Laura Hanske

Decline in Presumptively Protective Gut Bacterial Species and Metabolites Are Paradoxically Associated with Disease Improvement in Pediatric Crohn’s Disease During Enteral Nutrition

Background:The gut microbiota is implicated in the pathogenesis of Crohn’s disease (CD). Exclusive enteral nutrition (EEN) is a successful treatment, but its mode of action remains unknown. This study assessed serial changes in the fecal microbiota milieu during EEN. Methods:Five fecal samples were collected from CD children: 4 during EEN (start, 15, 30, end EEN approximately 60 days) and the fifth on habitual diet. Two samples were collected from healthy control subjects. Fecal pH, bacterial metabolites, global microbial diversity abundance, composition stability, and quantitative changes of total and 7 major bacterial groups previously implicated in CD were measured. Results:Overall, 68 samples were from 15 CD children and 40 from 21 control subjects. Fecal pH and total sulfide increased and butyric acid decreased during EEN (all P < 0.05). Global bacterial diversity abundance decreased (P < 0.05); a higher degree of microbiota composition stability was seen in control subjects than in CD children during EEN (at P ⩽ 0.008). Faecalibacterium prausnitzii spp concentration significantly decreased after 30 days on EEN (P < 0.05). In patients who responded to EEN, the magnitude of the observed changes was greater and the concentration of Bacteroides/Prevotella group decreased (P < 0.05). All these changes reverted to pretreatment levels on free diet, and EEN microbiota diversity increased when the children returned to their free diet. Conclusions:EEN impacts on gut microbiota composition and changes fecal metabolic activity. It is difficult to infer a causative association between such changes and disease improvement, but the results do challenge the current perception of a protective role for F. prausnitzii in CD.

The Bioavailability of Apigenin-7-Glucoside Is Influenced by Human Intestinal Microbiota in Rats

We investigated the impact of human intestinal microbiota on bioavailability of the flavone apigenin-7-glucoside (A7G) by comparing germ-free and human microbiota-associated (HMA) rats. First, the ability of the human intestinal microbiota to convert A7G was proven in vitro by incubating A7G with fecal suspensions. Apigenin, naringenin, and 3-(4-hydroxyphenyl)propionic acid were formed as main metabolites. After application of A7G to germ-free rats, apigenin, luteolin, and their conjugates were detected in urine and feces. In HMA rats, naringenin, eriodictyol, phloretin, 3-(3,4-dihydroxyphenyl)propionic acid, 3-(4-hydroxyphenyl)propionic acid, 3-(3-hydroxyphenyl)propionic acid, and 4-hydroxycinnamic acid in their free and conjugated forms were additionally formed. In whole-blood samples from germ-free and HMA rats, only apigenin conjugates and phloretin, respectively, were detected. The total excretion of A7G and its metabolites within 48 h was similarly low in both germ-free and HMA rats, with 11 and 13% of the A7G dose, respectively. In germ-free rats, A7G metabolites dominated by apigenin and its conjugates were mainly excreted with feces. In contrast, the compounds in HMA rats were predominantly recovered from urine, 3-(4-hydroxyphenyl)propionic acid being the main metabolite. The ability of selected gut bacteria and the host intestinal mucosa to deglycosylate A7G was tested using cell extracts. Apigenin was formed by cytosolic extracts of Eubacterium ramulus and Bacteroides distasonis and by the microsomal fraction of the small intestinal mucosa of rats. Overall, human intestinal microbiota largely contributed to A7G metabolism, indicating its influence on the bioactivity of flavones.

Recovery and metabolism of xanthohumol in germ-free and human microbiota-associated rats

The impact of human intestinal bacteria on the bioavailability of the prenylflavonoid xanthohumol (XN) was studied by comparing germ-free (GF) and human microbiota-associated (HMA) rats. After XN application, XN, XN conjugates, and isoxanthohumol (IX) conjugates occurred in blood samples of GF and HMA rats, whereas IX was detected only in the blood of HMA rats. Overall excretion of XN and its metabolites within 48 h was only 4.6% of the ingested dose in GF rats and 4.2% in HMA rats, feces being the major route of excretion. While both GF and HMA rats excreted XN, IX, and their conjugates with urine and feces, 8-prenylnaringenin and its corresponding conjugates were exclusively observed in the feces of HMA rats. The microbial formation of 8-prenylnaringenin was confirmed by incubation of XN and IX with human fecal slurries. The amount of conjugates excreted in urine and feces was lower in HMA rats compared to GF rats indicating their hydrolysis by human intestinal microbiota. Thus, the impact of bacteria on the XN metabolism in the gut may affect the in vivo effects of ingested XN.

Intestinimonas butyriciproducens gen. nov., sp. nov., a butyrate-producing bacterium from the mouse intestine.

A Gram-positive, spore-forming, non-motile, strictly anaerobic rod-shaped bacterium was isolated from the caecal content of a TNF(deltaARE) mouse. The isolate, referred to as strain SRB-521-5-I(T), was originally cultured on a reduced agar medium containing yeast extract, rumen fluid and lactic acid as main energy and carbon sources. Phylogenetic analysis of partial 16S rRNA genes revealed that the species most closely related to strain SRB-521-5-I(T) were Flavonifractor plautii and Pseudoflavonifractor capillosus (<95 % sequence similarity; 1436 bp). In contrast to F. plautii and P. capillosus, strain SRB-521-5-I(T) contained a substantial amount of C18 : 0 dimethylacetal. Additional major fatty acids were C14 : 0 methyl ester, C16 : 0 dimethylacetal and C18 : 0 aldehyde. Strain SRB-521-5-I(T) differed in its enzyme profile from F. plautii and P. capillosus by being positive for dextrin, maltotriose, turanose, dl-lactic acid and d-lactic acid methyl ester but negative for d-fructose. In reduced Wilkins-Chalgren-Anaerobe broth, strain SRB-521-5-I(T) produced approximately 8 mM butyrate and 4 mM acetate. In contrast to F. plautii, the strain did not metabolize flavonoids. It showed intermediate resistance towards the antibiotics ciprofloxacin, colistin and tetracycline. Based on genotypic and phenotypic characteristics, we propose the name Intestinimonas butyriciproducens gen. nov., sp. nov. to accommodate strain SRB-521-5-I(T) ( = DSM 26588(T) = CCUG 63529(T)) as the type strain.

Contribution of gut bacteria to the metabolism of cyanidin 3-glucoside in human microbiota-associated rats

Cyanidin 3-glucoside (C3G) is one of the major dietary anthocyanins implicated in the prevention of chronic diseases. To evaluate the impact of human intestinal bacteria on the fate of C3G in the host, we studied the metabolism of C3G in human microbiota-associated (HMA) rats in comparison with germ-free (GF) rats. Urine and faeces of the rats were analysed for C3G and its metabolites within 48 h after the application of 92 μmol C3G/kg body weight. In addition, we tested the microbial C3G conversion in vitro by incubating C3G with human faecal slurries and selected human gut bacteria. The HMA rats excreted with faeces a three times higher percentage of unconjugated C3G products and a two times higher percentage of conjugated C3G products than the GF rats. These differences were mainly due to the increased excretion of 3,4-dihydroxybenzoic acid, 2,4,6-trihydroxybenzaldehyde and 2,4,6-trihydroxybenzoic acid. Only the urine of HMA rats contained peonidin and 3-hydroxycinnamic acid and the percentage of conjugated C3G products in the urine was decreased compared with the GF rats. Overall, the presence of intestinal microbiota resulted in a 3·7% recovery of the C3G dose in HMA rats compared with 1·7% in GF rats. Human intestinal bacteria rapidly degraded C3G in vitro. Most of the C3G products were also found in the absence of bacteria, but at considerably lower levels. The higher concentrations of phenolic acids observed in the presence of intestinal bacteria may contribute to the proposed beneficial health effects of C3G.

A derivatization method for the simultaneous detection of glucosinolates and isothiocyanates in biological samples

Various analytical methods have been established to quantify isothiocyanates (ITCs) that derive from glucosinolate hydrolysis. However, to date there is no valid method applicable to pharmacokinetic studies that detects both glucosinolates and ITCs. A specific derivatization procedure was developed for the determination of ITCs based on the formation of a stable N-(tert-butoxycarbonyl)-L-cysteine methyl ester derivative, which can be measured by high-performance liquid chromatography with ultraviolet detection after extraction with ethylacetate. The novel method, which is also applicable to the indirect determination of glucosinolates after their hydrolysis by myrosinase, was established for the simultaneous determination of glucoraphanin and sulforaphane. By derivatization, the sensitivity of ITC detection was increased 2.5-fold. Analytical recoveries from urine and plasma were greater than 75% and from feces were approximately 50%. The method showed intra- and interday variations of less than 11 and 13%, respectively. Applicability of the method was demonstrated in mice that received various doses of glucoraphanin or that were fed a glucoraphanin-rich diet. Besides glucoraphanin and sulforaphane, glucoerucin and erucin were detected in urine and feces of mice. The novel method provides an essential tool for the analysis of bioactive glucosinolates and their hydrolysis products and, thus, will contribute to the elucidation of their bioavailability.

Alleviation of high fat diet‐induced obesity by oligofructose in gnotobiotic mice is independent of presence of Bifidobacterium longum

Scope Diet‐induced obesity is associated with changes in the gut microbiota and low‐grade inflammation. Oligofructose was reported to ameliorate high fat diet‐induced metabolic disorders in mice by restoring the number of intestinal bifidobacteria. However, this has not been experimentally demonstrated. Methods and results We fed conventional mice, germfree mice, mice associated with a simplified human gut microbiota composed of eight bacterial species including Bifidobacterium longum (SIHUMI), and mice associated with SIHUMI without B. longum a low fat diet (LFD), a high fat diet (HFD), or a HFD containing 10% oligofructose (HFD + OFS) for five weeks. We assessed body composition, bacterial cell numbers and metabolites, markers of inflammation, and gut permeability. Conventional mice fed HFD or HFD + OFS did not differ in body weight gain and glucose tolerance. The gnotobiotic mouse groups fed LFD or HFD + OFS gained less body weight and body fat, and displayed an improved glucose tolerance compared with mice fed HFD. These differences were not affected by the presence of B. longum. Mice fed HFD showed no signs of inflammation or increased intestinal permeability. Conclusion The ability of oligofructose to reduce obesity and to improve glucose tolerance in gnotobiotic mice fed HFD was independent of the presence of B. longum.

Increased bacterial putrescine has no impact on gut morphology and physiology in gnotobiotic adolescent mice

Gut bacteria influence host anatomy and physiology. It has been proposed that bacterial metabolites including polyamines are responsible for intestinal maturation and mucosal growth. We have hypothesised that bacterially produced polyamines act as trophic factors and thereby influence large intestinal crypt depth and thickness of the different gut layers. For that purpose, germ-free mice were associated with two different microbial consortia. One group was colonised with a simplified human microbiota (SIHUMI). The second group was associated with SIHUMI + Fusobacterium varium (SIHUMI + Fv), which is known to produce high amounts of polyamines. Polyamine concentrations were measured by HPLC and morphological parameters were determined microscopically. Germ-free and conventional mice served as controls. The caecal putrescine concentration of the SIHUMI + Fv was 61.8 μM (47.6-75.5 μM), whereas that of conventional and SIHUMI mice was 28.8 μM (1.3-41.7 μM) and 24.5 μM (16.8-29.1 μM), respectively. The caecal putrescine concentration of germ-free mice was only 0.6 μM (0-1.0 μM). Caecal crypt depth and thickness of the different caecal layers revealed no significant differences between SIHUMI and SIHUMI + Fv mice. However, the crypt depth in the caeca of conventional, SIHUMI and SIHUMI + Fv mice was increased by 48.6% (P<0.001), 39.7% (P<0.001) and 28.5% (P<0.05), respectively, compared to germ-free mice. These findings indicate that increased intestinal putrescine concentrations do not influence gut morphology in our gnotobiotic adolescent mice.

Glucosinolates Are Mainly Absorbed Intact in Germfree and Human Microbiota-Associated Mice

Chemoprotective or genotoxic effects of glucosinolates occurring in Brassica vegetables are attributed to their hydrolysis products formed upon tissue damage by plant myrosinase. Since Brassica vegetables, in which myrosinase has been heat-inactivated, still display bioactivity, glucosinolate activation has been attributed to intestinal bacteria. The aim of this study was to investigate whether this is true. Glucoraphanin (172 mg/kg body weight) and neoglucobrassicin (297 mg/kg body weight) were administered intragastrically to germ free and human microbiota associated (HMA) mice. Approximately 30% of the applied doses of glucoraphanin and neoglucobrassicin were excreted unchanged in the urine of both germ free and HMA mice. Isothiocyanates, sulforaphane, and erucin, formed from glucoraphanin, were mainly excreted as urinary N-acetyl-l-cysteine conjugates. N-Methoxyindole-3-carbinol formed from neoglucobrassicin was observed in small amounts in both germ free and HMA mice. Formation of DNA adducts from neoglucobrassicin was also independent from bacterial colonization of the mice. Hence, intestinal bacteria are involved in the bioactivation of glucosinolates in the gut, but their contribution to glucosinolate transformation in HMA mice is apparently very small.

Murimonas intestini gen. nov., sp. nov., an acetate-producing bacterium of the family Lachnospiraceae isolated from the mouse gut.

Three strains of an anaerobic, Gram-stain-positive coccobacillus were isolated from the intestines of mice. These strains shared 100 % similarity in their 16S rRNA gene sequences, but were distantly related to any described members of the family Lachnospiraceae (<94 %). The most closely related species with names that have standing in nomenclature were Robinsoniella peoriensis, Ruminococcus gnavus, Blautia producta and Clostridium xylanolyticum. Phylogenetic relationships based on 16S rRNA gene sequence analysis were confirmed by partial sequencing of hsp60 genes. The use of an in-house database search pipeline revealed that the new isolates are most prevalent in bovine gut samples when compared with human and mouse samples for Ruminococcus gnavus and B. producta. All three isolated strains shared similar cellular fatty acid patterns dominated by C16 : 0 methyl ester. Differences in the proportions of C12 : 0 methyl ester, C14 : 0 methyl ester and C18 : 1 cis-11 dimethyl acetal were observed when compared with phylogenetically neighbouring species. The major short-chain fatty acid produced by strain SRB-530-5-H(T) was acetic acid. This strain tested positive for utilization of d-fructose, d-galacturonic acid, d-malic acid, l-alanyl l-threonine and l-glutamic acid but was negative for utilization of amygdalin, arbutin, α-d-glucose, 3-methyl d-glucose and salicin, in contrast to the type strain of the closest related species Robinsoniella peoriensis. The isolates were not able to use mannitol for growth. Based on genotypic, phenotypic and chemotaxonomic characteristics, we propose to create the new genus and species Murimonas intestini gen. nov., sp. nov. to accommodate the three strains SRB-530-5-H(T) ( = DSM 26524(T) = CCUG 63391(T)) (the type strain of Murimonas intestini), SRB-509-4-S-H ( = DSM 27577 = CCUG 64595) and SRB-524-4-S-H ( = DSM 27578 = CCUG 64594).