microbiome, yet their study within the gastrointestinal tract (GIT) is limited by the scarcity of cultured representatives. Our study presents a method for the targeted enrichment and isolation of methanogenic archaea from human fecal samples. The procedure combines methane breath testing, in silico metabolic modeling, media optimization, FACS, dilution series, and genomic sequencing through Nanopore technology. Additional analyzes include the co-cultured bacteriome, comparative genomics of archaeal genomes, functional comparisons, and structure-based protein function prediction of unknown differential traits. Successful establishment of stable archaeal cultures from 14 out of 16 fecal samples yielded nine previously uncultivated strains, eight of which are absent from a recent archaeome genome catalog. Comparative genomic and functional assessments of Methanobrevibacter smithii and Candidatus Methanobrevibacter intestini strains from individual donors revealed features potentially associated with gastrointestinal diseases. Our work broadens available archaeal representatives for GIT studies, and offers insights into Candidatus Methanobrevibacter intestini genomes' adaptability in critical microbiome contexts.

Effects of an oral synbiotic on the gastrointestinal immune system and microbiota in patients with diarrhea-predominant irritable bowel syndrome.

PURPOSE: Diarrhea-predominant irritable bowel syndrome (IBS-D) is a common functional gastrointestinal disorder. Probiotics and synbiotics have been shown to improve symptoms of IBS, although mechanisms of action are currently not understood. METHODS: We investigated the effects of a 4-week oral synbiotic treatment (OMNi-BiOTiC® Stress Repair) in ten IBS-D patients on gastrointestinal mucosal and fecal microbiota, mucosa-associated immune cells, and fecal short-chain fatty acids. The upper and lower gastrointestinal tracts were compared before and after a 4-week synbiotic treatment using endoscopic evaluation to collect mucosal specimens for FACS analysis and mucosal 16S rRNA gene analysis. In stool samples, analysis for fecal SCFAs using GC-MS, fecal zonulin using ELISA, and fecal 16S rRNA gene analysis was performed. RESULTS: Synbiotics led to an increased microbial diversity in gastric (p = 0.008) and duodenal (p = 0.025) mucosal specimens. FACS analysis of mucosal immune cells showed a treatment-induced reduction of CD4+ T cells (60 vs. 55%, p = 0.042) in the ascending colon. Short-chain fatty acids (acetate 101 vs. 202 µmol/g; p = 0.007) and butyrate (27 vs. 40 µmol/g; p = 0.037) were elevated in fecal samples after treatment. Furthermore, treatment was accompanied by a reduction of fecal zonulin concentration (67 vs. 36 ng/ml; p = 0.035) and disease severity measured by IBS-SSS (237 vs. 54; p = 0.002). CONCLUSIONS: Our findings indicate that a short-course oral synbiotic trial may influence the human gastrointestinal tract in IBS-D patients on different levels which are region specific.

Mucosal biopsy shows immunologic changes of the colon in patients with early MS.

OBJECTIVE: To investigate immune cells of the colonic mucosa and fecal short-chain fatty acids (SCFAs) in treatment-naive patients with a clinically isolated syndrome (CIS) or early relapsing MS. METHODS: In this cross-sectional proof-of-concept study, we obtained mucosal specimens during ileocolonoscopy from 15 untreated patients with CIS/MS and 10 controls. Mucosal immune cells were analyzed by FACS, and gas chromatography-mass spectrometry measurements of stool samples served to determine SCFA. RESULTS: The number of total dendritic cells (DCs), CD103+ tolerogenic DCs, and CD4+25+127-regulatory T cells (Tregs) was significantly reduced in the distal colon of patients with CIS/MS compared with controls, whereas we found no differences in the proximal colon. The patients' fecal samples also showed a substantially lower content of SCFA and especially lower levels of butyrate and acetate. CONCLUSIONS: Our findings indicate a disturbed homeostasis of colonic DCs and Tregs in patients with MS which could be associated with colonic SCFA depletion. Although not implying causality, these findings confirm parallel abnormalities of the gut in MS and warrant further research if modulation of the colonic SCFA profile or the colonic Treg pool can serve to modify the course of MS.

Increased steroidogenesis promotes early-onset and severe vision loss in females with OPA1 dominant optic atrophy.

OPA1 mutations are responsible for autosomal dominant optic atrophy (ADOA), a progressive blinding disease characterized by retinal ganglion cell (RGC) degeneration and large phenotypic variations, the underlying mechanisms of which are poorly understood. OPA1 encodes a mitochondrial protein with essential biological functions, its main roles residing in the control of mitochondrial membrane dynamics as a pro-fusion protein and prevention of apoptosis. Considering recent findings showing the importance of the mitochondrial fusion process and the involvement of OPA1 in controlling steroidogenesis, we tested the hypothesis of deregulated steroid production in retina due to a disease-causing OPA1 mutation and its contribution to the visual phenotypic variations. Using the mouse model carrying the human recurrent OPA1 mutation, we disclosed that Opa1 haploinsufficiency leads to very high circulating levels of steroid precursor pregnenolone in females, causing an early-onset vision loss, abolished by ovariectomy. In addition, steroid production in retina is also increased which, in conjunction with high circulating levels, impairs estrogen receptor expression and mitochondrial respiratory complex IV activity, promoting RGC apoptosis in females. We further demonstrate the involvement of Muller glial cells as increased pregnenolone production in female cells is noxious and compromises their role in supporting RGC survival. In parallel, we analyzed ophthalmological data of a multicentre OPA1 patient cohort and found that women undergo more severe visual loss at adolescence and greater progressive thinning of the retinal nerve fibres than males. Thus, we disclosed a gender-dependent effect on ADOA severity, involving for the first time steroids and Müller glial cells, responsible for RGC degeneration.