Major Short-chain Fatty Acids Research Articles

Microbial Metabolite Butyrate Suppresses Murine Mast Cell Activation

BackgroundMast cell is known for its roles in mediating food allergy and regulating gut inflammation. Butyrate, a major short chain fatty acid produced by gut microbiota, exerts beneficial effects on intestinal homeostasis. However, its role in modulating mast cell function remain undefined. Methods: Murine mastocytoma P815 cells (P815) or murine bone marrow derived mast cells (BMMC) were treated with sodium butyrate (NaB). The proliferation, cell cycle, apoptosis, degranulation and cytokine secretion were assessed.ResultsIn P815 cells, NaB inhibited cell proliferation, increased acetylation of both histone 3 and α‐tubulin. The growth inhibition effect of NaB is probably through reducing c‐kit expression and inducing cell cycle arrest and apoptosis, which is indicated by enhanced annexin V/PI staining, caspase‐3 and PARP cleavage. In BMMC, NaB treatment dramatically suppressed FcεRI dependent TNFα and IL‐6 release without affecting degranulation. Furthermore, NaB enhanced histone 3 acetylation but diminished mRNA expression of Tnf ‐α and Il‐6, possibly through inhibiting the transcription initiation as revealed by reduced RNA polymerase II binding to the promoters of Tnf‐α and Il‐6.ConclusionsNaB modulates murine mast cell function possibly through enhancing histone acetylation, which is of importance in developing therapeutics for diseases involving mast cells (NIH R15HD073864).

Prebiotic Potential of Xylooligosaccharides Derived from Corn Cobs and Their In Vitro Antioxidant Activity When Combined with Lactobacillus.

In the present work, the in vitro prebiotic activity of xylooligosaccharides (XOS) derived from corn cobs combined with Lactobacillus plantarum, a probiotic microorganism, was determined. These probiotics exhibited different growth characteristics depending on strain specificity. L. plantarum S2 cells were denser and their growth rates were higher when cultured on XOS. Acetate was found to be the major short-chain fatty acid produced as the end-product of fermentation, and its amount varied from 1.50 to 1.78 mg/ml. The antimicrobial activity of XOS combined with L. plantarum S2 was determined against gastrointestinal pathogens. The results showed that XOS proved to be an effective substrate, enhancing antimicrobial activity for L. plantarum S2. In vivo evaluation of the influence of XOS and L. plantarum S2, used both alone and together, on the intestinal microbiota in a mouse model showed that XOS combined with L. plantarum S2 could increase the viable lactobacilli and bifidobacteria in mice feces and decrease the viable Enterococcus, Enterobacter, and Clostridia spp. Furthermore, in the in vitro antioxidant assay, XOS combined with L. plantarum S2 possessed significant 2,2-diphenyl-1- picrylhydrazyl, 2,2'-azino-bis, and superoxide anion radical-scavenging activities, and the combinations showed better antioxidant activity than either XOS or L. plantarum S2 alone.

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).

Comparison of the in vitro efficiency of supplementary bee propolis extracts of different origin in enhancing the ruminal degradability of organic matter and mitigating the formation of methane

The current study was conducted to compare the effects of ethanolic extracts of Brazilian red propolis (BRP) and Egyptian brown propolis (EBP) characterized by different individual compounds on the ruminal degradation of nutrients and methanogenesis. The utility of propolis as a natural alternative to the ionophore antibiotic monensin, which was used as a positive control, was also tested. The propolis extract was incubated with 0 (negative control), 125, 250, or 500μg per 500mg of dietary dry matter. A semi-automatic in vitro gas production (GP) system was operated with three batches of rumen fluid in three replicates per batch for each treatment. The major compounds of the BRP extract were isoflavonoids and chalcones, which represented 0.43 of the total. Isoflavonoids and saturated fatty acids made up 0.53 of the EBP extract. Extract of BRP25μg enhanced the true organic matter degradability (TDOM) (P<0.05) compared with negative control. All doses of BRP and EBP25μg enhanced (P<0.05) TDOM compared with monensin while no differences were observed among the propolis extract treatments. Compared with the negative control, the treatment suppressing (P<0.05) methane (CH4) and total GP was monensin. The propolis extracts were only half or less than half as effective as monensin against CH4 production. Both the BRP and EBP extracts increased (P<0.05) the production of total and all major (acetate, propionate, butyrate) short-chain fatty acids (SCFA). Monensin enhanced (P<0.05) only propionate production, clearly reduced (P<0.05) the acetate-to-propionate ratio compared with the negative control. Ruminal ammonia remained unaffected by the treatments, but the propolis extracts reduced (P<0.05) the protozoal count. This study demonstrated the efficiency of two types of propolis extracts, although they were less effective than monensin in mitigating CH4 emissions. It also showed that propolis with very different compositions seemed to act in a similar way and that the efficiency of the two types of propolis in modifying the ruminal degradation of nutrients and mitigating methane formation was comparable. The most active component(s) in propolis still have to be identified.

Effect of butyrate on immune response of a chicken macrophage cell line

Butyric acid is a major short chain fatty acid (SCFA), produced in the gastrointestinal tract by anaerobic bacterial fermentation, that has beneficial health effects in many species including poultry. To understand the immunomodulating effects of butyrate on avian macrophage, we treated a naturally transformed line of chicken macrophage cells named HTC with Na-butyrate in the absence or presence of Salmonella typhimurium lipopolysaccharide (LPS) or phorbol-12-myristate-13-acetate (PMA), a metabolic activator, evaluating its various functional parameters. The results demonstrate that, butyrate by itself had no significant effect on variables such as nitric oxide (NO) production and the expression of genes associated with various inflammatory cytokines but it inhibited NO production, and reduced the expression of cytokines such as IL-1β, IL-6, IFN-γ, and IL-10 in LPS-stimulated cells. Butyrate decreased the expression of TGF-β3 in the presence or absence of LPS, while it had no effect on IL-4, Tβ4, and MMP2 gene expression. In addition, butyrate augmented PMA induced oxidative burst indicated by DCF-DA oxidation and restored LPS induced attenuation of tartrate resistant acid phosphatase (TRAP) activity. Although butyrate had no significant effect on phagocytosis or matrix metalloproteinase (MMP) activities of resting macrophages, it significantly suppressed the effects induced by their respective stimulants such as LPS induced phagocytosis and PMA induced MMP expression. These results suggest that butyrate has immunomodulatory property in the presence of agents that incite the cells thus, has potential to control inflammation and restore immune homeostasis.

Oral administration of butyrate protects against sensitization to cow milk protein in a murine model of cow milk allergy

Background: Butyrate is a major short chain fatty acids produced by gut microbiota fermentation and exerts a broad range of biological effects from the intestine to peripheral tissue. We aimed to investigate whether if butyrate is able to protect against sensitization to cow milk.

LeuO enhances butyrate-induced virulence expression through a positive regulatory loop in enterohaemorrhagic Escherichia coli.

Enterohaemorrhagic Escherichia coli (EHEC) causes bloody diarrhoea and other severe symptoms such as haemorrhagic uraemic syndrome. The expression of virulence genes on the locus for enterocyte effacement (LEE) and associated genes is regulated by a variety of factors, including transcriptional regulators and environmental signals. Butyrate, one of the major short-chain fatty acids present in the intestine, enhances expression of LEE genes and flagella biosynthesis genes in EHEC O157:H7, resulting in increased bacterial adherence and motility. Here, we show that expression of the leuO gene, which encodes a LysR-type transcriptional regulator, is enhanced by butyrate via Lrp, which is also necessary for butyrate-induced responses of LEE genes. LeuO expression induces prolonged activation of the promoter of LEE1 operon, including the ler gene, as well as virulence mechanisms such as microcolony formation. Activation of the LEE1 promoter by LeuO depends on another regulator, called Pch. The response of the leuO promoter to butyrate requires two virulence regulators, Pch and Ler, in addition to Lrp. Pch, Ler and Lrp bind the upstream region of the leuO promoter. Thus, leuO is involved in butyrate-enhanced expression of LEE genes through a positive feedback mechanism, but its expression and action on the LEE1 promoter are dependent on the virulence regulators Pch and Ler.

Suppressive action of acetate on interleukin-8 production via tubulin-α acetylation.

Acetate is the major short-chain fatty acid produced by commensal bacteria in the gut and is known as a nutrient source for epithelial cells of the mucosa. Acetate also suppresses interleukin (IL)-2 production in T cells by inhibiting nuclear factor of activated T cells (NFAT) nuclear translocation via tubulin-α acetylation. Using acetylation of tubulin-α as a biomarker, we have examined the influence of acetate in the large intestine. Because of high concentrations of acetate in fecal material, tubulin-α acetylation is dominant in the proximal large intestine relative to other sections of the large intestine and is induced in epithelial cells of the colonic mucosa. Flagellin stimulation induces IL-8 production in epithelial cells and acetate suppresses this IL-8 production via tubulin-α acetylation. Flagellin stimulation activates nuclear factor-κB, CREB and AP-1, but not NFAT. Of these transcription factors, acetate specifically inhibits AP-1 activation. Acetate impairs flagellin-induced activation of the Rap1-MEK-ERK-Elk-1 pathway with acetylation of tubulin-α that is bound to Rap1, resulting in reduced expression of c-Fos, a subunit of AP-1. These findings reveal a novel action of acetate via tubulin-α acetylation in epithelial cells of the colonic mucosa.

Impact of diet and individual variation on intestinal microbiota composition and fermentation products in obese men.

There is growing interest in understanding how diet affects the intestinal microbiota, including its possible associations with systemic diseases such as metabolic syndrome. Here we report a comprehensive and deep microbiota analysis of 14 obese males consuming fully controlled diets supplemented with resistant starch (RS) or non-starch polysaccharides (NSPs) and a weight-loss (WL) diet. We analyzed the composition, diversity and dynamics of the fecal microbiota on each dietary regime by phylogenetic microarray and quantitative PCR (qPCR) analysis. In addition, we analyzed fecal short chain fatty acids (SCFAs) as a proxy of colonic fermentation, and indices of insulin sensitivity from blood samples. The diet explained around 10% of the total variance in microbiota composition, which was substantially less than the inter-individual variance. Yet, each of the study diets induced clear and distinct changes in the microbiota. Multiple Ruminococcaceae phylotypes increased on the RS diet, whereas mostly Lachnospiraceae phylotypes increased on the NSP diet. Bifidobacteria decreased significantly on the WL diet. The RS diet decreased the diversity of the microbiota significantly. The total 16S ribosomal RNA gene signal estimated by qPCR correlated positively with the three major SCFAs, while the amount of propionate specifically correlated with the Bacteroidetes. The dietary responsiveness of the individual's microbiota varied substantially and associated inversely with its diversity, suggesting that individuals can be stratified into responders and non-responders based on the features of their intestinal microbiota.

The synergistic effect of 1’‐acetoxychavicol acetate and sodium butyrate on the death of human hepatocellular carcinoma cells (644.17)

It has been suggested that the combined effect of natural products may improve the effect of treatment against the proliferation of cancer cells. We evaluated the combination of 1’‐acetoxychavicol acetate (ACA), obtained from Alpinia galangal, and sodium butyrate (NaB), a major short chain fatty acid, on the growth of HepG2 cells and found that treatment had a synergistic inhibitory effect.The number of HepG2 cells was synergistically decreased via apoptosis induction with the combined treatment of ACA and NaB. In ACA‐ and NaB‐treated cells, intracellular reactive oxygen species (ROS) levels and NADPH oxidase activities were increased significantly. The decrease in cell number after combined treatment of ACA and NaB was improved with the treatment of catalase. These results suggest that an increase in intracellular ROS levels is involved in cancer cell death. AMP‐activated protein kinase (AMPK) plays an essential role in controlling processes related to tumor development. In ACA‐ and NaB‐treated cells, AMPK phosphorylation was induced significantly, and this induction improved when cells were pretreated with catalase. These results suggest that the increase in intracellular ROS is involved in the increase of AMPK phosphorylation.In conclusion, combined treatment with ACA and NaB synergistically induced apoptotic cell death via an increase in intracellular ROS and phosphorylation of AMPK.

The synergistic effect of 1′-acetoxychavicol acetate and sodium butyrate on the death of human hepatocellular carcinoma cells

It has been suggested that the combined effect of natural products may improve the effect of treatment against the proliferation of cancer cells. In this study, we evaluated the combination of 1′-acetoxychavicol acetate (ACA), obtained from Alpinia galangal, and sodium butyrate, a major short chain fatty acid, on the growth of HepG2 human hepatocellular carcinoma cells and found that treatment had a synergistic inhibitory effect. The number of HepG2 cells was synergistically decreased via apoptosis induction when cells were treated with both ACA and sodium butyrate. In ACA- and sodium butyrate-treated cells, intracellular reactive oxygen species (ROS) levels and NADPH oxidase activities were increased significantly. The decrease in cell number after combined treatment of ACA and sodium butyrate was diminished when cells were pretreated with catalase. These results suggest that an increase in intracellular ROS levels is involved in cancer cell death. AMP-activated protein kinase (AMPK), a cellular energy sensor, plays an essential role in controlling processes related to tumor development. In ACA- and sodium butyrate-treated cells, AMPK phosphorylation was induced significantly, and this induction improved when cells were pretreated with catalase. These results suggest that the increase in intracellular ROS is involved in the increase of AMPK phosphorylation. In normal hepatocyte cells, treatment with ACA and sodium butyrate did not decrease cell numbers or increase ROS levels. In conclusion, combined treatment with ACA and sodium butyrate synergistically induced apoptotic cell death via an increase in intracellular ROS and phosphorylation of AMPK. Our findings may provide new insight into the development of novel combination therapies against hepatocellular carcinoma.

Strong Stability and Host Specific Bacterial Community in Faeces of Ponies

The horse, as a hindgut fermenter, is reliant on its intestinal bacterial population for efficient diet utilisation. However, sudden disturbance of this population can result in severe colic or laminitis, both of which may require euthanasia. This study therefore aimed to determine the temporal stability of the bacterial population of faecal samples from six ponies maintained on a formulated high fibre diet. Bacterial 16S rRNA terminal restriction fragment length polymorphism (TRFLP) analyses of 10 faecal samples collected from 6 ponies at regular intervals over 72 hour trial periods identified a significant pony-specific profile (P<0.001) with strong stability. Within each pony, a significantly different population was found after 11 weeks on the same diet (P<0.001) and with greater intra-individual similarity. Total short chain fatty acid (SCFA) concentration increased in all ponies, but other changes (such as bacterial population diversity measures, individual major SCFA concentration) were significant and dependent on the individual. This study is the first to report the extent of stability of microbes resident in the intestinal tract as represented with such depth and frequency of faecal sampling. In doing so, this provides a baseline from which future trials can be planned and the extent to which results may be interpreted.

Analysis of fermentation selectivity of purified galacto-oligosaccharides by in vitro human faecal fermentation

The in vitro fermentation of several purified galacto-oligosaccharides (GOS), specifically the trisaccharides 4'-galactosyl-lactose and 6'-galactosyl-lactose and a mixture of the disaccharides 6-galactobiose and allolactose, was carried out. The bifidogenic effect of GOS at 1% (w/v) was studied in a pH-controlled batch culture fermentation system inoculated with healthy adult human faeces. Results were compared with those obtained with a commercial GOS mixture (Bimuno-GOS). Changes in bacterial populations measured through fluorescence in situ hybridization and short-chain fatty acid (SCFA) production were determined. Bifidobacteria increased after 10-h fermentation for all the GOS substrates, but the changes were only statistically significant (P<0.05) for the mixture of disaccharides and Bimuno-GOS. Acetic acid, whose formation is consistent with bifidobacteria metabolism, was the major SCFA synthesized. The acetate concentration at 10 h was similar with all the substrates (45-50 mM) and significantly higher than the observed for formic, propionic and butyric acids. All the purified GOS could be considered bifidogenic under the assayed conditions, displaying a selectivity index in the range 2.1-3.0, which was slightly lower than the determined for the commercial mixture Bimuno-GOS.

The synergistic effect of 1′‐acetoxychavicol acetate and sodium butyrate on cell death of human hepatocellular carcinoma cells

Introduction1′‐Acetoxychavicol acetate (ACA), obtained from alpinia galangal, exhibits chemopreventive effects on chemically induced tumor formation. Sodium butyrate (NaB), one of the major short chain fatty acids, is known to inhibit cancer cell proliferation in tumor cells. The interaction between ACA and NaB is still unclear. Here, we examined the effect of ACA and NaB on cell death of HepG2 cells.MethodsThe cell number was measured by neutral red assay. Intercellular reactive oxygen species (ROS) was measured by DCFH‐DA assay.ResultsThe cell number of HepG2 cells was synergistically decreased with the combined treatment of ACA and NaB, resulting from apoptotic cell death. In ACA and NaB simultaneously treated cells, intercellular ROS also increased significantly. However, the cell number in ACA and NaB treated cells was improved with the treatment of catalase. The increase in intercellular ROS was prevented with DPI, the inhibitor of NADPH oxidase. These events weren't observed in normal cells.ConclusionACA and NaB synergistically induce apoptotic cell death via the activation of NADPH oxidase and the increase in the intercellular ROS, and the effect is specific to carcinoma cells.

Activities of free and encapsulated Lactobacillus acidophilusLA5 or Lactobacillus casei 01 in processed longan juices on exposure to simulated gastrointestinal tract

Fruit drinks containing probiotics are gaining interest in the global marketplace. For example, longan juice, containing carbohydrate and various bioactive components, is a potentially health-promoting beverage as well as probiotic carrier for human consumption. In this study, high-pressure and thermal processes were applied to eliminate competitive micro-organisms in longan juice prior to the addition of Lactobacillus acidophilus LA5 or Lactobacillus casei 01. The activities of these probiotics in a simulated gastrointestinal tract were also investigated. Encapsulated probiotics could survive in the acidic environment of the stomach and small intestine, while the free cells were completely eliminated. In the colon experiment, the influence of encapsulated L. casei 01 on colon lactobacilli was significantly greater than that of encapsulated L. acidophilus LA5. Both encapsulated probiotics suspended in processed longan juices led to extensive increases in the formation of lactic acid and short-chain fatty acids (SCFA). Acetate was the major SCFA produced by colon bacteria, followed by propionate and butyrate. The discernible clear zone suggested that L. casei 01 provided greater antibacterial activity than L. acidophilus LA5. Both encapsulated probiotics along with processed longan juice led to significant increases in colon lactobacilli, lactic acid and SCFA formation.

Fluctuations in butyrate-producing bacteria in ulcerative colitis patients of North India

To study the interplay between butyrate concentration and butyrate-producing bacteria in fecal samples of ulcerative colitis (UC) patients vs control individuals. Fecal samples were collected from 14 control individuals (hemorrhoid patients only) and 26 UC patients (severe: n = 12, moderate: n = 6, remission: n = 8), recruited by the gastroenterologist at the Department of Gastroenterology, All India Institute of Medical Sciences, New Delhi, India. Disease activity in UC patients was determined by clinical colitis activity index. We employed fluorescent in situ hybridization in combination with flow cytometry to enumerate the clostridium cluster population targeted by 16S rRNA gene probe. Major butyrate-producing species within this cluster were quantified to see if any change existed in control vs UC patients with different disease activity. This observed change was further validated by quantitative polymerase chain reaction. In addition to this, we carried out gas chromatography to evaluate the changes in concentration of major short chain fatty acids (SCFAs), namely acetate, n-butyrate, iso-butyrate, in the above samples. Student t test and Graph pad prism-6 were used to compare the data statistically. There was a significant decrease of Clostridium coccoides (control, 25.69% ± 1.62% vs severe, 9.8% ± 2.4%, P = 0.0001) and Clostridium leptum clusters (control, 13.74% ± 1.05% vs severe, 6.2% ± 1.8%, P = 0.0001) in fecal samples of UC patients. Furthermore, we demonstrated that some butyrate-producing members of the clostridial cluster, like Fecalibacterium prausnitzii (control, 11.66% ± 1.55% vs severe, 6.01% ± 1.6%, P = 0.0001) and Roseburia intestinalis (control, 14.48% ± 1.52% vs severe, 9% ± 1.83%, P = 0.02) were differentially present in patients with different disease activity. In addition, we also demonstrated decreased concentrations of fecal SCFAs, especially of n-butyrate (control, 24.32 ± 1.86 mmol/μL vs severe, 12.74 ± 2.75 mmol/μL, P = 0.003), iso-butyrate (control, 1.70 ± 0.41 mmol/μL vs severe, 0.68 ± 0.24 mmol/μL, P = 0.0441) and acetate (control, 39.51 ± 1.76 mmol/μL vs severe, 32.12 ± 2.95 mmol/μL, P = 0.047), in the fecal samples of UC patients. The observed decrease of predominant butyrate producers of clostridial clusters correlated with the reduced SCFA levels in active UC patients. This was further confirmed by the restoration in the population of some butyrate producers with simultaneous increase in the level of SCFA in remission samples. Our observations indicate that decreases in members of the clostridial cluster resulting in reduced butyrate levels contribute to the etiology of UC.

A novel nutrient sensing mechanism underlies substrate-induced regulation of monocarboxylate transporter-1

Monocarboxylate transporter isoform-1 (MCT1) plays an important role in the absorption of short-chain fatty acids (SCFAs) in the colon. Butyrate, a major SCFA, serves as the primary energy source for the colonic mucosa, maintains epithelial integrity, and ameliorates intestinal inflammation. Previous studies have shown substrate (butyrate)-induced upregulation of MCT1 expression and function via transcriptional mechanisms. The present studies provide evidence that short-term MCT1 regulation by substrates could be mediated via a novel nutrient sensing mechanism. Short-term regulation of MCT1 by butyrate was examined in vitro in human intestinal C2BBe1 and rat intestinal IEC-6 cells and ex vivo in rat intestinal mucosa. Effects of pectin feeding on MCT1, in vivo, were determined in rat model. Butyrate treatment (30-120 min) of C2BBe1 cells increased MCT1 function {p-(chloromercuri) benzene sulfonate (PCMBS)-sensitive [(14)C]butyrate uptake} in a pertussis toxin-sensitive manner. The effects were associated with decreased intracellular cAMP levels, increased V(max) of butyrate uptake, and GPR109A-dependent increase in apical membrane MCT1 level. Nicotinic acid, an agonist for the SCFA receptor GPR109A, also increased MCT1 function and decreased intracellular cAMP. Pectin feeding increased apical membrane MCT1 levels and nicotinate-induced transepithelial butyrate flux in rat colon. Our data provide strong evidence for substrate-induced enhancement of MCT1 surface expression and function via a novel nutrient sensing mechanism involving GPR109A as a SCFA sensor.

1058 Crosstalk Between Commensal Bacteria and Colonic Epithelium in Gnotobiotic Rats: A Key Role of Acetate

The gastrointestinal tract (GIT) of mammals is colonized by a community of micro-organisms reaching levels as high as 1011 bacteria/g of contents in the colon. This microbiota is involved in intestinal homeostasis and epithelium structure through the control of proliferation and differentiation processes (Cherbuy et al., 2010). Bacteroides thetaiotaomicron and Faecalibacterium prausnitzii, dominant members of microbiota, are commensal model bacteria to study host-microbiota interactions. The objective of our work was to understand the effects of these two bacteria on colonic epithelium homeostasis in gnotobiotic rats and underlying this crosstalk using In Vitro models. Germ-free rats (GF) were orally inoculated with B. thetaiotaomicron either alone or with a mix of B. thetaiotaomicron and F. prausnitzii. Colonic epithelium morphology, crypt depth, cell cycle-related proteins and goblet cells markers were analyzed by histological staining, immunohistochemistry and Western blot in GF (n= 6), mono-associated (Ino-Bt; n=12) or di-associated (Ino-Bt+Fp; n=6) rats 30 days after inoculation. In Ino-Bt rats, no effect was observed neither on crypt depth nor on proliferation markers whereas Kruppel Like Factor 4 protein (KLF4), a transcription factor involved in goblet cells differentiation, was significantly increased (2.5 ±0.8-fold) compared to GF rats. In parallel with this KLF4 induction, a significant increase of goblet cells per crypt in the colon of Ino-Bt compared to GF rats was observed. In Ino-Bt+Fp rats, there was no induction of KLF4 protein, suggesting that the presence of F. prausnitzii impaired the mucus-stimulatory effect triggered by B. thetaiotaomicron. Short Chain Fatty Acids (SCFA) composition was determined on caecal contents by gas chromatograph to assess bacterial metabolic activity. In Ino-Bt rats, acetate was the major SCFA detected (5.8 μmol/g) with a low level of propionate (1.0 μmol/g) and no butyrate. In Ino-Bt+Fp rats, butyrate was the major SCFA (1.3 μmol/g) and acetate concentration was significantly decreased (two-fold) compared to Ino-Bt rats (3.0 μmol/g). Using HT29-MTX cells, a cell line producing mucus, we showed that acetate was able to increase significantly KLF4 protein whereas butyrate and propionate have no effect on KLF4 level. All of these results suggested that acetate, a bacterial metabolite produced by B. thetaiotaomicron, could mediate a bacterial signal to promote goblet cells differentiation pathway in the GIT of mono-associated rats. In Crohn's disease patients, a diminished prevalence of F. prausnitzii (Martinez-Medina et al., 2006) and an increase of mucus production (Gersemann et al., 2009) have been often reported. Our observations in gnotobiotic rats are coherent with reports made in patients and may provide mechanistic clues to understand the pathophysiology of inflammatory bowel diseases.

Evaluation of probiotic bacteria for their effects on the growth performance and intestinal microbiota of newly-weaned pigs fed fermented high-moisture maize

The effects of feeding high-moisture maize fermented with Lactobacillus acidophilus or Pediococcus acidilactici on the growth performance and intestinal microflora of newly weaned pigs were investigated. A total of 270 newly weaned pigs (equal numbers of castrated males and females) were allocated to 3 dietary treatments (5 pens per treatment and 18 pigs per pen) in a randomized complete block design on the basis of body weight (BW) and litter. The dietary treatments included a basal diet supplemented with: 1) naturally fermented high-moisture maize without any microbial inoculants (Control); 2) high-moisture maize fermented with L. acidophilus (LA); and 3) high-moisture maize fermented with P. acidilactici (PA). Pigs were fed diets in liquid form for a 5-wk period. Quantitative polymerase chain reaction (QPCR) analysis with PCR primers specific to L. acidophilus or P. acidilactici indicated a well-developed bacterial population of each inoculated bacterium in the fermented high-moisture maize. Lactic acid was the major short-chain fatty acid (SCFA) produced from the fermentation. Among the 3 treatments and on most sampling days, LA-fermented maize had the numerically highest concentration of lactic acid. Dietary treatments over the 5-wk period did not impact pig growth performance. However, during last 3wks of the trial, pigs on the LA treatment had higher BW gains than pigs on PA. Pigs fed LA-fermented high-moisture maize showed fewer coliform bacteria in fecal samples when compared to pigs fed PA-fermented high-moisture maize (P<0.05). The PCR and denaturing gradient gel electrophoresis (DGGE) analysis on the relatedness of bacterial profiles of colonic microbiota revealed 3 bacterial clusters and the microbiota from PA-treated pigs formed 1 cluster. In addition, feeding LA- or PA-fermented maize reduced the diversity and richness of bacterial microbiota in the colon (P<0.05). Both L. acidophilus and P. acidilactici colonized poorly in the intestine of pigs regardless of the dietary treatments. These results indicate that feeding the probiotic-fermented, high-moisture maize can modulate the intestinal microbiota, and LA has the potential to impact pig growth performance.

Association Between Low Colonic Short-Chain Fatty Acids and High Bile Acids in High Colon Cancer Risk Populations

We propose that the influence of diet on colon cancer risk is mediated by the microbiota. To investigate how dietary fat influences risk, we compared the colonic contents of 12 adult high-risk African Americans (AAs) and 10 Caucasian Americans (CAs) who consumed a high-fat diet (123 ± 11 g/d and 129 ± 17 g/d, respectively) to 13 native Africans (NAs) who subsisted on a low-fat (38 ± 3.0 g/d) diet, all aged 50–60 yr. The colonic bile acids were measured by LC-MS and the short-chain fatty acids (SCFAs) by GC. The chief secondary colonic bile acids, deoxycholic acid and lithocholic acid, were correlated with fat intake and similar between AAs and CAs, but 3–4 times higher than in AAs (p < 0.05). The major SCFAs were lower in AAs (p < 0.001) and CAs (p < 0.001) compared to AAs, but conversely, the branched chain fatty acids (BFCA) were higher. Our results suggest that the higher risk of colon cancer in Americans may be partly explained by their high-fat and high-protein, low complex carbohydrate diet, which produces colonic residues that promote microbes to produce potentially carcinogenic secondary bile acids and less antineoplastic SCFAs. The role of BCFA in colonic carcinogenesis deserves further study.