Effect of Dietary Ceramide and Glucosylceramide on the Alleviation of Experimental Inflammatory Bowel Disease in Mice.

Ceramide prepared from glucosylceramide (GlcCer) with Gluceribacter canis NATH-2371T was administrated to inflammatory bowel disease (IBD) model mice. Dietary ceramide significantly suppressed the decrease in final body weight, and the increase in the disease activity index and myeloperoxidase activity more greatly than GlcCer in IBD mice. Intestinal microbiome profiles were found to be altered in IBD mice, but ceramide counteracted the changes. These results suggest that dietary plant-based ceramide may alleviate symptoms of IBD in mice.

Augmentation of dietary glucosylceramide hydrolysis by the novel bacterium Glucerabacter canisensis NATH-2371T.

The purpose of this study was to evaluate the effects of intragastrical administration of Glucerabacter canisensis NATH-2371T on glucosylceramide (GluCer) digestion in mice. Although G. canisensis was unable to utilize starch and cellulose, coculture of G. canisensis with mouse fecal bacteria greatly increased GluCer hydrolysis in polysaccharide medium, indicating that G. canisensis grew in competition with other intestinal bacteria. Although most of the administered G. canisensis cells were detected in feces, some cells were present in the colorectum contents, which had GluCer-hydrolyzing activity. These results indicate that G. canisensis can viably transit through the mouse gut. Administration of G. canisensis to mice fed diets supplemented with GluCer or GluCer-containing foods significantly enhanced GluCer hydrolysis. Since G. canisensis did not show acute toxicity, it may be useful as a probiotic to augment GluCer hydrolysis in the large intestine. Abbreviations: GluCer: glucosylceramide; KPi: potassium phosphate buffer; C-M: chloroform-methanol.

Glucerabacter canisensis gen. nov., sp. nov., isolated from dog feces and its effect on the hydrolysis of plant glucosylceramide in the intestine of dogs.

A Gram-positive, obligately anaerobic, oval-rod shaped, non-spore-forming, and non-pigmented bacterium, designated strain NATH-2371T (= JCM31739T = DSM105698T), was isolated from dog feces. Comparative 16S rRNA gene sequence analysis revealed that strain NATH-2371T belongs to Clostridium cluster XIVa, and the closest strains were Coprococcus comes ATCC 27758T (94.8% 16S rRNA gene sequence similarity) and Clostridium nexile DSM 1787T (94.0%). Strain NATH-2371T produced acetate, formate, and ethanol from glucose. Predominant cellular fatty acids are C16:0 and C16:0 DMA. On the basis of the phenotypic and genotypic differences, strain NATH-2371T represents a novel species in a new genus of the family Lachnospiraceae, for which the name Glucerabacter canisensis gen. nov., sp. nov., is proposed. This strain was found to efficiently hydrolyze plant glucosylceramide (GluCer). The abundance of strain NATH-2371T in dog feces was higher in young dogs than in old dogs. Incubation of dog fecal bacteria showed that GluCer-hydrolyzing activity decreased with the age of dogs. The cell number of strain NATH-2371T in dog feces appeared to be correlated with GluCer hydrolysis. Thus, this bacterium is likely to play a major role in GluCer hydrolysis in the dog intestine.

Effects of nitrate addition to a diet on fermentation and microbial populations in the rumen of goats, with special reference to Selenomonas ruminantium having the ability to reduce nitrate and nitrite.

This study investigated the effects of dietary nitrate addition on ruminal fermentation characteristics and microbial populations in goats. The involvement of Selenomonas ruminantium in nitrate and nitrite reduction in the rumen was also examined. As the result of nitrate feeding, the total concentration of ruminal volatile fatty acids decreased, whereas the acetate : propionate ratio and the concentrations of ammonia and lactate increased. Populations of methanogens, protozoa and fungi, as estimated by real-time PCR, were greatly decreased as a result of nitrate inclusion in the diet. There was modest or little impact of nitrate on the populations of prevailing species or genus of bacteria in the rumen, whereas Streptococcus bovis and S. ruminantium significantly increased. Both the activities of nitrate reductase (NaR) and nitrite reductase (NiR) per total mass of ruminal bacteria were increased by nitrate feeding. Quantification of the genes encoding NaR and NiR by real-time PCR with primers specific for S. ruminantium showed that these genes were increased by feeding nitrate, suggesting that the growth of nitrate- and nitrite-reducing S. ruminantium is stimulated by nitrate addition. Thus, S. ruminantium is likely to play a major role in nitrate and nitrite reduction in the rumen.

Effects of oral administration of Butyrivibrio fibrisolvens MDT-1 on the development and healing of atopic dermatitis in NC/Nga mice.

The purpose of this study was to evaluate the effects of oral administration of Butyrivibrio fibrisolvens MDT-1 (MDT-1) on mite antigen-induced atopic dermatitis (AD) in NC/Nga mice. When administration of the freeze-dried cells of MDT-1 was initiated one week before mite-antigen swabbing, the development of AD-like skin lesions was alleviated. The cell homogenate and membrane fraction had similar effects, showing that cell components are effective. Freeze-dried cells were found to be replaceable by the crude lipopolysaccharide extracted from MDT-1. Alleviation of AD was concomitant with a decrease in the serum IgE level. Determination of cytokines produced by Peyer's patch cells showed that MDT-1 administration increased IL-12, tended to increase IFN-γ, and tended to decrease IL-10, but did not affect IL-4. When MDT-1 was administered after the skin lesions had progressed, the symptoms were ameliorated, suggesting that MDT-1 has AD-healing effects. MDT-1 was as effective as prednisolone, but different from prednisolone in that MDT-1 showed no detectable side effects such as body weight loss. We previously reported that MDT-1 administration prevented and alleviated colorectal cancer, sarcoma, and enterocolitis in mice. Taken together, MDT-1 might be beneficial for maintaining the health of pet animals and humans.

Responses in digestion, rumen fermentation and microbial populations to inhibition of methane formation by a halogenated methane analogue.

The effects of the anti-methanogenic compound, bromochloromethane (BCM), on rumen microbial fermentation and ecology were examined in vivo. Japanese goats were fed a diet of 50 % Timothy grass and 50 % concentrate and then sequentially adapted to low, mid and high doses of BCM. The goats were placed into the respiration chambers for analysis of rumen microbial function and methane and H2 production. The levels of methane production were reduced by 5, 71 and 91 %, and H2 production was estimated at 545, 2941 and 3496 mmol/head per d, in response to low, mid and high doses of BCM, respectively, with no effect on maintenance feed intake and digestibility. Real-time PCR quantification of microbial groups showed a significant decrease relative to controls in abundance of methanogens and rumen fungi, whereas there were increases in Prevotella spp. and Fibrobacter succinogenes, a decrease in Ruminococcus albus and R. flavefaciens was unchanged. The numbers of protozoa were also unaffected. Denaturing gradient gel electrophoresis and quantitative PCR analysis revealed that several Prevotella spp. were the bacteria that increased most in response to BCM treatment. It is concluded that the methane-inhibited rumen adapts to high hydrogen levels by shifting fermentation to propionate via Prevotella spp., but the majority of metabolic hydrogen is expelled as H2 gas.

Molecular characterization and significance of phosphoenolpyruvate carboxykinase in a ruminal bacterium, Streptococcus bovis.

To gain knowledge about the significance of phosphoenolpyruvate (PEP) carboxykinase (PCK) in Streptococcus bovis, the sequence of the gene encoding PCK (pck) was determined. Transcriptional analysis indicated that the pck is transcribed in a monocistronic fashion. The level of pck-mRNA was higher when cells were grown on lactose than on glucose, suggesting that PCK synthesis increases when the growth rate is low. The pck-mRNA level was higher in a mutant lacking ccpA, which encodes the catabolite control protein A (CcpA), than in the parent strain, suggesting that pck transcription is suppressed by CcpA. S. bovis PCK showed oxaloacetate (OAA)-decarboxylating activity, but no PEP-carboxylating activity (reverse reaction). In S. bovis, OAA was speculated to be produced from PEP via pyruvate. Disruption of pck in S. bovis resulted in decreased growth rate and cell yield. When a pck-disrupted mutant was grown in a medium lacking amino acids, the lag phase was longer and the cell yield was lower than the case of the parent strain. These results suggest that pck is involved in the initiation of growth, including the induction of amino acid synthesis and energy metabolism.

Involvement of two-component signal transduction system, ComDE, in the regulation of growth and genetic transformation, in the ruminal bacterium Streptococcus bovis.

In ruminants, Streptococcus bovis is considered to be associated with acute rumen acidosis. To elucidate the regulatory mechanisms of S. bovis growth, we investigated the function of the two components of the peptide pheromone-signaling system, ComD and ComE, which are encoded by comD and comE, respectively, via the competence-stimulating peptide ComC, which is encoded by comC. Deletion of entire comC and two-thirds of comD resulted in decreased growth rate, which may be related to the change in the expression of several proteins, as shown by two-dimensional gel electrophoresis. The transcript level of comED was decreased by the disruption of comCD, suggesting that the transcription of comED might be stimulated by ComC. The transformation frequency was decreased by the disruption of comCD. Addition of recombinant ComC to S. bovis cultures increased the growth rate and transformation frequency. In the cultures of mixed ruminal microbes, addition of mature ComC peptide increased the number of S. bovis per total bacterial counts as estimated by the cDNA amounts of 16SrRNA. Thus, the peptide pheromone-signaling system via ComC, D, and E might be involved in the control of S. bovis growth in addition to competence development. This is the first report suggesting that an autoinducing peptide functions in the ruminal ecosystem.

Isolation of a novel bacterium, Blautia glucerasei sp. nov., hydrolyzing plant glucosylceramide to ceramide.

A bacterial strain that is capable of hydrolyzing plant glucosylceramide (GluCer) was newly isolated from dog feces. The novel strain, designated as strain HFTH-1(T), hydrolyzed plant GluCer with a variety of chemical structures, but did not hydrolyze glucosylsphingosine, lactosylceramide, or monosialoganglioside GM(3), indicating that strain HFTH-1(T) produces GluCer-specific glucosylceramidase. Strain HFTH-1(T) was Gram-positive, anaerobic, oval-spore-forming, rod-shaped, lecithinase-negative, and lipase-negative. It fermented a wide variety of carbohydrates and produced mainly acetate, formate, and lactate from glucose. The G + C content of its DNA was 40.7 mol%. The phylogenetic analysis of 16S rRNA sequence revealed that strain HFTH-1(T) is placed in the clostridial rRNA cluster XIVa, with Ruminococcus obeum as the nearest relative. Pairwise comparison revealed approximately 5.0% sequence divergence between strain HFTH-1(T) and the type strain of R. obeum. On the basis of its phenotypic characteristics and phylogenetic divergence, it is proposed that the hitherto unknown rod-shaped bacterial strain HFTH-1(T) (= DSM 22028(T) = NBRC 104932(T)) should be placed in the genus Blautia as a novel species, Blautia glucerasei sp. nov, the only currently known isolate of the species.

Molecular and biochemical characterization of phosphoenolpyruvate carboxykinase in the ruminal bacterium Ruminococcus albus.

Molecular properties and transcriptional control of phosphoenolpyruvate carboxykinase (PCK; EC 4.1.1.32) in Ruminococcus albus were examined. The putative 537-amino acid PCK polypeptide has a predicted mass of 59.4 kDa and an isoelectric point of 4.82. RT-PCR and Northern blot analyses of pck mRNA suggest that the transcript is monocistronic and that pck transcription is not affected by changes in sugar sources present in growth medium. PCK enzymatic activity requires either Mg(2+) or Mn(2+) and an optimal pH of 7.0. R. albus PCK phosphorylated ADP more readily than GDP. Apparent K ( m ) values of PCK for PEP and ADP were considerably lower than those for OAA and ATP, suggesting that the reaction from PEP to OAA is favored in R. albus. The enzyme properties of PCK in R. albus appear to be more similar to Selenomonas ruminantium PCK than to Ruminococcus flavefacience, although R. albus and R. flavefacience belong to the same genus. The specific activity of PCK, representing the amount of enzyme per cell, in R. albus was much lower than that in S. ruminantium. The amount of succinate produced in R. albus from one unit of cellobiose was also much lower than the sum of succinate and propionate produced in S. ruminantium. Based on these results, we propose enhancement of PCK activity by stimulating PCK transcription as a method to decrease R. albus H(2) production without suppressing growth.

Properties and role of glyceraldehyde-3-phosphate dehydrogenase in the control of fermentation pattern and growth in a ruminal bacterium, Streptococcus bovis.

To clarify the control of glycolysis and the fermentation pattern in Streptococcus bovis, the molecular and enzymatic properties of NAD(+)-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were examined. The GAPDH gene (gapA) was found to cluster with several others, including those that encode phosphoglycerate kinase and translation elongation factor G, however, gapA was transcribed in a monocistronic fashion. Since biochemical properties, such as optimal pH and affinity for glyceraldehyde-3-phosphate (GAP), were not very different between GAPDH- and NADP(+)-specific glyceraldehyde-3-phosphate dehydrogenase (GAPN), the flux from GAP may be greatly influenced by the relative amounts of these two enzymes. Using S. bovis JB1 as a parent, JB1gapA and JB1ldh, which overproduce GAPDH and lactate dehydrogenase (LDH), respectively, were constructed to examine the control of the glycolytic flux and lactate production. There were no significant differences in growth rates and formate-to-lactate ratios among JB1, JB1gapA, and JB1ldh grown on glucose. When grown on lactose, JB1ldh showed a much lower formate-to-lactate ratio than JB1gapA, which showed the highest NADH-to-NAD(+) ratio. However, growth rates did not differ among JB1, JB1gapA, and JB1ldh. These results suggest that GAPDH is not involved in the control of the glycolytic flux and that lactate production is mainly controlled by LDH activity.

Dietary sphingomyelin alleviates experimental inflammatory bowel disease in mice.

The effect of dietary sphingomyelin (SM) on inflammatory bowel disease (IBD) induced with dextran sodium sulfate (DSS) was examined in mice. Although the severity of IBD as expressed by the disease activity index (DAI) markedly increased with DSS administration, feeding a diet containing SM lowered the DAI value significantly. Myeloperoxidase (MPO) activity in colonic tissue also increased with DSS administration, suggesting the development of inflammation. Because simultaneous administration of SM with DSS prevented the MPO activity increase, we concluded that SM could suppress the development of inflammation. These results provide novel evidence that dietary supplementation with SM can alleviate the symptoms of IBD in mice. Dietary SM also increased the amount of IgA in the large intestine, suggesting that SM promotes IgA secretion into the large intestine. These results suggest that the mechanism of IBD mitigation by SM is complex and involves the immune system.

Molecular properties and transcriptional control of the phosphofructokinase and pyruvate kinase genes in a ruminal bacterium, Streptococcus bovis.

Molecular properties of pyruvate kinase (PYK) and phosphofructokinase (PFK) in Streptococcus bovis and transcriptional control of the two enzymes were examined. Sequence analysis indicated that the PYK gene (pyk) clusters with the PFK gene (pfk) and several other genes. It was demonstrated that the pyk and pfk are cotranscribed and their transcription appeared to be regulated at the transcriptional level in response to the sugars supplied. The intracellular pyk-mRNA level was lower in a catabolite control protein A (CcpA)-disrupted mutant than in its parent strain, and a binding site of CcpA was found in the upstream region of pfk. These results suggest that pfk-pyk transcription is enhanced by CcpA. A recombinant pyk-overexpressing strain showed approximately five-fold higher PYK activity, but it did not affect the growth rate or formate-to-lactate ratio significantly, suggesting that the flux in the glycolytic pathway is not altered by an increase in PYK activity.

Effect of oral administration of Butyrivibrio fibrisolvens MDT-1, a gastrointestinal bacterium, on 3-methylcholanthrene-induced tumor in mice.

Butyrivibrio fibrisolvens MDT-1 was evaluated for use as a probiotic to prevent tumor formation. Oral administration of MDT-1 (10(9) cfu/dose, 3 times a wk for 15 wk) delayed the onset of tumors induced by 3-methylcholanthrene and also reduced tumor incidence in mice. Furthermore, the numbers of natural killer (NK) and NKT cells in the spleen increased markedly in response to MDT-1 administration. Increased numbers of NK and NKT cells may contribute to the alleviation of carcinogenesis by MDT-1 administration. Cultured splenic lymphocytes isolated from mice fed MDT-1 produced higher levels of interferon-gamma than control mice but showed no change in interleukin-4 production. These results suggest that MDT-1 administration skews the ratio of helper T (Th) cells type 1 (Th1) to Th2 toward Th1 dominance. MDT-1 administration resulted in increased immunoglobulin A secretion into the intestine. These results suggest that probiotic B. fibrisolvens MDT-1 may contribute to the prevention or alleviation of cancers, allergic disorders, and infectious diseases.

Effect of oral administration of Butyrivibrio fibrisolvens MDT-1 on experimental enterocolitis in mice.

Butyrivibrio fibrisolvens MDT-1, a butyrate-producing strain, was evaluated for use as a probiotic to prevent enterocolitis. Oral administration of the MDT-1 strain (10(9) CFU/dose) alleviated the symptoms of colitis (including body weight loss, diarrhea, bloody stool, organic disorder, and mucosal damage) that are induced in mice drinking water that contains 3.0% dextran sulfate sodium. In addition, myeloperoxidase (MPO) activity levels in colonic tissue were reduced, suggesting that MDT-1 mitigates bowel inflammation. The addition of MDT-1 culture supernatant inhibited the growth of nine clinical isolates of Campylobacter jejuni and Campylobacter coli that could potentially cause enterocolitis. Infection of mice with C. coli 11580-3, one of the isolates inhibited by MDT-1 in vitro, resulted in diarrhea, mucosal damage, increased MPO activity levels in colonic tissue, increased numbers of C. coli in the cecum, and decreased body weight gain. However, administration of MDT-1 to mice, prior to and during C. coli infection, reduced these effects. These results suggest that Campylobacter-induced enterocolitis can be alleviated by using B. fibrisolvens as a probiotic.

Presence of NAD+-specific glyceraldehyde-3-phosphate dehydrogenase and CcpA-dependent transcription of its gene in the ruminal bacterium Streptococcus bovis.

To know the role of NADP+-specific glyceraldehyde-3-phosphate dehydrogenase (GAPN) in Streptococcus bovis, the molecular properties and transcriptional control of the gene encoding GAPN (gapN) were examined. The GAPN in S. bovis was deduced to consist of 476 amino acids with a molecular mass of 51.1 kDa. The gapN gene was transcribed in a monocistronic fashion. GAPN synthesis appeared to be regulated at the transcriptional level in response to changes in growth conditions. In a mutant that lacks the ccpA gene encoding catabolite control protein A (CcpA), the gapN-mRNA level was lower than in the parent strain. A binding site of CcpA was found in the upper region of gapN. These results suggest that transcription of gapN is regulated through CcpA. Overexpression of GAPN in S. bovis did not affect the growth rate or formate-to-lactate ratio, suggesting that the flux in the glycolytic pathway is unlikely to be altered by GAPN activity. Streptococcus bovis GAPN was NADP+ dependent, but not phosphate dependent. In addition, S. bovis did not have other NADPH-producing systems such as the hexose monophosphate pathway and NADPH:NAD+ oxidoreductase. Therefore, GAPN may play an important role in NADPH production in S. bovis.

Oral administration of butyrivibrio fibrisolvens, a butyrate-producing bacterium, decreases the formation of aberrant crypt foci in the colon and rectum of mice.

Butyrivibrio fibrisolvens, a butyrate-producing ruminal bacterium, was evaluated for use as a probiotic to prevent colorectal cancer. Oral administration to Jcl:ICR mice of a new strain of B. fibrisolvens (MDT-1) that produces butyrate at a high rate (10(9) cfu/dose) increased the rate of butyrate production by fecal microbes, suggesting that MDT-1 can grow in the gut. The number of colorectal aberrant crypt foci (ACF), putative preneoplastic lesions induced by 1,2-dimethylhydrazine, was reduced after MDT-1 administration (10(9) cfu/dose, 3 times/wk for 4 wk). The number of aberrant crypts (ACs), number of foci having 3 or 4 ACs per focus, and the percentage of mice having 3 or 4 ACs per focus were also reduced, suggesting that the progress of lesions was suppressed by MDT-1. Interestingly, the MDT-1 cell homogenate did not have a similar beneficial effect. MDT-1 had low beta-glucuronidase activity, and administration of MDT-1 reduced the beta-glucuronidase activity in the colorectal contents. The numbers of natural killer (NK) and NKT cells in the spleen were markedly enhanced in response to MDT-1. Decreased beta-glucuronidase activity and increased numbers of NK and NKT cells and butyrate production may explain in part why MDT-1 administration suppressed ACF formation. These results suggest that colorectal cancer may be prevented or suppressed by the utilization of MDT-1 as a probiotic. Administration of MDT-1 had no harmful effect on the health of mice at least for 3 mo.

Characterization and transcription of the genes involved in butyrate production in Butyrivibrio fibrisolvens type I and II strains.

The genes in Butyrivibrio fibrisolvens that encode the enzymes involved in butyrate production were sequenced. In a type I strain (ATCC 19171(T)), the genes coding for the enzymes that catalyze the conversion from acetyl-CoA to butyryl-CoA, thl (thiolase), crt (crotonase), hbd (beta-hydroxybutyryl-CoA dehydrogenase), bcd (butyryl-CoA dehydrogenase), etfB (electron transfer flavoprotein [ETF]-beta), and etfA (ETF-alpha), were found to be clustered and arranged in this order. A type II strain (ATCC 51255) had the same clustered genes with the same arrangement, except that crt was not present in the clustered genes. The deduced amino acid sequences of these enzymes did not greatly differ between the two strains, and even between B. fibrisolvens and clostridia. Amino acid identity appeared to be higher within the same type than between types I and II. The clustered genes were shown to be cotranscribed, and constitutively transcribed without being affected significantly by culture conditions.

A new strain of Butyrivibrio fibrisolvens that has high ability to isomerize linoleic acid to conjugated linoleic acid.

A new strain of Butyrivibrio fibrisolvens (TH1) that has high potential to produce conjugated linoleic acid (CLA) was isolated. Strain TH1 had higher LA isomerase (LA-I) activity, and was much more tolerant to linoleic acid (LA) than other strains examined. However, high CLA reductase (CLA-R) activity resulted in the temporary accumulation of CLA and subsequent conversion to trans-vaccenic acid (t-VA). When LA was added to growing TH1 cultures in a solution with dimethylsulfoxide (LA/DMSO), CLA produced was greater than when LA was added in a mixture with bovine serum albumin (BSA). The number of viable cells decreased upon addition of LA/DMSO, but then increased as the CLA decreased upon its conversion to t-VA. This result suggests that B. fibrisolvens can resume growing by the removal of CLA from the cells. Most CLA was released from B. fibrisolvens cells by gentle washing with BSA, suggesting that CLA bound to the cells might be removed in the rumen and large intestine. Thus, CLA production by B. fibrisolvens in the digestive tract could be increased by a reduction in CLA-R activity without accompanying an overall decrease in the cell number of B. fibrisolvens. Fatty acids (FAs) with 18 carbon backbone inducted LA-I activity, whereas unsaturated FAs induced CLA-R activity, suggesting that FAs stimulate the synthesis of LA-I and CLA-R. Providing a diet with a low ratio of unsaturated to saturated FAs may favor CLA production.