Alvaro Belenguer

Reduced Dietary Intake of Carbohydrates by Obese Subjects Results in Decreased Concentrations of Butyrate and Butyrate-Producing Bacteria in Feces

ABSTRACT Weight loss diets for humans that are based on a high intake of protein but low intake of fermentable carbohydrate may alter microbial activity and bacterial populations in the large intestine and thus impact on gut health. In this study, 19 healthy, obese (body mass index range, 30 to 42) volunteers were given in succession three different diets: maintenance (M) for 3 days (399 g carbohydrate/day) and then high protein/medium (164 g/day) carbohydrate (HPMC) and high protein/low (24 g/day) carbohydrate (HPLC) each for 4 weeks. Stool samples were collected at the end of each dietary regimen. Total fecal short-chain fatty acids were 114 mM, 74 mM, and 56 mM (P < 0.001) for M, HPMC, and HPLC diets, respectively, and there was a disproportionate reduction in fecal butyrate (18 mM, 9 mM, and 4 mM, respectively; P < 0.001) with decreasing carbohydrate. Major groups of fecal bacteria were monitored using nine 16S rRNA-targeted fluorescence in situ hybridization probes, relative to counts obtained with the broad probe Eub338. No significant change was seen in the relative counts of the bacteroides (Bac303) (mean, 29.6%) or the clostridial cluster XIVa (Erec482, 23.3%), cluster IX (Prop853, 9.3%), or cluster IV (Fprau645, 11.6%; Rbro730 plus Rfla729, 9.3%) groups. In contrast, the Roseburia spp. and Eubacterium rectale subgroup of cluster XIVa (11%, 8%, and 3% for M, HPMC, and HPLC, respectively; P < 0.001) and bifidobacteria (4%, 2.1%, and 1.9%, respectively; P = 0.026) decreased as carbohydrate intake decreased. The abundance of butyrate-producing bacteria related to Roseburia spp. and E. rectale correlated well with the decline in fecal butyrate.

Two Routes of Metabolic Cross-Feeding between Bifidobacterium adolescentis and Butyrate-Producing Anaerobes from the Human Gut

ABSTRACT Dietary carbohydrates have the potential to influence diverse functional groups of bacteria within the human large intestine. Of 12 Bifidobacterium strains of human gut origin from seven species tested, four grew in pure culture on starch and nine on fructo-oligosaccharides. The potential for metabolic cross-feeding between Bifidobacterium adolescentis and lactate-utilizing, butyrate-producing Firmicute bacteria related to Eubacterium hallii and Anaerostipes caccae was investigated in vitro. E. hallii L2-7 and A. caccae L1-92 failed to grow on starch in pure culture, but in coculture with B. adolescentis L2-32 butyrate was formed, indicating cross-feeding of metabolites to the lactate utilizers. Studies with [13C]lactate confirmed carbon flow from lactate, via acetyl coenzyme A, to butyrate both in pure cultures of E. hallii and in cocultures with B. adolescentis. Similar results were obtained in cocultures involving B. adolescentis DSM 20083 with fructo-oligosaccharides as the substrate. Butyrate formation was also stimulated, however, in cocultures of B. adolescentis L2-32 grown on starch or fructo-oligosaccharides with Roseburia sp. strain A2-183, which produces butyrate but does not utilize lactate. This is probably a consequence of the release by B. adolescentis of oligosaccharides that are available to Roseburia sp. strain A2-183. We conclude that two distinct mechanisms of metabolic cross-feeding between B. adolescentis and butyrate-forming bacteria may operate in gut ecosystems, one due to consumption of fermentation end products (lactate and acetate) and the other due to cross-feeding of partial breakdown products from complex substrates.

Phylogenetic distribution of three pathways for propionate production within the human gut microbiota

Propionate is produced in the human large intestine by microbial fermentation and may help maintain human health. We have examined the distribution of three different pathways used by bacteria for propionate formation using genomic and metagenomic analysis of the human gut microbiota and by designing degenerate primer sets for the detection of diagnostic genes for these pathways. Degenerate primers for the acrylate pathway (detecting the lcdA gene, encoding lactoyl-CoA dehydratase) together with metagenomic mining revealed that this pathway is restricted to only a few human colonic species within the Lachnospiraceae and Negativicutes. The operation of this pathway for lactate utilisation in Coprococcus catus (Lachnospiraceae) was confirmed using stable isotope labelling. The propanediol pathway that processes deoxy sugars such as fucose and rhamnose was more abundant within the Lachnospiraceae (based on the pduP gene, which encodes propionaldehyde dehydrogenase), occurring in relatives of Ruminococcus obeum and in Roseburia inulinivorans. The dominant source of propionate from hexose sugars, however, was concluded to be the succinate pathway, as indicated by the widespread distribution of the mmdA gene that encodes methylmalonyl-CoA decarboxylase in the Bacteroidetes and in many Negativicutes. In general, the capacity to produce propionate or butyrate from hexose sugars resided in different species, although two species of Lachnospiraceae (C. catus and R. inulinivorans) are now known to be able to switch from butyrate to propionate production on different substrates. A better understanding of the microbial ecology of short-chain fatty acid formation may allow modulation of propionate formation by the human gut microbiota.

Impact of pH on Lactate Formation and Utilization by Human Fecal Microbial Communities

ABSTRACT The human intestine harbors both lactate-producing and lactate-utilizing bacteria. Lactate is normally present at <3 mmol liter−1 in stool samples from healthy adults, but concentrations up to 100 mmol liter−1 have been reported in gut disorders such as ulcerative colitis. The effect of different initial pH values (5.2, 5.9, and 6.4) upon lactate metabolism was studied with fecal inocula from healthy volunteers, in incubations performed with the addition of dl-lactate, a mixture of polysaccharides (mainly starch), or both. Propionate and butyrate formation occurred at pH 6.4; both were curtailed at pH 5.2, while propionate but not butyrate formation was inhibited at pH 5.9. With the polysaccharide mix, lactate accumulation occurred only at pH 5.2, but lactate production, estimated using l-[U-13C]lactate, occurred at all three pH values. Lactate was completely utilized within 24 h at pH 5.9 and 6.4 but not at pH 5.2. At pH 5.9, more butyrate than propionate was formed from l-[U-13C]lactate in the presence of polysaccharides, but propionate, formed mostly by the acrylate pathway, was the predominant product with lactate alone. Fluorescent in situ hybridization demonstrated that populations of Bifidobacterium spp., major lactate producers, increased approximately 10-fold in incubations with polysaccharides. Populations of Eubacterium hallii, a lactate-utilizing butyrate-producing bacterium, increased 100-fold at pH 5.9 and 6.4. These experiments suggest that lactate is rapidly converted to acetate, butyrate, and propionate by the human intestinal microbiota at pH values as low as 5.9, but at pH 5.2 reduced utilization occurs while production is maintained, resulting in lactate accumulation.

Rates of production and utilization of lactate by microbial communities from the human colon

Lactate metabolism was studied in mixed bacterial communities using single-stage continuous flow fermentors inoculated with faecal slurries from four different volunteers and run for 6 days at pH 5.5 and 6.0, using carbohydrates, mainly starch, as substrates. A continuous infusion of [U-(13) C]starch and l-[3-(13) C]lactate was performed on day 5 and a bolus injection of l-[3-(13) C]lactate plus dl-lactate on day 6. Short-chain fatty acids and lactate concentrations plus enrichments and numbers of lactate-producing and -utilizing bacteria on day 5 were measured. Faecal samples were also collected weekly over a 3-month period to inoculate 24-h batch culture incubation at pH 5.9 and 6.5 with carbohydrates alone or with 35 mmol L(-1) lactate. In the fermentors, the potential lactate disposal rates were more than double the formation rates, and lactate concentrations usually remained below detection. Lactate formation was greater (P<0.05) at the lower pH, with a similar tendency for utilization. Up to 20% of butyrate production was derived from lactate. In batch cultures, lactate was also efficiently used at both pH values, especially at 6.5, although volunteer and temporal variability existed. Under healthy gut environmental conditions, bacterial lactate disposal seems to exceed production markedly.

Protein recycling in growing rabbits: contribution of microbial lysine to amino acid metabolism

To study the absorption of microbial lysine in growing rabbits, a labelled diet (supplemented with (15)NH4Cl) was administered to six animals (group ISOT); a control group (CTRL, four rabbits) received a similar, but unlabelled, diet. Diets were administered for 30 d. An additional group of six animals were fed the unlabelled diet for 20 d and then the labelled diet for 10 d while wearing a neck collar to avoid caecotrophy (group COLL), in order to discriminate it from direct intestinal absorption. At day 30 animals were slaughtered and caecal bacteria and liver samples taken. The (15)N enrichment in amino acids of caecal bacteria and liver were determined by GC-combustion/isotope ratio MS. Lysine showed a higher enrichment in caecal microflora (0.925 atom% excess, APE) than liver (0.215 APE) in group ISOT animals, confirming the double origin of body lysine: microbial and dietary. The COLL group showed a much lower enrichment in tissue lysine (0.007 (se 0.0029) APE for liver). Any enrichment in the latter animals was due to direct absorption of microbial lysine along the digestive tract, since recycling of microbial protein (caecotrophy) was avoided. In such conditions liver enrichment was low, indicating a small direct intestinal absorption. From the ratio of [(15)N]lysine enrichment between liver and bacteria the contribution of microbes to body lysine was estimated at 23 %, with 97 % of this arising through caecotrophy. Absorption of microbial lysine through caecotrophy was 119 (se 4.0) mg/d, compared with 406 (se 1.8) mg/d available from the diet. This study confirms the importance of caecotrophy in rabbit nutrition (15 % of total protein intake).

Urinary excretion of purine derivatives as an index of microbial protein synthesis in the camel (Camelus dromedarius).

Five experiments were carried out to extend knowledge of purine metabolism in the camel (Camelus dromedarius) and to establish a model to enable microbial protein outflow from the forestomachs to be estimated from the urinary excretion of purine derivatives (PD; i.e. xanthine, hypoxanthine, uric acid, allantoin). In experiment 1, four camels were fasted for five consecutive days to enable endogenous PD excretion in urine to be determined. Total PD excretion decreased during the fasting period to 267 (SE 41.5) micromol/kg body weight (W)0.75 per d. Allantoin and xanthine + hypoxanthine were consistently 86 and 6.1 % of total urinary PD during this period but uric acid increased from 3.6 % to 7.4 %. Xanthine oxidase activity in tissues (experiment 2) was (micromol/min per g fresh tissue) 0.038 in liver and 0.005 in gut mucosa but was not detected in plasma. In experiment 3, the duodenal supply of yeast containing exogenous purines produced a linear increase in urinary PD excretion rate with the slope indicating that 0.63 was excreted in urine. After taking account of endogenous PD excretion, the relationship can be used to predict purine outflow from the rumen. From the latter prediction, and also the purine:protein ratio in bacteria determined in experiment 5, we predicted the net microbial outflow from the rumen. In experiment 4, with increasing food intake, the rate of PD excretion in the urine increased linearly by about 11.1 mmol PD/kg digestible organic matter intake (DOMI), equivalent to 95 g microbial protein/kg DOMI.

Urinary excretion of purine derivatives in Bos indicus × Bos taurus crossbred cattle

Four experiments were performed to study the kinetics of purine metabolism and urinary excretion in Zebu crossbred cattle. Fasting excretion was established in Expt 1, using eighteen male Bos indicus x Bos taurus crossbred cattle (261 (SE 9.1) kg body weight), six of each of the following genotypes: 5/8 Bos indicus, 1/2 Bos indicus and 3/8 Bos indicus. No significant differences were observed among genotypes in fasting purine derivative excretion (277.3 (SE 35.43) micromol/metabolic body weight). In a second experiment we measured the xanthine oxidase activity, which was higher in liver than in duodenal mucosa (0.64 and 0.06 (SE 0.12) units/g wet tissue per min respectively; P>0.05) being in plasma 0.60 (SE 0.36) units/l per min. The kinetics of uric acid were measured by intravenous pulse dose of [1,3-15N]uric acid (Expt 3). The cumulative recovery of the isotope in urine was 82 (SE 6.69) %, and uric acid plasma removal, pool size and mean retention time were 0.284 (SE 0.051) per h, 5.45 (SE 0.823) mmol and 3.52 (SE 0.521) h, respectively. Allantoin was removed from plasma at an estimated fractional rate of 0.273 (SE 0.081) per h and mean retention was 3.66 (SE 1.08) h. In Expt 4, the relationship between urinary purine derivative excretion (Y; mmol/d) and digestible organic matter intake (X, kg/d) was defined by the equation: Y=7.69 (SE 4.2)+5.69 (SE 1.68) X; n 16, Se 1.31, r 0.67.

Quantitative Analysis of Microbial Metabolism in the Human Large Intestine

Microbial metabolism in the human colon impacts on health and disease. Production of intermediate metabolites and end-products depends largely on the supply of dietary carbohydrates, including prebiotics (fructooligosaccharides) and functional foods (resistant starch), that resist small intestinal digestion. Colonic bacteria ferment these substrates to a wide range of products, predominantly short-chain fatty acids, including metabolites that can be either deleterious (e.g. D-lactate, sulphides) or beneficial (e.g. butyrate) to gut health. Lactate accumulation in the colon has been associated with gastrointestinal disturbance, for example in severe ulcerative colitis, whereas in the healthy state lactate is efficiently utilised by gut bacteria. Understanding the interaction between microbial metabolism and dietderived nutrient supply is crucial for maintaining a healthy metabolic balance in the colon. Prediction of such nutritional responses can be achieved by integrating in mathematical models information from stable isotope studies, that quantify metabolite flows, and molecular techniques, that accurately determine changes in microbial composition diversity. From such approaches, better nutritional advice can be provided in order to improve gut health. Furthermore, such understanding can be used to manipulate and improve the action of prebiotics and probiotics.

Contribution of gut microbial lysine to liver and milk amino acids in lactating does

The contribution of microbial amino acids through caecotrophy to tissue protein metabolism was investigated in lactating does. Attempts were made to vary microbial supply through a dietary antibiotic, Zn bacitracin, and to vary tissue demand through manipulation of litter size. Three groups of eight New Zealand does were fed different experimental diets from day 28 of pregnancy to day 26 of lactation. The control group received the basal diet formulated to meet requirements with grass hay, wheat, soybean meal and barley grain. The second (no antibiotic) group and the third (bacitracin; BAC) group ingested the basal diet supplemented with ammonium sulfate (5 g/kg), initially unlabelled (day 1 to day 8) then labelled with 15N (day 9 to day 30), while the BAC diet was also supplemented throughout with antibiotic (Zn bacitracin; 100 mg/kg). From just after birth each group of does was subdivided into two groups, each of four females, with the litter size either five (LS5) or nine (LS9) pups. The 15N enrichment in liver, milk and caecal bacteria amino acids was determined by GC-combustion-isotope ratio MS. All amino acids in bacterial protein were enriched with the (15 NH 4)2SO4 treatment, with lysine 15N enrichment significantly greater in caecal bacteria (0.23 (SE 0.0063) atom % excess (ape)) than in liver (0.04 (SE 0.0004) ape) or milk protein (0.05 (SE 0.0018) ape), confirming the double origin (bacterial and dietary) of tissue lysine. The contribution of microbes to tissue lysine was 0.23 (SE 0.006) when milk protein was used as reference.