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Growth requirements and fermentation products of Fusobacterium prausnitzii, and a proposal to reclassify it as Faecalibacterium prausnitzii gen. nov., comb. nov.

Two newly isolated strains of obligately anaerobic bacteria from human faeces are shown here to be related to Fusobacterium prausnitzii, which is regarded as one of the most abundant colonizers of the human colon. These strains, along with Fusobacterium prausnitzii ATCC 27768(T) and 27766, are non-motile and produce butyrate, formate and lactate, but not hydrogen as fermentation products. A new finding is that all four strains produce D-lactate, but not L-lactate. The strains have a requirement for acetate in the growth medium and this may account for the previously reported requirement for rumen fluid. The DNA G+C content of the four strains is 47-57 mol%. Together with phylogenetic analysis based on 16S rRNA sequencing, this establishes that Fusobacterium prausnitzii strains are only distantly related to Fusobacterium sensu stricto and are more closely related to members of Clostridium cluster IV (the Clostridium leptum group). It is proposed that a new genus, Faecalibacterium gen. nov. be created; this genus should include Faecalibacterium prausnitzii gen. nov., comb. nov. ATCC 27768(T) (= NCIMB 13872(T)) (formerly Fusobacterium prausnitzii) as the type species together with ATCC 27766 and the newly isolated strains A2-165 and L2-6.

Base Composition↗

Gut microbiota and metabolic alterations in participants with flatulence identify Faecalibacterium prausnitzii as a key microbial target for clinical intervention.

Flatulence is closely associated with gut dysbiosis, yet the characteristic microbial signatures, metabolic alterations, and actionable intervention targets remain unclear. This limited mechanistic understanding has hindered the development of precise microbiota-based strategies for managing flatulence. Here, we found that participants with flatulence exhibited marked shifts in gut microbial functions and fecal metabolic profiles compared with healthy controls, characterized by enhanced abnormal fermentation, enrichment of oxidative stress-related functions, elevated low-grade inflammatory signatures, and reduced anti-inflammatory and mucosal-protective metabolic features. Faecalibacterium prausnitzii was significantly negatively associated with the high-gas-producing phenotype. In vitro replenishment experiments further validated the role of F. prausnitzii in reducing gas production, promoting butyrate generation, and remodeling butyrate-associated microbial communities. Based on microbial interaction analysis, we identified Bifidobacterium longum CCFM1319 as a candidate strain for targeting F. prausnitzii. In a double-blind, randomized, placebo-controlled clinical trial, supplementation with B. longum CCFM1319 significantly increased intestinal F. prausnitzii abundance and improved flatulence-related symptoms. Collectively, these findings reveal the microbiota and metabolic dysbiosis underlying flatulence, highlight the key regulatory role of F. prausnitzii, and lays the foundation for targeted microbiota-based intervention strategies for flatulence.

Humans↗

Faecalibacterium prausnitzii-derived L-arginine ameliorates insomnia by inhibiting POMC-ACTH-cortisol axis.

Insomnia is associated with gut microbial dysbiosis, but the specific microbial metabolites mediating gut-brain communication remain elusive. Here, we integrate metagenomic sequencing from 171 individuals (primary insomnia, post-COVID insomnia, and controls) with functional pathway analysis and preclinical validation. We identify Faecalibacterium prausnitzii depletion and reduced L-arginine biosynthesis as consistent features in both insomnia subtypes, accompanied by elevated cortisol levels. Genomic and in vitro analyses confirm that F. prausnitzii is a key microbial contributor to L-arginine production. In a chronic mild stress mouse model, administration of either F. prausnitzii or L-arginine restores sleep duration, normalizes corticosterone levels, and reverses stress-induced gut dysbiosis. Mechanistically, L-arginine suppresses POMC gene expression and dampens adrenocorticotropic hormone (ACTH)-stimulated corticosterone release, implicating the POMC-ACTH-cortisol axis as a key target. These findings uncover a gut-brain axis driven by F. prausnitzii-derived L-arginine that modulates sleep through endocrine signaling, positioning this metabolite as a potential therapeutic avenue for insomnia.

Arginine↗

Identification of a novel human gut microbes and microbial metabolites related genes signature for prognostic implication in head and neck squamous carcinomas.

BACKGROUND: The gut microbiota acts as a critical driver influencing the pathogenesis, therapeutic response, and clinical outcomes across various cancer types. This study aimed to investigate the prognostic value of human gut microbes and microbial metabolites related genes (HGMMMRGs) in head and neck squamous cell carcinoma (HNSCC). METHODS: We constructed a prognostic risk model comprising 19 core HGMMMRGs using LASSO penalized regression and a multivariate Cox proportional hazards model. The predictive performance of the model was evaluated through Kaplan-Meier analysis, receiver operating characteristic (ROC) curves, nomograms, and concordance index. In addition, functional enrichment analysis was performed on the differentially expressed risk genes. Furthermore, the relationship between the immune microenvironment of HNSCC and the risk diagnostic model was examined. Western blot analysis was used to assess the expression levels of IL10 in both HNSCC tissues and adjacent normal tissues. Finally, the correlation between IL10 and the gut microbiota was explored. RESULTS: This study developed a risk score model integrating 19 HGMMMRG genes, which can serve as a tool to guide prognosis and immune microenvironment assessment in HNSCC patients. Survival analysis showed that patients in the high-risk group had significantly worse outcomes (P&#x2009;<&#x2009;0.05). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed significant enrichment of differentially expressed genes (DRLs) and immune-related pathways. Western blot analysis further confirmed that IL10 was highly expressed in HNSCC, and the abundance of Faecalibacterium prausnitzii and Enterococcus durans colonies was correlated with IL10 expression. CONCLUSION: We developed a prognostic model for HGMMMRGs that can be effectively used to predict OS in patients with HNSCC. Second, Faecalibacterium prausnitzii and Enterococcus durans can influence the prognosis of patients with HNSCC by mediating the expression IL10 and thereby affecting the prognosis of HNSCC patients. Thus, human gut microbes and microbial metabolite-related genes may be another promising strategy for the treatment of patients with HNSCC.

HNSCC↗

Oligonucleotide probes that detect quantitatively significant groups of butyrate-producing bacteria in human feces.

16S rRNA-targeted oligonucleotide probes were designed for butyrate-producing bacteria from human feces. Three new cluster-specific probes detected bacteria related to Roseburia intestinalis, Faecalibacterium prausnitzii, and Eubacterium hallii at mean populations of 2.3, 3.8, and 0.6%, respectively, in samples from 10 individuals. Additional species-level probes accounted for no more than 1%, with a mean of 7.7%, of the total human fecal microbiota identified as butyrate producers in this study. Bacteria related to E. hallii and the genera Roseburia and Faecalibacterium are therefore among the most abundant known butyrate-producing bacteria in human feces.

Adult↗

Subdoligranulum variabile gen. nov., sp. nov. from human feces.

During studies on the microflora of human feces we have isolated a strictly anaerobic, non-spore-forming, Gram-negative staining organism which exhibits a somewhat variable coccus-shaped morphology. Comparative 16S ribosomal RNA gene sequencing studies show the unidentified organism is phylogenetically a member of the Clostridium leptum supra-generic rRNA cluster and displays a close affinity to some rDNA clones derived from human and pig feces. The nearest named relatives of the unidentified isolate corresponded to Faecalibacterium prausnitzii (formerly Fusobacterium prausnitzii) displaying a 16S rRNA sequence divergence of approximately 9%, with Anaerofilum agile and A. pentosovorans the next closest relatives of the unidentified bacterium (sequence divergence approximately 10%). Based on phenotypic and phylogenetic considerations, it is proposed that the unusual coccoid-shaped organism be classified as a new genus and species, Subdoligranulum variabile. The type strain of S. variabile is BI 114(T) (=CCUG 47106(T)=DSM 15176(T)).

Journal Article↗

Gut metagenome and plasma metabolome profiles in older adults suggest pyruvate metabolism as a link between sleep quality and frailty.

Poor sleep quality is associated with increased frailty in older adults, but the role of the gut microbiome in this relationship remains unclear. Here, gut metagenome and plasma metabolome were profiled in 1,225 individuals aged 62-96 years. Poor sleep quality was associated with reduced abundances of potential probiotics such as Faecalibacterium prausnitzii and elevated abundances of pathobionts. A gut microbiome sleep quality index (GMSI) was developed to quantify microbial balance related to better sleep quality; higher GMSI scores were inversely associated with frailty and related clinical traits. Pyruvate metabolism emerged as a key microbial pathway linking sleep quality to frailty, with features such as F. prausnitzii abundance and microbial pyridoxal 5'-phosphate biosynthesis implicated in this connection. These findings deepen our understanding of microbiome-metabolome pathways related to sleep quality and frailty in aging and provide a valuable resource for future longitudinal and interventional studies.

Humans↗

Gut Microbiome Composition Is Associated With Response to CD38 Antibody (Daratumumab) Treatment Among Relapsed Multiple Myeloma Patients.

INTRODUCTION: Growing data support interactions between host-gut microbes and treatment responses in multiple myeloma (MM), where a higher abundance of Eubacterium hallii in stool samples has been found among MM patients with negative minimal residual disease after induction therapy. Here, we evaluated changes in the gut microbiome associated with daratumumab (dara) based therapy in 40 MM patients, before and after therapy. PATIENTS AND METHODS: Patients with relapsed MM and prior autologous transplantation who had received 1 to 4 prior lines of therapy were eligible. Two stool samples were collected, one within 1 week prior to dara (predara) and one immediately after 4 doses of dara (postdara). Metagenomics sequencing was conducted. Microbiome taxonomic analyses were performed using MetaPhlAn4, and microbial functional pathway analyses were conducted using HUMAnN3.6. QIIME2 was used for compositional and statistical analyses. RESULTS: Of 40 participants enrolled, there were 5 nonresponders; 35 patients achieved partial response (PR) or better (responders). Among responders, 10 patients achieved complete remission (CR), and 25 patients achieved either very good partial response (VGPR) or PR. There were no statistically significant differences between overall pre and postdara gut microbiomes. Differential abundance analysis (ANCOM-BC) showed statistically significant (q &#x2264; 0.05) overgrowth of Alistipes finegoldii and Acidaminococcus intestini species in responders and Ruminococcus torques, Sellimonas intestinalis and Clostridium symbiosum in nonresponders. Compared to non-CR, CR samples showed enrichment of Faecalibacterium prausnitzii; non-CR samples were enriched in Segatella copri and Faecalimonas umbilicata. DISCUSSION/CONCLUSION: Our results suggest differences in species between clinical responders and nonresponders, but larger prospective studies are needed to confirm these results.

Clinical response↗

Characterisation of intestinal bacteria in infant stools using real-time PCR and northern hybridisation analyses.

Real-time PCR and northern hybridisations were used to quantify bacterial populations in the large gut of infants. PCR primers for rapid, sensitive, high throughput detection of bifidobacteria, bacteroides, sulphate-reducing bacteria and Enterococcus faecalis, based on analysis of 16S rRNA genes were used. Bacterial populations were analysed in faeces from 40 infants aged 0-6, 7-12 and 13-24 months. The effects of breast versus bottle feeding was also investigated. Real-time PCR indicated that bacteroides and desulfovibrio numbers increased markedly in the 7-12 and 13-24 month age groups, and that the reverse occurred with Ent. faecalis. With the exception of desulfovibrios, this was seen with northern hybridisations, which also showed increased colonisation by the Clostridium coccoides group and Faecalibacterium prausnitzii after 6 months. Both methodologies indicated increased bifidobacteria in breast-fed babies, and higher levels of desulfovibrios in bottle-fed children.

Age Factors↗

Validation of fluorescent in situ hybridization combined with flow cytometry for assessing interindividual variation in the composition of human fecal microflora during long-term storage of samples.

This work was conducted to assess the accuracy of in situ hybridization to show differences in human microflora composition between volunteers and to optimize the storage of fecal samples to allow delayed analysis of gut microflora composition in humans. Fecal samples from 25 healthy subjects (14 women, 11 men aged 24-51) were collected. The samples were fixed in 4% Paraformaldehyde (PFA) solution at 4 degrees C overnight and stored at -70 degrees C. Twenty samples were analysed to quantify the variation due to interindividual differences in the composition of fecal microflora. The five remaining samples were stored either after PFA fixation or directly frozen at -70 degrees C and were monitored on a 12-month period. The fecal microflora was analysed by in situ hybridization combined with flow cytometry detection. Ribosomal RNA-targeted probes were used to assess the relative proportions of four phylogenetic groups: Clostridium coccoides-Eubacterium rectale (Erec 482), Bacteroides (Bac 303), Faecalibacterium prausnitzii (Fprau 645) and Bifidobacterium (Bif 164). Our results demonstrated that the method used is adapted to detect significant differences in fecal microflora composition in humans. Moreover, samples stored in PFA solution demonstrated a stable composition even after 8 months of storage. Conversely, frozen samples were less stable as the Bifidobacterium and C. coccoides-E. rectale groups showed significant differences after 2 months of storage. In conclusion, the fecal microflora composition can be analysed up to 8 months after 4% PFA fixation and storage at -70 degrees C. It represents an extended time compared with the 2-month period currently recommended. This will give more flexibility for applying this technology in epidemiological studies including a large number of samples.

Adult↗

Temporal stability and lack of variance in microbiome composition and functionality in fit recreational athletes.

Human gut microbiome composition and function is influenced by environmental and lifestyle factors, including exercise and fitness. We studied the composition and functionality of the faecal microbiome of recreational (non-elite) runners (n&#x2009;=&#x2009;62) with serial shotgun metagenomics, at 4 time points over a 7-week period. Gut microbiome composition and function was stable over time. Grouping of samples on the basis of their fitness level (fair, good, excellent, and superior) or habitual training (low (4-6&#xa0;h/week), medium (7-9&#xa0;h/week), high (10-12&#xa0;h/week), and extreme (13&#x2009;+&#x2009;hours/week)) revealed no significant microbiome-related differences. Overall, the species Faecalibacterium prausnitzii, Blautia wexlerae, and Prevotella copri were the most abundant members of the gut microbiome. Analysis of co-abundance groups (CAGs) revealed no significant relationship between CAGs and fitness levels or training subgroups. Functional pathways were similar across all samples and timepoints with no clustering based on associated metadata. The most abundant genes identified within samples corresponded to pathways for nucleoside and nucleotide biosynthesis, amino acid biosynthesis, and cell wall biosynthesis. Collectively, these results describe the microbiome of active recreational runners and note temporal stability amongst participants.

Humans↗

Contribution of acetate to butyrate formation by human faecal bacteria.

Acetate is normally regarded as an endproduct of anaerobic fermentation, but butyrate-producing bacteria found in the human colon can be net utilisers of acetate. The butyrate formed provides a fuel for epithelial cells of the large intestine and influences colonic health. [1-(13)C]Acetate was used to investigate the contribution of exogenous acetate to butyrate formation. Faecalibacterium prausnitzii and Roseburia spp. grown in the presence of 60 mm-acetate and 10 mm-glucose derived 85-90 % butyrate-C from external acetate. This was due to rapid interchange between extracellular acetate and intracellular acetyl-CoA, plus net acetate uptake. In contrast, a Coprococcus-related strain that is a net acetate producer derived only 28 % butyrate-C from external acetate. Different carbohydrate-derived energy sources affected butyrate formation by mixed human faecal bacteria growing in continuous or batch cultures. The ranking order of butyrate production rates was amylopectin > oat xylan > shredded wheat > inulin > pectin (continuous cultures), and inulin > amylopectin > oat xylan > shredded wheat > pectin (batch cultures). The contribution of external acetate to butyrate formation in these experiments ranged from 56 (pectin) to 90 % (xylan) in continuous cultures, and from 72 to 91 % in the batch cultures. This is consistent with a major role for bacteria related to F. prausnitzii and Roseburia spp. in butyrate formation from a range of substrates that are fermented in the large intestine. Variations in the dominant metabolic type of butyrate producer between individuals or with variations in diet are not ruled out, however, and could influence butyrate supply in the large intestine.

Acetates↗

Isoflavones and functional foods alter the dominant intestinal microbiota in postmenopausal women.

Dietary phytoestrogens, such as isoflavones, are used as food additives to prevent menopause-related disorders. In addition to other factors, their bioavailability strongly depends on the activity of intestinal bacteria but the underlying interactions remain poorly understood. A randomized, double-blind, placebo-controlled study was undertaken with 39 postmenopausal women to characterize changes in the dominant microbial communities of the intestinal tract after 2 mo of isoflavone supplementation with and without pro- or prebiotic. The diversity and composition of the dominant microbiota were analyzed by temporal temperature-gradient gel electrophoresis (TTGE) and fluorescent in situ hybridization. Isoflavones alone stimulated dominant microorganisms of the Clostridium coccoides-Eubacterium rectale cluster, Lactobacillus-Enterococcus group, Faecalibacterium prausnitzii subgroup, and Bifidobacterium genus. The stimulation of the Clostridium coccoides-Eubacterium rectale cluster depended on the women's equol excretion and was transient, with the exception of a prolonged bifidogenic effect. Lasting changes in the diversity of the dominant species were also observed. The probiotic strain supplied could be detected by TTGE during its passage through the intestinal tract, and ingestion of fructooligosaccharides triggered a marked and specific bifidogenic effect. In conclusion, this is the first human study that shows changes in the diversity and composition of dominant bacterial communities in response to dietary supplementation with hormone-related compounds combined with functional foods.

Aged↗

Microbial community composition of the ileum and cecum of broiler chickens as revealed by molecular and culture-based techniques.

The microbial communities of the ileum and cecum of broiler chickens from a conventional and an organic farm were investigated using conventional culture techniques as well as cloning and sequencing of 16S rRNA genes. Eighty-five percent of the 557 cloned sequences were <97% related to known cultured species. The chicken ileum was dominated by lactobacilli, whereas the cecum harbored a more diverse microbial community. The cecum was dominated by a large group of bacteria with hitherto no close cultured relatives but most closely related to Faecalibacterium prausnitzii. Approximately 49 and 20% of the cecal clones belonged to this cluster in conventional and organic broiler chickens, respectively. We were, however, able to recover a number of these phylotypes by cultivation, and the isolates were shown to be butyric acid producers. The investigation was a descriptive rather than a comparative study of 2 different rearing systems; however, several differences were observed. For instance, Clostridium perfringens was found in significantly higher numbers in the birds from the organic farm compared with the conventional broilers, probably due to the addition of salinomycin to the conventional feed. In the ileum, the abundance of the different Lactobacillus species differed between the 2 broiler types. The culture-based and culture-independent techniques complemented each other well. Strengths and limitations of the different methods are discussed.

Animal Feed↗

Design and validation of 16S rRNA probes to enumerate members of the Clostridium leptum subgroup in human faecal microbiota.

Among human faecal bacteria, many members of the Clostridium leptum subgroup are fibrolytic and butyrate producing microorganisms thereby contributing to processes important to colonic health. Yet this phylogenetic subgroup remains poorly described to date. To improve detection and description of members of the C. leptum subgroup, the Clep 866 group probe was developed. Its association with probes targeting the Clostridium viride cluster (Cvir 1414) and Eubacterium desmolans species (Edes 635) allowed for the first time the detection of all members found in this phylogenetic group in human faecal microbiota. A species-specific probe was also designed to detect members of the Ruminococcus callidus species (Rcal 733). The design of signature regions was based on alignment of 16S rRNA sequences isolated from faeces of five healthy adults. Furthermore, an oligonucleotide competitor strategy was developed in order to improve the specificity of the probes formerly validated or designed in this study. The oligonucleotide probes were tested using a collection of target and non-target strains using FISH combined with flow cytometry. These new probes were added to a panel of 18 phylogenetic probes selected to describe faecal microbiota composition in 21 human faeces of healthy adults. Clostridium leptum subgroup represented 22% of the total faecal bacteria and codominated with members of Clostridium coccoides group. The cluster Faecalibacterium prausnitzii was the dominant component of the C. leptum subgroup and 20% of the latter subgroup remained unidentified at the species level.

Adult↗

Studies on the effect of system retention time on bacterial populations colonizing a three-stage continuous culture model of the human large gut using FISH techniques.

Fluorescence in situ hybridization was used to quantitate bacteria growing in a three-stage continuous culture system inoculated with human faeces, operated at two system retention times (60 and 20 h). Twenty-three different 16S rRNA gene oligonucleotide probes of varying specificities were used to detect bacteria. Organisms belonging to genera Bacteroides and Bifidobacterium, together with the Eubacterium rectale/Clostridium coccoides group, the Atopobium, Faecalibacterium prausnitzii and Eubacterium cylindroides groups, as well as the segmented filamentous bacteria, the Roseburia intestinalis group and lactic acid bacteria, were all present in high numbers in the continuous culture system. Other groups and species such as Ruminococci and Enterobacteria also persisted in the model, though not always at levels that allowed reliable quantitation. Some organisms such as Streptococci and Corynebacteria, present in the faecal inoculum, did not colonize the system. Other probes specific for Eubacterium lentum and for members of the genus Desulfovibrio did not detect these organisms at any time. Short chain fatty acid production was always highest in vessel I of the continuous culture system, however, a marked increase in acetate formation and a reduction in butyrate production occurred when system retention time was reduced to 20 h, which correlated with reductions in the numbers of butyrate-producing Roseburia.

Acetates↗

16S rRNA gene-based analysis of mucosa-associated bacterial community and phylogeny in the chicken gastrointestinal tracts: from crops to ceca.

Mucosa-associated microbiota from different regions of the gastrointestinal (GI) tract of adult broilers was studied by analysis of 16S rRNA gene sequences. The microbiota mainly comprised Gram-positive bacteria along the GI tract. Fifty-one operational taxonomic units (OTUs) (from 98 clones) were detected in the ceca, as compared with 13 OTUs (from 49 clones) in the crops, 11 OTUs (from 51 clones) in the gizzard, 14 OTUs (from 52 clones) in the duodenum, 12 OTUs (from 50 clones) in the jejunum and nine OTUs (from 50 clones) in the ileum. Ceca were dominantly occupied by clostridia-related sequences (40%) with other abundant sequences being related to Faecalibacterium prausnitzii (14%), Escherichia coli (11%), lactobacilli (7%) and Ruminococcus (6%). Lactobacilli were predominant in the upper GI tract and had the highest diversity in the crop. Both Lactobacillus aviarius and Lactobacillus salivarius were the predominant species among lactobacilli. Candidatus division Arthromitus was also abundant in the jejunum and ileum.

Animals↗