The gut microbiome modulates the impact of Anaerobutyricum soehngenii supplementation on glucose homeostasis in mice.

Background There is growing interest in the development of next-generation probiotics to prevent or treat metabolic syndrome. Previous studies suggested that Anaerobutyricum soehngenii may represent a promising probiotic candidate. A recent human study showed that while A . soehngenii supplementation is well tolerated and safe, it resulted in variable responses among individuals with a subset of the subjects significantly benefiting from the treatment. We hypothesized that gut microbiome variation is linked to the heterogeneous responses to A . soehngenii treatment observed in humans. Results We colonized germ-free mice with fecal microbiota from human subjects that responded to A . soehngenii treatment (R65 and R55) and non-responder subjects (N96 and N40). Colonized mice were fed a high-fat diet (45% kcal from fat) to induce insulin resistance, and orally treated with either live A . soehngenii culture or heat-killed culture. We found that R65-colonized mice received a benefit in glycemic control with live A . soehngenii treatment while mice colonized with microbiota from the other donors did not. The glucose homeostasis improvements observed in R65-colonized mice were positively correlated with levels of cecal propionate, an association that was reversed in N40-colonized mice. To test whether the microbiome modulates the effects of propionate, R65- or N40-colonized mice were treated with tripropionin (TP, glycerol tripropionate), a pro-drug of propionate, or glycerol (control). TP supplementation showed a similar response pattern as that observed in live A . soehngenii treatment, suggesting that propionate may mediate the effects of A . soehngenii . We also found that TP supplementation to conventional mice reduces adiposity, improves glycemic control, and reduces plasma insulin compared to control animals supplemented with glycerol. Conclusions These findings highlight the importance of the microbiome on glycemic control and underscore the need to better understand personal microbiome-by-therapeutic interactions to develop more effective treatment strategies.

Comparative genomics and proteomics of Eubacterium maltosivorans: functional identification of trimethylamine methyltransferases and bacterial microcompartments in a human intestinal bacterium with a versatile lifestyle.

Eubacterium maltosivorans YIT is a human intestinal isolate capable of acetogenic, propionogenic and butyrogenic growth. Its 4.3-Mb genome sequence contains coding sequences for 4227 proteins, including 41 different methyltransferases. Comparative proteomics of strain YIT showed the Wood-Ljungdahl pathway proteins to be actively produced during homoacetogenic growth on H2 and CO2 while butyrogenic growth on a mixture of lactate and acetate significantly upregulated the production of proteins encoded by the recently identified lctABCDEF cluster and accessory proteins. Growth on H2 and CO2 unexpectedly induced the production of two related trimethylamine methyltransferases. Moreover, a set of 16 different trimethylamine methyltransferases together with proteins for bacterial microcompartments were produced during growth and deamination of the quaternary amines, betaine, carnitine and choline. Growth of strain YIT on 1,2-propanediol generated propionate with propanol and induced the formation of bacterial microcompartments that were also prominently visible in betaine-grown cells. The present study demonstrates that E. maltosivorans is highly versatile in converting low-energy fermentation end-products in the human gut into butyrate and propionate whilst being capable of preventing the formation of the undesired trimethylamine by converting betaine and other quaternary amines in bacterial microcompartments into acetate and butyrate.

Conversion of dietary inositol into propionate and acetate by commensal Anaerostipes associates with host health.

We describe the anaerobic conversion of inositol stereoisomers to propionate and acetate by the abundant intestinal genus Anaerostipes. A inositol pathway was elucidated by nuclear magnetic resonance using [13C]-inositols, mass spectrometry and proteogenomic analyses in A. rhamnosivorans, identifying 3-oxoacid CoA transferase as a key enzyme involved in both 3-oxopropionyl-CoA and propionate formation. This pathway also allowed conversion of phytate-derived inositol into propionate as shown with [13C]-phytate in fecal samples amended with A. rhamnosivorans. Metabolic and (meta)genomic analyses explained the adaptation of Anaerostipes spp. to inositol-containing substrates and identified a propionate-production gene cluster to be inversely associated with metabolic biomarkers in (pre)diabetes cohorts. Co-administration of myo-inositol with live A. rhamnosivorans in western-diet fed mice reduced fasting-glucose levels comparing to heat-killed A. rhamnosivorans after 6-weeks treatment. Altogether, these data suggest a potential beneficial role for intestinal Anaerostipes spp. in promoting host health.

Genomic diversity and ecology of human-associated Akkermansia species in the gut microbiome revealed by extensive metagenomic assembly.

BACKGROUND: Akkermansia muciniphila is a human gut microbe with a key role in the physiology of the intestinal mucus layer and reported associations with decreased body mass and increased gut barrier function and health. Despite its biomedical relevance, the genomic diversity of A. muciniphila remains understudied and that of closely related species, except for A. glycaniphila, unexplored. RESULTS: We present a large-scale population genomics analysis of the Akkermansia genus using 188 isolate genomes and 2226 genomes assembled from 18,600 metagenomes from humans and other animals. While we do not detect A. glycaniphila, the Akkermansia strains in the human gut can be grouped into five distinct candidate species, including A. muciniphila, that show remarkable whole-genome divergence despite surprisingly similar 16S rRNA gene sequences. These candidate species are likely human-specific, as they are detected in mice and non-human primates almost exclusively when kept in captivity. In humans, Akkermansia candidate species display ecological co-exclusion, diversified functional capabilities, and distinct patterns of associations with host body mass. Analysis of CRISPR-Cas loci reveals new variants and spacers targeting newly discovered putative bacteriophages. Remarkably, we observe an increased relative abundance of Akkermansia when cognate predicted bacteriophages are present, suggesting ecological interactions. A. muciniphila further exhibits subspecies-level genetic stratification with associated functional differences such as a putative exo/lipopolysaccharide operon. CONCLUSIONS: We uncover a large phylogenetic and functional diversity of the Akkermansia genus in humans. This variability should be considered in the ongoing experimental and metagenomic efforts to characterize the health-associated properties of A. muciniphila and related bacteria.

Next-generation therapeutic bacteria for treatment of obesity, diabetes, and other endocrine diseases.

The human gut microbiota has appeared as an important factor affecting host health and intestinal bacteria have recently emerged as potential therapeutics to treat diabetes and other endocrine diseases. These mainly anaerobic bacteria have been identified either via comparative "omics" analysis of the intestinal microbiota in healthy and diseased subjects or of data collected by fecal microbiota transplantation studies. Both approaches require advanced and in-depth sequencing technologies to perform massive genomic screening to select bacteria with potential benefits. It has been shown that these potentially therapeutic bacteria can either produce bioactive products that directly influence the host patho-physiology and endocrine systems or produce specific signaling molecules that may do so. These bioactive compounds can be formed via degradation of dietary or host-derived components or the conversion of intermediate compounds produced by fermentation of intestinal bacteria. Several of these bacteria have shown causality in preclinical models and entered clinical phase studies, while their mode of action is being analyzed. In this review, we summarize the research on the most promising bacterial candidates with therapeutic properties with a specific focus on diabetes.

Mutual Metabolic Interactions in Co-cultures of the Intestinal Anaerostipes rhamnosivorans With an Acetogen, Methanogen, or Pectin-Degrader Affecting Butyrate Production.

The human intestinal tract harbors diverse and complex microbial communities that have a vast metabolic capacity including the breakdown of complex carbohydrates into short chain fatty acids, acetate, propionate, and butyrate. As butyrate is beneficial for gut health there is much attention on butyrogenic bacteria and their role in the colonic anaerobic food chain. However, our understanding how production of butyrate by gut microorganisms is controlled by interactions between different species and environmental nutrient availability is very limited. To address this, we set up experimental in vitro co-culture systems to study the metabolic interactions of Anaerostipes rhamnosivorans, a butyrate producer with each of its partners; Blautia hydrogenotrophica, an acetogen; Methanobrevibacter smithii, a methanogen and Bacteroides thetaiotaomicron, a versatile degrader of plant cell wall pectins; through corresponding specific cross-feeding. In all co-cultures, A. rhamnosivorans was able to benefit from its partner for enhanced butyrate formation compared to monocultures. Interspecies transfer of hydrogen or formate from A. rhamnosivorans to the acetogen B. hydrogenotrophica and in turn of acetate from the acetogen to the butyrogen were essential for butyrate formation. A. rhamnosivorans grown on glucose supported growth of M. smithii via interspecies formate/hydrogen transfer enhancing butyrate formation. In the co-culture with pectin, lactate was released by B. thetaiotaomicron which was concomitantly used by A. rhamnosivorans for the production of butyrate. Our findings indicate enhanced butyrate formation through microbe-microbe interactions between A. rhamnosivorans and an acetogen, a methanogen or a pectin-degrader. Such microbial interactions enhancing butyrate formation may be beneficial for colonic health.

Anaerobic Degradation of N-ε-Carboxymethyllysine, a Major Glycation End-Product, by Human Intestinal Bacteria.

Modifications of lysine contribute to the amount of dietary advanced glycation end-products reaching the colon. However, little is known about the ability of intestinal bacteria to metabolize dietary N-ε-carboxymethyllysine (CML). Successive transfers of fecal microbiota in growth media containing CML were used to identify and isolate species able to metabolize CML under anaerobic conditions. From our study, only donors exposed to processed foods degraded CML, and anaerobic bacteria enrichments from two of them used 77 and 100% of CML. Oscillibacter and Cloacibacillus evryensis increased in the two donors after the second transfer, highlighting that the bacteria from these taxa could be candidates for anaerobic CML degradation. A tentative identification of CML metabolites produced by a pure culture of Cloacibacillus evryensis was performed by mass spectrometry: carboxymethylated biogenic amines and carboxylic acids were identified as CML degradation products. The study confirmed the ability of intestinal bacteria to metabolize CML under anoxic conditions.

Reclassification of Eubacterium hallii as Anaerobutyricum hallii gen. nov., comb. nov., and description of Anaerobutyricum soehngenii sp. nov., a butyrate and propionate-producing bacterium from infant faeces.

A bacterial strain designated L2-7T, phylogenetically related to Eubacterium hallii DSM 3353T, was previously isolated from infant faeces. The complete genome of strain L2-7T contains eight copies of the 16S rRNA gene with only 98.0-98.5 % similarity to the 16S rRNA gene of the previously described type strain E. hallii. The next closest validly described species is Anaerostipes hadrus DSM 3319T (90.7 % 16S rRNA gene similarity). A polyphasic taxonomic approach showed strain L2-7T to be a novel species, related to type strain E. hallii DSM 3353T. The experimentally observed DNA-DNA hybridization value between strain L2-7T and E. hallii DSM 3353T was 26.25 %, close to that calculated from the genomes (34.3 %). The G+C content of the chromosomal DNA of strain L2-7T was 38.6 mol%. The major fatty acids were C16 : 0, C16 : 1cis9 and a component with summed feature 10 (C18 : 1c11/t9/t6c). Strain L2-7T had higher amounts of C16 : 0 (30.6 %) compared to E. hallii DSM 3353T (19.5 %) and its membrane contained phosphatidylglycerol and phosphatidylethanolamine, which were not detected in E. hallii DSM 3353T. Furthermore, 16S rRNA gene phylogenetic analysis advocates that E. hallii DSM 3353T is misclassified, and its reclassification as a member of the family Lachnospiraceae is necessary. Using a polyphasic approach, we propose that E. hallii (=DSM 3353T=ATCC 27751T) be reclassified as the type strain of a novel genus Anaerobutyricum sp. nov., comb. nov. and we propose that strain L2-7T should be classified as a novel species, Anaerobutyricum soehngenii sp. nov. The type strain is L2-7T (=DSM 17630T=KCTC 15707T).

Comparative genomics and physiology of the butyrate-producing bacterium Intestinimonas butyriciproducens.

Intestinimonas is a newly described bacterial genus with representative strains present in the intestinal tract of human and other animals. Despite unique metabolic features including the production of butyrate from both sugars and amino acids, there is to date no data on their diversity, ecology, and physiology. Using a comprehensive phylogenetic approach, Intestinimomas was found to include at least three species that colonize primarily the human and mouse intestine. We focused on the most common and cultivable species of the genus, Intestinimonas butyriciproducens, and performed detailed genomic and physiological comparison of strains SRB521T and AF211, isolated from the mouse and human gut respectively. The complete 3.3-Mb genomic sequences of both strains were highly similar with 98.8% average nucleotide identity, testifying to their assignment to one single species. However, thorough analysis revealed significant genomic rearrangements, variations in phage-derived sequences, and the presence of new CRISPR sequences in both strains. Moreover, strain AF211 appeared to be more efficient than strain SRB521T in the conversion of the sugars arabinose and galactose. In conclusion, this study provides genomic and physiological insight into Intestinimonas butyriciproducens, a prevalent butyrate-producing species, differentiating strains that originate from the mouse and human gut.

Production of butyrate from lysine and the Amadori product fructoselysine by a human gut commensal.

Human intestinal bacteria produce butyrate, which has signalling properties and can be used as energy source by enterocytes thus influencing colonic health. However, the pathways and the identity of bacteria involved in this process remain unclear. Here we describe the isolation from the human intestine of Intestinimonas strain AF211, a bacterium that can convert lysine stoichiometrically into butyrate and acetate when grown in a synthetic medium. Intestinimonas AF211 also converts the Amadori product fructoselysine, which is abundantly formed in heated foods via the Maillard reaction, into butyrate. The butyrogenic pathway includes a specific CoA transferase that is overproduced during growth on lysine. Bacteria related to Intestinimonas AF211 as well as the genetic coding capacity for fructoselysine conversion are abundantly present in colonic samples from some healthy human subjects. Our results indicate that protein can serve as a source of butyrate in the human colon, and its conversion by Intestinimonas AF211 and related butyrogens may protect the host from the undesired side effects of Amadori reaction products.

Anaerostipes rhamnosivorans sp. nov., a human intestinal, butyrate-forming bacterium.

A novel butyrate-producing bacterium, strain 1y-2(T), was isolated from a stool sample of a 1-year-old, healthy Dutch infant. The isolate was obtained by using lactate and acetate as sources of carbon and energy. The strain was Gram-variable, strictly anaerobic and spore-forming and formed curly rod-shaped cells that fermented glucose into butyrate, lactate, formate and acetate as main products. The DNA G+C content of the strain was 44.5 mol% and its major cellular fatty acids were C12:0, iso-C19:1 I and C16:0. Strain 1y-2(T) was related to Anaerostipes caccae DSM 14662(T) based on 16S rRNA gene sequence analysis, with 3% divergence, but hybridization studies of their genomic DNA revealed only 33% relatedness. Moreover, strain 1y-2(T) showed marked physiological and biochemical differences from known species of the genus Anaerostipes. Based on phylogenetic, chemotypic and phenotypic criteria, we propose that strain 1y-2(T) should be classified in the genus Anaerostipes within a novel species, Anaerostipes rhamnosivorans sp. nov. The type strain is 1y-2(T) ( = DSM 26241(T) = KCTC 15316(T)).

Lactobacillus koreensis sp. nov., isolated from the traditional Korean food kimchi.

A lactic acid bacterium, strain DCY50(T), isolated from the traditional Korean food kimchi, was studied to determine its taxonomic position. The strain was Gram-stain-positive, catalase-negative, facultatively anaerobic, rod-shaped and motile. The genomic DNA G+C content was 49 mol% and the peptidoglycan structure was of the A4α (l-Lys-d-Asp) type. Chemotaxonomic markers of the strain were consistent with its classification in the genus Lactobacillus. Comparisons of 16S rRNA and rpoA gene sequences showed that strain DCY50(T) was most closely related to the type strains of Lactobacillus parabrevis (98.4 and 91.6 % similarity, respectively, for the 16S rRNA and rpoA genes), L. hammesii (98.0 and 91.2 %), L. brevis (97.6 and 93.3 %) and L. senmaizukei (97.4 and 90.5 %). DNA-DNA relatedness of strain DCY50(T) to these type strains was below 36 %. According to the genotypic and phenotypic data, strain DCY50(T) could be differentiated from all known Lactobacillus species and should be classified in a novel species, for which the name Lactobacillus koreensis sp. nov. is proposed; the type strain is DCY50(T) ( = KCTC 13530(T)  = JCM 16448(T)).

Rhodanobacter soli sp. nov., isolated from soil of a ginseng field.

Strain DCY45(T) was isolated from soil of a ginseng field in Pocheon Province, Korea. Strain DCY45(T) was Gram-negative, oxidase- and catalase-positive, motile and rod-shaped and produced yellow pigments on R2A agar. The organism grew optimally at 30 °C and at pH 7.0. The G+C content of the genomic DNA was 65.4 mol%. The predominant respiratory quinone was Q-8. The major fatty acids were iso-C(17 : 1)ω9c, iso-C(16 : 0) and iso-C(15 : 0). Phylogenetic analysis based on the 16S rRNA gene sequence was used to determine the taxonomic position of strain DCY45(T), which is most closely related to species of the genus Rhodanobacter, with similarity levels of 96.0-98.4 %; DNA-DNA relatedness with related strains was lower than 60 %. Strain DCY45(T) differed significantly from related type strains in phenotypic characteristics. On the basis of these phenotypic, genotypic and chemotaxonomic studies, strain DCY45(T) represents a novel species of the genus Rhodanobacter, for which the name Rhodanobacter soli sp. nov. is proposed. The type strain is DCY45(T) (=KCTC 22620(T) =JCM 16126(T)).

Solibius ginsengiterrae gen. nov., sp. nov., isolated from soil of a ginseng field, and emended description of the genus Sediminibacterium and of Sediminibacterium salmoneium.

A novel Gram-staining-negative bacterial strain, designated DCY13T, was isolated from soil of a ginseng field in Korea and characterized by using a polyphasic approach. The phylogenetic analysis based on 16S rRNA gene sequences revealed that strain DCY13T forms a cluster with members of the genera Sediminibacterium, Flavisolibacter, Niabella, Terrimonas, Niastella and Chitinophaga in the family 'Chitinophagaceae', phylum Bacteroidetes, and shares highest sequence similarity (95.2 %) with Sediminibacterium salmoneum NJ-44T. Sequence similarity with other members of the family is 87.6-91.4 %. Cells are non-spore-forming rods, catalase and oxidase-positive, motile by gliding that grow under strictly aerobic conditions. The predominant respiratory quinone is menaquinone MK-7, and the major fatty acids are anteiso-C15:0, iso C16:0, anteiso-C17:0, and iso-C15:0. The G+C content of the genomic DNA is 47.8 mol%. The major cell wall amino acids are D-aspartic acid, D-glycine, D-serine, L-lysine, D-glucosamine, and D-alanine. The major cell wall sugars are ribose, xylose, and galactose. The major polar lipids are phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. It is proposed that strain DCY13T represents a novel genus and species in the family 'Chitinophagaceae' for which the name Solibius ginsengiterrae gen. nov., sp. nov., is proposed. The type strain is DCY13T (= KCTC 12833T = JCM 15794T). Emended descriptions of the genus Sediminibacterium and of S. salmoneum NJ44T are also proposed.

Microbacterium ginsengiterrae sp. nov., a beta-glucosidase-producing bacterium isolated from soil of a ginseng field.

Strain DCY37(T) was isolated from a soil sample of a ginseng field in the Republic of Korea and characterized in order to determine its taxonomic position. Cells were Gram-staining-positive, heterotrophic, strictly aerobic, non-motile short rods. 16S rRNA gene sequence analysis revealed that strain DCY37(T) belongs to the genus Microbacterium. According to 16S rRNA gene sequence analysis, it is closely related to Microbacterium aerolatum DSM 14217(T) (98.8 %), Microbacterium hydrocarbonoxydans DSM 16089(T) (98.5 %), Microbacterium natoriense JCM 12611(T) (98.5 %), Microbacterium foliorum (98.4 %) and Microbacterium phyllosphaerae (98.3 %). However, DNA-DNA hybridization studies showed reassociation values of less than 70 % between representative strains and DCY37(T). The DNA G+C content was 64.5 mol%. Strain DCY37(T) possessed chemotaxonomic markers that were consistent with classification in the genus Microbacterium, i.e. MK-12 and MK-13 as the major menaquinones and anteiso-C(15 : 0), anteiso-C(17 : 0) and iso-C(16 : 0) as the predominant cellular fatty acids. The major cell wall sugars were ribose, xylose and galactose. The diamino acid in cell-wall hydrolysates of strain DCY37(T) was ornithine and major cell-wall amino acids were alanine, glycine, d-glutamic acid and serine. The major polar lipids were glycolipid, phosphatidylglycerol, diphosphatidylglycerol and unknown aminolipids. Based on these data, DCY37(T) (=KCTC 19526(T) =JCM 15516(T)) should be classified as the type strain of a novel species of the genus Microbacterium, for which the name Microbacterium ginsengiterrae sp. nov. is proposed.

Stenotrophomonas ginsengisoli sp. nov., isolated from a ginseng field.

A Gram-negative, non-spore-forming, rod-shaped bacterium, designated strain DCY01(T), was isolated from soil from a ginseng field in South Korea and was characterized in order to determine its taxonomic position. 16S rRNA gene sequence analysis revealed that strain DCY01(T) belonged to the Gammaproteobacteria and was most closely related to Stenotrophomonas koreensis KCTC 12211(T) (98.4 % similarity), Stenotrophomonas humi R-32729(T) (97.2 %), Stenotrophomonas terrae R-32768 (97.1 %), Stenotrophomonas maltophilia DSM 50170(T) (96.9 %) and Stenotrophomonas nitritireducens DSM 12575(T) (96.8 %). Chemotaxonomic analyses revealed that strain DCY01(T) possessed a quinone system with Q-8 as the predominant compound, and iso-C(15 : 0) (28.2 %), C(16 : 0) 10-methyl (13.2 %), iso-C(15 : 1) F (10.8 %) and C(15 : 0) (7.5 %) as major fatty acids, corroborating assignment of strain DCY01(T) to the genus Stenotrophomonas. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The results of DNA-DNA hybridization and physiological and biochemical tests clearly demonstrated that strain DCY01(T) represents a species distinct from recognized Stenotrophomonas species. Based on these data, DCY01(T) (=KCTC 12539(T)=NBRC 101154(T)) should be classified as the type strain of a novel species of the genus Stenotrophomonas, for which the name Stenotrophomonas ginsengisoli sp. nov. is proposed.