Evelyne Forano

Microbial degradation of complex carbohydrates in the gut

Bacteria that colonize the mammalian intestine collectively possess a far larger repertoire of degradative enzymes and metabolic capabilities than their hosts. Microbial fermentation of complex non-digestible dietary carbohydrates and host–derived glycans in the human intestine has important consequences for health. Certain dominant species, notably among the Bacteroidetes, are known to possess very large numbers of genes that encode carbohydrate active enzymes and can switch readily between different energy sources in the gut depending on availability. Nevertheless, more nutritionally specialized bacteria appear to play critical roles in the community by initiating the degradation of complex substrates such as plant cell walls, starch particles and mucin. Examples are emerging from the Firmicutes, Actinobacteria and Verrucomicrobium phyla, but more information is needed on these little studied groups. The impact of dietary carbohydrates, including prebiotics, on human health requires understanding of the complex relationship between diet composition, the gut microbiota and metabolic outputs.

Quantification by real-time PCR of cellulolytic bacteria in the rumen of sheep after supplementation of a forage diet with readily fermentable carbohydrates: effect of a yeast additive

Aim:  To examine the effect of concentrate and yeast additive on the number of cellulolytic bacteria in the rumen of sheep.

Long-term defaunation increases the abundance of cellulolytic ruminococci and methanogens but does not affect the bacterial and methanogen diversity in the rumen of sheep.

Protozoa are commensal eukaryotes in the rumen of herbivores. Protozoa are large producers of hydrogen, which is utilized by methanogenic archaea to produce methane, a greenhouse gas. The removal of protozoa from the rumen (defaunation) decreases methanogenesis, but also negatively affects fiber digestion, which is the main function of the rumen. The aim of this study was to examine the effect of long-term defaunation on the structure of the microbiota and particularly methanogenic archaea and fibrolytic bacteria to better understand the microbial mechanisms responsible for the decrease in methanogenesis and fibrolysis. The trial was conducted in 5 adult sheep subjected successively to long-term defaunation (2 yr), refaunation (12 wk), and short-term defaunation (10 wk). Methanogens were enumerated by quantitative PCR targeting the rrs (16S ribosomal RNA subunit) and mcrA (methyl coenzyme-M reductase) genes. The rrs gene was used to quantify the 3 major culturable rumen cellulolytic bacterial species (i.e., Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens) and total bacteria. Bacterial and methanogen diversity was also examined by PCR-DGGE (PCR-denaturing gradient gel electrophoresis) analysis targeting the rrs and mcrA genes, respectively. Total rumen bacterial density estimated as rrs copies per gram of DM of rumen content increased in response to long- and short-term defaunation (+1 log, P < 0.001), but without noticeable shifts in diversity. Defaunation increased the rrs copies per gram of DM of rumen content of R. albus and R. flavefaciens (+2 log, P < 0 0.001), but did not affect that of F. succinogenes. Despite a 20% reduction in methane emission in the 2 defaunated periods, the mcrA and rrs copies of methanogens per gram of DM of rumen content increased (+1 log, P < 0.001) in the absence of protozoa, whereas the diversity of the dominant methanogenic community was not modified. This study shows no major difference between long- and short-term defaunation in abundance and diversity of bacteria and archaea. It also provides evidence that monitoring the abundance and diversity of methanogens is not sufficient to comprehend the microbial mechanisms leading to a reduction in methane emissions by ruminants. This study also reports for the first time in sheep a selective effect of defaunation on the abundance of cellulolytic bacterial species.

Fiber-degrading systems of different strains of the genus Fibrobacter

ABSTRACT The S85 type strain of Fibrobacter succinogenes, a major ruminal fibrolytic species, was isolated 49 years ago from a bovine rumen and has been used since then as a model for extensive studies. To assess the validity of this model, we compared the cellulase- and xylanase-degrading activities of several other F. succinogenes strains originating from different ruminants, including recently isolated strains, and looked for the presence of 10 glycoside hydrolase genes previously identified in S85. The NR9 F. intestinalis type strain, representative of the second species of the genus, was also included in this study. DNA-DNA hybridization and 16S rRNA gene sequencing first classified the strains and provided the phylogenetic positions of isolates of both species. Cellulase and xylanase activity analyses revealed similar activity profiles for all F. succinogenes strains. However, the FE strain, phylogenetically close to S85, presented a poor xylanolytic system and weak specific activities. Furthermore, the HM2 strain, genetically distant from the other F. succinogenes isolates, displayed a larger cellulolytic profile on zymograms and higher cellulolytic specific activity. F. intestinalis NR9 presented a higher cellulolytic specific activity and a stronger extracellular xylanolytic activity. Almost all glycoside hydrolase genes studied were found in the F. succinogenes isolates by PCR, except in the HM2 strain, and few of them were detected in F. intestinalis NR9. As expected, the fibrolytic genes of strains of the genus Fibrobacter as well as the cellulase and xylanase activities are better conserved in closely related phylogenetic isolates.

Degradation of Wheat Straw by Fibrobacter succinogenes S85: a Liquid- and Solid-State Nuclear Magnetic Resonance Study

ABSTRACT Wheat straw degradation by Fibrobacter succinogenes was monitored by nuclear magnetic resonance (NMR) spectroscopy and chemolytic methods to investigate the activity of an entire fibrolytic system on an intact complex substrate. In situ solid-state NMR with 13C cross-polarization magic angle spinning was used to monitor the modification of the composition and structure of lignocellulosic fibers (of 13C-enriched wheat straw) during the growth of bacteria on this substrate. There was no preferential degradation either of amorphous regions of cellulose versus crystalline regions or of cellulose versus hemicelluloses in wheat straw. This suggests either a simultaneous degradation of the amorphous and crystalline parts of cellulose and of cellulose and hemicelluloses by the enzymes or degradation at the surface at a molecular scale that cannot be detected by NMR. Liquid-state two-dimensional NMR experiments and chemolytic methods were used to analyze in detail the various sugars released into the culture medium. An integration of NMR signals enabled the quantification of oligosaccharides produced from wheat straw at various times of culture and showed the sequential activities of some of the fibrolytic enzymes of F. succinogenes S85 on wheat straw. In particular, acetylxylan esterase appeared to be more active than arabinofuranosidase, which was more active than α-glucuronidase. Finally, cellodextrins did not accumulate to a great extent in the culture medium.

Dietary fibre degradation and fermentation by two xylanolytic bacteria Bacteroides xylanisolvens XB1AT and Roseburia intestinalis XB6B4 from the human intestine

AimsZ:  To characterize fibre degradation, colonization and fermentation, and xylanase activity of two xylanolytic bacteria Bacteroides xylanisolvens XB1AT and Roseburia intestinalis XB6B4 from the human colon.

Carbohydrate utilization by enterohaemorrhagic Escherichia coli O157:H7 in bovine intestinal content

The bovine gastrointestinal (GI) tract is the main reservoir for enterohaemorrhagic Escherichia coli (EHEC) responsible for food-borne infections. Characterization of nutrients preferentially used by EHEC in the bovine intestine would help to develop ecological strategies to reduce EHEC carriage. However, the carbon sources that support the growth of EHEC in the bovine intestine are poorly documented. In this study, a very low concentration of glucose, the most abundant monomer included in the cattle dietary polysaccharides, was detected in bovine small intestine contents (BSIC) collected from healthy cows at the slaughterhouse. Six carbohydrates reported to be included in the mucus layer covering the enterocytes [galactose, N-acetyl-glucosamine (GlcNAc), N-acetyl- galactosamine (GalNAc), fucose, mannose and N-acetyl neuraminic acid (Neu5Ac)] have been quantified for the first time in BSIC and accounted for a total concentration of 4.2 mM carbohydrates. The genes required for enzymatic degradation of the six mucus-derived carbohydrates are highly expressed during the exponential growth of the EHEC strain O157:H7 EDL933 in BSIC and are more strongly induced in EHEC than in bovine commensal E. coli. In addition, EDL933 consumed the free monosaccharides present in the BSIC more rapidly than the resident microbiota and commensal E. coli, indicating a competitive ability of EHEC to catabolize mucus-derived carbohydrates in the bovine gut. Mutations of EDL933 genes required for the catabolism of each of these sugars have been constructed, and growth competitions of the mutants with the wild-type strain clearly demonstrated that mannose, GlcNAc, Neu5Ac and galactose catabolism confers a high competitive growth advantage to EHEC in BSIC and probably represents an ecological niche for EHEC strains in the bovine small intestine. The utilization of these mucus-derived monosaccharides by EDL933 is apparently required for rapid growth of EHEC in BSIC, and for maintaining a competitive growth rate as compared with that of commensal E. coli. The results suggest a strategy for O157:H7 E. coli survival in the bovine intestine, whereby EHEC rapidly consumes mucus-derived carbohydrates that are poorly consumed by bacteria belonging to the resident intestinal microbiota, including commensal E. coli.

Characterization of XYN10B, a modular xylanase from the ruminal protozoan Polyplastron multivesiculatum, with a family 22 carbohydrate-binding module that binds to cellulose

A new xylanase gene, xyn10B, was isolated from the ruminal protozoan Polyplastron multivesiculatum and the gene product was characterized. XYN10B is the first protozoan family 10 glycoside hydrolase characterized so far and is a modular enzyme comprising a family 22 carbohydrate-binding module (CBM) preceding the catalytic domain. The CBM22 was shown to be a true CBM. It showed high affinity for soluble arabinoxylan and is the first example of a CBM22 that binds strongly to celluloses of various crystallinities. The enzymic properties of XYN10B were also analysed. Its optimal temperature and pH for activity were 39 degrees C and 7.0 respectively; these values being close to those of the ruminal ecosystem. The phylogenetic relationships between the XYN10B CBM22 or catalytic domain and related sequences from ruminal and non-ruminal bacteria and eukaryotes are reported. The xyn10B gene is shown to lack introns.

Effect of camelina oil or live yeasts (Saccharomyces cerevisiae) on ruminal methane production, rumen fermentation, and milk fatty acid composition in lactating cows fed grass silage diets

The potential of dietary supplements of 2 live yeast strains (Saccharomyces cerevisiae) or camelina oil to lower ruminal methane (CH4) and carbon dioxide (CO2) production and the associated effects on animal performance, rumen fermentation, rumen microbial populations, nutrient metabolism, and milk fatty acid (FA) composition of cows fed grass silage-based diets were examined. Four Finnish Ayrshire cows (53±7 d in milk) fitted with rumen cannula were used in a 4×4 Latin square with four 42-d periods. Cows received a basal total mixed ration (control treatment) with a 50:50 forage-to-concentrate ratio [on a dry matter (DM) basis] containing grass silage, the same basal total mixed ration supplemented with 1 of 2 live yeasts, A or B, administered directly in the rumen at 10(10) cfu/d (treatments A and B), or supplements of 60g of camelina oil/kg of diet DM that replaced concentrate ingredients in the basal total mixed ration (treatment CO). Relative to the control, treatments A and B had no effects on DM intake, rumen fermentation, ruminal gas production, or apparent total-tract nutrient digestibility. In contrast, treatment CO lowered DM intake and ruminal CH4 and CO2 production, responses associated with numerical nonsignificant decreases in total-tract organic matter digestibility, but no alterations in rumen fermentation characteristics or changes in the total numbers of rumen bacteria, methanogens, protozoa, and fungi. Compared with the control, treatment CO decreased the yields of milk, milk fat, lactose, and protein. Relative to treatment B, treatment CO improved nitrogen utilization due to a lower crude protein intake. Treatment A had no influence on milk FA composition, whereas treatment B increased cis-9 10:1 and decreased 11-cyclohexyl 11:0 and 24:0 concentrations. Treatment CO decreased milk fat 8:0 to 16:0 and total saturated FA, and increased 18:0, 18:1, 18:2, conjugated linoleic acid, 18:3n-3, and trans FA concentrations. Decreases in ruminal CH4 production to treatment CO were related, at least in part to lowered DM intake, whereas treatments had no effect on ruminal CH4 emission intensity (g/kg of digestible organic matter intake or milk yield). Results indicated that live yeasts A and B had no influence on animal performance, ruminal gas production, rumen fermentation, or nutrient utilization in cows fed grass silage-based diets. Dietary supplements of camelina oil decreased ruminal CH4 and CO2 production, but also lowered the yields of milk and milk constituents due to an adverse effect on intake.

Influence of the Composition of the Cellulolytic Flora on the Development of Hydrogenotrophic Microorganisms, Hydrogen Utilization, and Methane Production in the Rumens of Gnotobiotically Reared Lambs

ABSTRACT We investigated the influence of the composition of the fibrolytic microbial community on the development and activities of hydrogen-utilizing microorganisms in the rumens of gnotobiotically reared lambs. Two groups of lambs were reared. The first group was inoculated with Fibrobacter succinogenes, a non-H2-producing species, as the main cellulolytic organism, and the second group was inoculated with Ruminococcus albus, Ruminococcus flavefaciens, and anaerobic fungi that produce hydrogen. The development of hydrogenotrophic bacterial communities, i.e., acetogens, fumarate and sulfate reducers, was monitored in the absence of methanogens and after inoculation of methanogens. Hydrogen production and utilization and methane production were measured in rumen content samples incubated in vitro in the presence of exogenous hydrogen (supplemented with fumarate or not supplemented with fumarate) or in the presence of ground alfalfa hay as a degradable substrate. Our results show that methane production was clearly reduced when the dominant fibrolytic species was a non-H2-producing species, such as Fibrobacter succinogenes, without significantly impairing fiber degradation and fermentations in the rumen. The addition of fumarate to the rumen contents stimulated H2 utilization only by the ruminal microbiota inoculated with F. succinogenes, suggesting that these communities could play an important role in fumarate reduction in vivo.