Increasing carbohydrate availability in the hindgut promotes hypothalamic neurotransmitter synthesis: aromatic amino acids linking the microbiota-brain axis.

The gut microbiota is increasingly recognized to modulate brain function by recent studies demonstrating the central effects of various gut microbial manipulation strategies. Our previous study demonstrated that antibiotic-induced alterations of hindgut microbiota are associated with changes in aromatic amino acid (AAA) metabolism and hypothalamic neurochemistry, while the underlying mechanistic insight is limited. Given that the microbial AAA metabolism can be affected by luminal carbohydrate availability, here we hypothesize that increasing hindgut carbohydrate availability affects the expression of neurotransmitters in the porcine hypothalamus. A hindgut microbiota-targeted strategy was adopted by increasing hindgut carbohydrate availability in a cecal-cannulated piglet model. Mechanistic involvement of AAAs along the gut microbiota-brain axis was further investigated in mice and neuronal cells. Increasing carbohydrate availability by cecal starch infusion led to a decrease in hindgut AAA metabolism, and an increase in systemic AAA availability, central AAA-derived neurotransmitters (5-HT, dopamine), and neurotrophin BDNF in piglets, indicating that hindgut microbiota affect hypothalamic neurochemistry in an AAA-dependent manner. Single AAA i.p. injection in mice revealed that an increase in circulating tryptophan and tyrosine elevated their concentrations in brain and finally promoted the expressions of 5-HT, dopamine, and BDNF in a time-dependent manner. Neuronal cells treated with single AAAs in vitro further demonstrated that tryptophan and tyrosine enhanced 5-HT and dopamine synthesis, respectively, and promoted BDNF expression partly through the 5-HT1A/DRD1-CREB pathway. Our study reveals that increasing hindgut carbohydrate availability promotes hypothalamic neurotransmitter synthesis and that AAAs act as potential mediators between hindgut microbiota and brain neurochemistry.

Antibiotics-induced modulation of large intestinal microbiota altered aromatic amino acid profile and expression of neurotransmitters in the hypothalamus of piglets.

The evidence of gut microbiota-mediated modulation of brain function has been widely recognized from studies using germ-free rodents or animals with oral antibiotic-induced microbiota depletion. Since the large intestine harbors greater numbers and more diverse of microbes than in the small intestine, large intestinal microbiota may play a crucial role in the modulation of brain function. In this study, a large intestinal microbiota-targeted strategy was used to investigate the impact of large intestinal microbiota on brain function. Twelve piglets (12.08 ± 0.28 kg) fitted with a T-cannula at the distal ileum were fed a standard diet and randomly assigned to two groups (n = 6) for ileal infusion of either saline or antibiotics. After 25 days of infusion, ileal and fecal microbiota, serum amino acids and neurotransmitters, and hypothalamic transcriptomics were analyzed. While the antibiotic infusion did not change the proximal ileal microbial composition, it markedly altered the fecal microbial composition and increased aromatic amino acid (AAAs) metabolism (p < 0.05), suggesting the infusion specifically targeted large intestinal microbes. Concentrations of AAAs were likewise decreased in the blood and hypothalamus (p < 0.05) by antibiotic infusion. Antibiotic infusion further decreased concentrations of hypothalamic 5-hydroxytryptamine (5-HT) and dopamine, in line with AAAs being their precursors. An up-regulation in gene expressions of neurotransmitter transporters and synthetases was observed (q < 0.001). In conclusion, the distalileal-antibiotic infusion altered neurotransmitter expression in the porcine hypothalamus and this effect occurred simultaneously with changes in both the large intestinal microbiota, and AAAs in the large intestine, blood and hypothalamus. These findings indirectly indicate that large intestinal microbiota affects hypothalamic neurotransmitter expressions. Read the Editorial Highlight for this article on page 208.

Time-course responses of ileal and fecal microbiota and metabolite profiles to antibiotics in cannulated pigs.

We investigated the time-course effects of therapeutic antibiotics on intestinal microbial composition and metabolism in an ileal-cannulated pig model. Sixteen ileal-cannulated piglets (12 ± 0.5 kg) were assigned to two groups (n = 8) and fed standard diets with or without antibiotics. At 4 days before, and at days 2, 7, and 13 after antibiotic administration, ileal and fecal samples were collected for analysis of microbiota composition via 16S rRNA MiSeq sequencing and metabolites (short-chain fatty acids, biogenic amines, and indole). It was found that Lactobacillus and Bifidobacterium had decreased by an average 2.68-fold and 508-fold in ileum on days 2-13, and by an average 45.08-fold and 71.50-fold in feces on days 7-13 (P < 0.05). Escherichia/Shigella had increased by an average 265-fold in ileum on days 2-13, and by an average 36.70-fold in feces on days 7-13 (P < 0.05). Acetate concentration had decreased in ileum by an average 2.88-fold on days 2-13, and by 1.83-fold in feces on day 7 (P < 0.05). Cadaverine concentration had increased by an average 7.03-fold in ileum on days 2-13, and by an average 9.96-fold in feces on days 7-13 (P < 0.05), and fecal indole concentration had increased by an average 2.51-fold on days 7-13 (P < 0.05). Correlation analysis between significant microbes and metabolites indicated that the antibiotic-induced microbiota shift appeared to result in the changes of intestinal metabolism. In conclusion, antibiotic administration led to dynamic changes in microbial communities and metabolism in ileum and feces, with ileal microbiota being more prone to shift than fecal microbiota.

Microbiome-metabolome analysis reveals unhealthy alterations in the composition and metabolism of ruminal microbiota with increasing dietary grain in a goat model.

Currently, knowledge about the impact of high-grain (HG) feeding on rumen microbiota and metabolome is limited. In this study, a combination of the 454 pyrosequencing strategy and the mass spectrometry-based metabolomics technique was applied to investigate the effects of increased dietary grain (0%, 25% and 50% maize grain) on changes in whole ruminal microbiota and their metabolites using goat as a ruminant model. We observed a significant influence of HG feeding in shaping the ruminal bacterial community structure, diversity and composition, with an overall dominance of bacteria of the phylum Firmicutes along with a low abundance of Bacteriodetes in the HG group. High-grain feeding increased the number of ciliate and methanogens, and decreased the density of anaerobic fungi and the richness of the archaeal community. The metabolomics analysis revealed that HG feeding increased the levels of several toxic, inflammatory and unnatural compounds, including endotoxin, tryptamine, tyramine, histamine and phenylacetate. Correlation analysis on the combined datasets revealed some potential relationships between ruminal metabolites and certain microbial species. Information about these relationships may prove useful in either direct (therapeutic) or indirect (dietary) interventions for ruminal disorders due to microbial compositional shifts, such as ruminal acidosis.

Bromochloromethane, a Methane Analogue, Affects the Microbiota and Metabolic Profiles of the Rat Gastrointestinal Tract.

Bromochloromethane (BCM), an inhibitor of methanogenesis, has been used in animal production. However, little is known about its impact on the intestinal microbiota and metabolic patterns. The present study aimed to investigate the effect of BCM on the colonic bacterial community and metabolism by establishing a Wistar rat model. Twenty male Wistar rats were randomly divided into two groups (control and treated with BCM) and raised for 6 weeks. Bacterial fermentation products in the cecum were determined, and colonic methanogens and sulfate-reducing bacteria (SRB) were quantified. The colonic microbiota was analyzed by pyrosequencing of the 16S rRNA genes, and metabolites were profiled by gas chromatography and mass spectrometry. The results showed that BCM did not affect body weight and feed intake, but it did significantly change the intestinal metabolic profiles. Cecal protein fermentation was enhanced by BCM, as methylamine, putrescine, phenylethylamine, tyramine, and skatole were significantly increased. Colonic fatty acid and carbohydrate concentrations were significantly decreased, indicating the perturbation of lipid and carbohydrate metabolism by BCM. BCM treatment decreased the abundance of methanogen populations, while SRB were increased in the colon. BCM did not affect the total colonic bacterial counts but significantly altered the bacterial community composition by decreasing the abundance of actinobacteria, acidobacteria, and proteobacteria. The results demonstrated that BCM treatment significantly altered the microbiotic and metabolite profiles in the intestines, which may provide further information on the use of BCM in animal production.

Discovery of a novel rumen methanogen in the anaerobic fungal culture and its distribution in the rumen as revealed by real-time PCR.

BACKGROUND: The novel archaea belonging to Rumen Cluster C (RCC), which may play an important role in methane production in the rumen have received increased attention. However, the present information on RCC in the rumen is limited by the unsuccessful isolation of axenic pure RCC from the rumen. In the present study, RCC grown in anaerobic fungal subcultures was identified by the molecular and culture methods. RESULTS: A novel RCC species existing in the fungal subcultures was identified and demonstrated by the 16S rRNA gene clone library. Interestingly, the novel RCC species survived in the fungal cultures over all the subculture transferring, even in the 62nd subculture, in contrast to the other methanogens, which disappeared during subcultures. Further work showed that subculture transfer frequency significantly affected the relative abundance of the novel RCC species in the fungal subcultures. The five-day and seven-day transfer frequencies increased the relative abundance of the RCC species (P<0.05). In addition, quantitative real-time PCR revealed that high concentrate diets did not affect the abundance of archaea, but numerically reduced the abundance of the novel RCC species in the rumen. In addition, the relative abundance of the RCC species was numerically higher in the rumen liquid fraction than in the rumen epithelium and solid fractions. Finally, a purified fungal culture containing the RCC species was successfully obtained. PCR and sequencing analysis showed that the novel RCC species contained a mcrA gene, which is known to play a crucial role in methanogenesis, and thus could be identified as a methanogen. CONCLUSION: In this study, a novel RCC species was identified as a methanogen and closely associated with anaerobic fungi. This novel approach by using co-culture with anaerobic fungi may provide a feasible way to culture and investigate not yet identified methanogens.

Important impacts of intestinal bacteria on utilization of dietary amino acids in pigs.

Bacteria in pig intestine can actively metabolize amino acids (AA). However, little research has focused on the variation in AA metabolism by bacteria from different niches. This study compared the metabolism of AA by microorganisms derived from the lumen and epithelial wall of the pig small intestine, aiming to test the hypothesis that the metabolic profile of AA by gut microbes was niche specific. Samples from the digesta, gut wall washes and gut wall of the jejunum and ileum were used as inocula. Anaerobic media containing single AA were used and cultured for 24 h. The 24-h culture served as inocula for the subsequent 30 times of subcultures. Results showed that for the luminal bacteria, all AA concentrations except phenylalanine in the ileum decreased during the 24-h in vitro incubation with a increase of ammonia concentration, while 4 AA (glutamate, glutamine, arginine and lysine) in the jejunum decreased, with the disappearance rate at 60-95 %. For tightly attached bacteria, all AA concentrations were generally increased during the first 12 h and then decreased coupled with first a decrease and then an increase of ammonia concentration, suggesting a synthesis first and then a catabolism pattern. Among them, glutamate in both segments, histidine in the jejunum and lysine in the ileum increased significantly during the first 12 h and then decreased at 24 h. The concentrations of glutamine and arginine did not change during the first 12 h, but significantly decreased at 24 h. Jejunal lysine and ileal threonine were increased for the first 6 or 12 h. For the loosely attached bacteria, there was no clear pattern for the entire AA metabolism. However, glutamate, methionine and lysine in the jejunum decreased after 24 h of cultivation, while glutamine and threonine in the jejunum and glutamine and lysine in the ileum increased in the first 12 h. During subculture, AA metabolism, either utilization or synthesis, was generally decreased with disappearance rate around 20-40 % for most of AA and negligible for branch chained AA (BCAA). However, the disappearance rate of lysine in each group was around 90 % throughout the subculture, suggesting a high utilization of lysine by bacteria from all three compartments. Analysis of the microbial community during the 24-h in vitro cultivation revealed that bacteria composition in most AA cultures varied between different niches (lumen and wall-adherent fractions) in the jejunum, while being relatively similar in the ileum. However, for isoleucine and leucine cultures, bacteria diversity was similar between the luminal fraction and tightly attached fraction, but significantly higher than in the loosely attached fraction. For glutamine and valine cultures, bacteria diversity was similar between the luminal and loosely attached fractions, but lower than that of tightly attached bacteria. After 30 subcultures, bacteria diversity in arginine, valine, glutamine, and leucine cultures varied between niches in the jejunum while being relatively stable in the ileum, consistent with those in the 24-h in vitro cultures. The findings may suggest that luminal bacteria tended to utilize free AA, while tightly attached adherent bacteria seemed in favor of AA synthesis, and that small intestinal microbes contributed little to BCAA metabolism.

Effects of Acarbose Addition on Ruminal Bacterial Microbiota, Lipopolysaccharide Levels and Fermentation Characteristics In vitro.

This study investigated the effects of acarbose addition on changes in ruminal fermentation characteristics and the composition of the ruminal bacterial community in vitro using batch cultures. Rumen fluid was collected from the rumens of three cannulated Holstein cattle fed forage ad libitum that was supplemented with 6 kg of concentrate. The batch cultures consisted of 8 mL of strained rumen fluid in 40 mL of an anaerobic buffer containing 0.49 g of corn grain, 0.21 g of soybean meal, 0.15 g of alfalfa and 0.15g of Leymus chinensis. Acarbose was added to incubation bottles to achieve final concentrations of 0.1, 0.2, and 0.4 mg/mL. After incubation for 24 h, the addition of acarbose linearly decreased (p<0.05) the total gas production and the concentrations of acetate, propionate, butyrate, total volatile fatty acids, lactate and lipopolysaccharide (LPS). It also linearly increased (p<0.05) the ratio of acetate to propionate, the concentrations of isovalerate, valerate and ammonia-nitrogen and the pH value compared with the control. Pyrosequencing of the 16S rRNA gene showed that the addition of acarbose decreased (p<0.05) the proportion of Firmicutes and Proteobacteria and increased (p<0.05) the percentage of Bacteroidetes, Fibrobacteres, and Synergistetes compared with the control. A principal coordinates analysis plot based on unweighted UniFrac values and molecular variance analysis revealed that the structure of the ruminal bacterial communities in the control was different to that of the ruminal microbiota in the acarbose group. In conclusion, acarbose addition can affect the composition of the ruminal microbial community and may be potentially useful for preventing the occurrence of ruminal acidosis and the accumulation of LPS in the rumen.

A high-grain diet causes massive disruption of ruminal epithelial tight junctions in goats.

Alterations in rumen epithelial tight junctions (TJs) at the tissue and molecular levels during high-grain (HG) diet feeding are unknown. Here, 10 male goats were randomly assigned to either a hay diet (0% grain; n = 5) or HG diet group (65% grain; n = 5) to characterize the changes in ruminal epithelial structure and TJ protein expression and localization using scanning and transmission electron microscopy, quantitative real-time PCR, Western blot analysis, and immunofluorescence. After 7 wk of feeding, ruminal free LPS in HG group increased significantly (P < 0.001) compared with the hay group, and free LPS in the peripheral blood was detectable with concentrations of 0.8 ± 0.20 EU/ml, while not detectable in the control, suggesting a leakage of LPS into the blood in the HG group. Correspondingly, the HG-fed goats showed profound alterations in ruminal epithelial structure and TJ proteins, depicted by marked epithelial cellular damage and intercellular junction erosion, down-regulation of TJ proteins claudin-4, occludin, and zonula occludens-1 mRNA and protein expression, as well as redistribution of claudin-1, claudin-4, and occludin. Furthermore, these changes in TJ proteins in the HG group were coupled with the upregulation of mRNA levels for the cytokines TNF-α and IFN-γ in the ruminal epithelia. These results demonstrated for the first time that the HG diet feeding caused disruption of ruminal epithelial TJs that was associated with a local inflammatory response in the rumen epithelium. These findings may provide new insights into understanding the role of TJ proteins in the ruminal epithelial immune homeostasis of ruminants.

L-Glutamine regulates amino acid utilization by intestinal bacteria.

Catabolism of amino acids (AA) by intestinal bacteria greatly affects their bioavailability in the systemic circulation and the health of animals and humans. This study tests the novel hypothesis that L-glutamine regulates AA utilization by luminal bacteria of the small intestine. Pure bacterial strains (Streptococcus sp., Escherichia coli and Klebsiella sp.) and mixed bacterial cultures derived from the jejunum or ileum of pigs were cultured in the presence of 0-5 mM L-glutamine under anaerobic conditions. After 3 h of incubation, samples were taken for the determination of AA utilization. Results showed concentration-dependent increases in the utilization of glutamine in parallel with the increased conversion of glutamine into glutamate in all the bacteria. Complete utilization of asparagine, aspartate and serine was observed in pure bacterial strains after the 3-h incubation. The addition of glutamine reduced the net utilization of asparagine by both jejunal and ileal mixed bacteria. Net utilization of lysine, leucine, valine, ornithine and serine by jejunal or ileal mixed bacteria decreased with the addition of glutamine in a concentration-dependent manner. Collectively, glutamine dynamically modulates the bacterial metabolism of the arginine family of AA as well as the serine and aspartate families of AA and reduced the catabolism of most AA (including nutritionally essential and nonessential AA) in jejunal or ileal mixed bacteria. The beneficial effects of glutamine on gut nutrition and health may involve initiation of the signaling pathways related to AA metabolism in the luminal bacteria of the small intestine.

Fecal microbiota of piglets prefer utilizing DL-lactate mixture as compared to D-lactate and L-lactate in vitro.

To compare the fermentation characteristics of D-lactate, L-lactate and DL-lactate mixture by fecal microbiota of pigs, in vitro fermentation test was conducted with D-lactate, L-lactate and DL-lactate mixture as substrates, feces of piglets as inocula. The concentrations of lactate and short chain fatty acid (SCFA) were quantified during the fermentation. The composition of bacterial communities in the inocula and 24 h fermentation fluids was analyzed by pyrosequencing. The results showed that, when 5 mmol/l lactate was used, there was no significant difference in utilizing efficiency among D-lactate, L-lactate and DL-lactate mixture, propionate and butyrate were the major end-products converted from lactate. However, when 25 mmol/l lactates were used, a higher utilizing efficiency of DL-lactate mixture and a slower utilizing rate of D-lactate were observed, acetate and propionate became the main end-products. The SCFA proportions were significantly affected by lactate source and its concentration. Pyrosequencing analysis showed that after 24 h fermentation, significant difference in the composition of bacterial communities (genus and OTU levels) was observed among different substrate groups. The results suggest that lactate accumulation in the hindgut is related to the cooperation between microbiota members, and regulating bacterial community may be a possible way to control lactate accumulation.

Individual and combined effects of genistein and hesperidin supplementation on meat quality in meat-type broiler chickens.

BACKGROUND: There is growing interest in improving the production and meat quality of farm animals through dietary supplementation with phytochemical (e.g. flavonoids)-rich plants and/or their extracts. This study was conducted to analyse the supplemental effects of two purified flavonoids (genistein and hesperidin) individually and in combination on the oxidative status, sensory score and quality of breast meat in meat-type broiler chickens. RESULTS: A significant increase (P < 0.05) in meat colour (L* score) and pH was observed for the group supplemented with 20 mg kg(-1) genistein and hesperidin. Water-holding capacity was also improved significantly (P < 0.01) for all genistein- and hesperidin-treated groups, while the sensory quality of breast meat remained unaffected. Lipid oxidation of breast meat was reduced significantly (P < 0.01) at 0 and 15 days of refrigeration in a dose-dependent manner for all supplemented groups. Meanwhile, some treated groups showed improved (P < 0.05) body weight, feed/gain ratio and hot carcass weight. CONCLUSION: Genistein and hesperidin supplementation to broilers improved meat quality in a dose-dependent fashion, with pronounced effects of combined treatment. The results indicated that purified flavonoids such as genistein and hesperidin could potentially be used as feed additives in broiler production to promote meat quality.

Development of Gut Microbiota in a Mouse Model of Ovalbumin-induced Allergic Diarrhea under Sub-barrier System.

This study aimed to present a mouse model of ovalbumin (OVA) induced allergic diarrhea under a sub-barrier system and investigate the development of gut microbiota in this model. Male BALB/c mice were systemically sensitized with OVA or sham-sensitized with saline, and followed by oral OVA intubation, leading to OVA-specific acute diarrhea. Compared with sham-sensitized mice, sera OVA-specific IgG1 and total IgE in OVA-sensitized mice were dramatically elevated, and the number of mast cells was greatly increased in the jejunum of the OVA-sensitized mice. Principle component analysis of the DGGE profile showed that samples from group of OVA-sensitized mice and group of sham-sensitized mice were scattered into two different regions. Real-time PCR analysis showed that the number of 16S rRNA gene copies of Lactobacillus in the colon of OVA-sensitized mice decreased significantly, while there was no significant difference in the number of Bifidobacterium and total bacteria. In conclusion, OVA-specific allergic diarrhea was successfully induced under a sub-barrier system, and changes of allergic reactions during induction was coupled with changes in gut microbiota, especially the number of colonic Lactobacillus, but the role of gut microbiota in the development of food allergic reactions needs to be further evaluated.

Production of Citrate by Anaerobic Fungi in the Presence of Co-culture Methanogens as Revealed by (1)H NMR Spectrometry.

The metabolomic profile of the anaerobic fungus Piromyces sp. F1, isolated from the rumen of goats, and how this is affected by the presence of naturally associated methanogens, was analyzed by nuclear magnetic resonance spectroscopy. The major metabolites in the fungal monoculture were formate, lactate, ethanol, acetate, succinate, sugars/amino acids and α-ketoglutarate, whereas the co-cultures of anaerobic fungi and associated methanogens produced citrate. This is the first report of citrate as a major metabolite of anaerobic fungi. Univariate analysis showed that the mean values of formate, lactate, ethanol, citrate, succinate and acetate in co-cultures were significantly higher than those in the fungal monoculture, while the mean values of glucose and α-ketoglutarate were significantly reduced in co-cultures. Unsupervised principal components analysis revealed separation of metabolite profiles of the fungal mono-culture and co-cultures. In conclusion, the novel finding of citrate as one of the major metabolites of anaerobic fungi associated with methanogens may suggest a new yet to be identified pathway exists in co-culture. Anaerobic fungal metabolism was shifted by associated methanogens, indicating that anaerobic fungi are important providers of substrates for methanogens in the rumen and thus play a key role in ruminal methanogenesis.

Effect of Saccharomyces cerevisiae Fermentation Product on Lactation Performance and Lipopolysaccharide Concentration of Dairy Cows.

To evaluate lactation performance and changes in plasma and fecal lipopolysaccharide (LPS) concentrations in response to the supplementation of Saccharomyces cerevisiae fermentation product (SC), two dairy farms were selected. On each farm, 32 cows in early to mid lactation (21 to 140 DIM) were blocked by parity and days in milk (DIM), and randomly assigned to one of the two treatments within block (Control or 56 g SC/cow/d). Effect of SC on lactation performance (daily) and changes in blood and fecal LPS level were examined on d 0 and 28 of supplementation. The results showed that SC supplementation increased lactation performance of dairy cows on both farms. On Farm 1, milk production, 3.5% fat corrected milk (FCM), and yield of milk fat and protein were greater (p<0.01) for cows supplemented with SC. Supplementation of SC increased percentage milk fat (p = 0.029) from 81 to 110 DIM. There was no significant effect (p>0.05) of SC supplementation on percentage of milk protein, dry matter intake and feed efficiency. On Farm 2, cows supplemented with SC had a greater (p<0.05) milk yield, percentage of milk fat and milk protein, yield of milk fat and protein, 3.5% FCM and feed efficiency. Supplemental SC had no effect on LPS concentrations in feces (p>0.05) while it trended to reduce (p = 0.07 or 0.207) the concentration in plasma. The results indicate that supplemental SC can increase lactation performance of dairy cattle and has potential for reducing plasma LPS concentration.

An immortal porcine preadipocyte cell strain for efficient production of cell-cultured fat.

Adding adipose cells to cell-cultured meat can provide a distinctive aroma and juicy texture similar to real meat. However, a significant challenge still exists in obtaining seed cells that can be propagated for long periods, maintain their adipogenic potential, and reduce production costs. In this study, we present a cell strain derived from immortalized porcine preadipocytes that can be subculture for over 40 passages without losing differentiation capacity. This cell strain can be differentiated within 3D bioscaffolds to generate cell-cultured fat using fewer chemicals and less serum. Additionally, it can be expanded and differentiated on microcarriers with upscaled culture to reduce costs and labor. Moreover, it can co-differentiate with muscle precursor cells, producing a pattern similar to real meat. Therefore, our cell strain provides an exceptional model for studying and producing cell-cultured fat.

Lean breed Landrace pigs harbor fecal methanogens at higher diversity and density than obese breed Erhualian pigs.

The diversity of fecal methanogens of Erhualian (obese type) and Landrace (lean type) pigs was examined using separate 16S rRNA gene libraries for each breed. A total of 763 clones were analyzed; 381 from the Erhualian library and 382 from the Landrace library were identified belonging to the genus Methanobrevibacter. Others were identified belonging to the genus Methanosphaera. The two libraries showed significant differences in diversity (P < 0.05) and composition (P < 0.0001). Only two operational taxonomic units (OTUs) were found in both libraries, whereas six OTUs were found only in the Erhualian library and 23 OTUs were found only in the Landrace library. Real-time PCR showed that the abundance of fecal methanogens in Landrace pigs was significantly higher than that in Erhualian pigs (P < 0.05). Results showed that the Landrace pig (lean) harbored a greater diversity and higher numbers of methanogen mcrA gene copies than the Erhualian pig (obese). These differences may be related to the fatness or leanness in these two pig breeds. The results provide new leads for further investigations on the fat storage of pigs or even humans.

Metabolism of select amino acids in bacteria from the pig small intestine.

This study investigated the metabolism of select amino acids (AA) in bacterial strains (Streptococcus sp., Escherichia coli and Klebsiella sp.) and mixed bacterial cultures derived from the jejunum and ileum of pigs. Cells were incubated at 37°C for 3 h in anaerobic media containing 0.5-5 mM select AA plus [U-14C]-labeled tracers to determine their decarboxylation and incorporation into bacterial protein. Results showed that all types of bacteria rapidly utilized glutamine, lysine, arginine and threonine. However, rates of the utilization of AA by pure cultures of E. coli and Klebsiella sp. were greater than those for mixed bacterial cultures or Streptococcus sp. The oxidation of lysine, threonine and arginine accounted for 10% of their utilization in these pure bacterial cultures, but values were either higher or lower in mixed bacterial cultures depending on AA, bacterial species and the gut segment (e.g., 15% for lysine in jejunal and ileal mixed bacteria; 5.5 and 0.3% for threonine in jejunal mixed bacteria and ileal mixed bacteria, respectively; and 20% for arginine in ileal mixed bacteria). Percentages of AA used for bacterial protein synthesis were 50-70% for leucine, 25% for threonine, proline and methionine, 15% for lysine and arginine and 10% for glutamine. These results indicate diverse metabolism of AA in small-intestinal bacteria in a species- and gut compartment-dependent manner. This diversity may contribute to AA homeostasis in the gut. The findings have important implications for both animal and human nutrition, as well as their health and well-beings.

Regulatory role for L-arginine in the utilization of amino acids by pig small-intestinal bacteria.

We recently reported that bacteria from the pig small intestine rapidly utilize and metabolize amino acids (AA). This study investigated the effect of L-arginine on the utilization of AA by pure bacterial strains (Streptococcus sp., Escherichia coli and Klebsiella sp.) and mixed bacterial cultures derived from the pig small intestine. Bacteria were incubated at 37°C for 3 h in anaerobic AA media containing 0-5 mmol/L of arginine to determine the effect of arginine on the bacterial utilization of AA. Amino acids in the medium plus cell extracts were analyzed by high-performance liquid chromatography. Results indicated concentration-dependent increases in the bacterial utilization of arginine and altered fluxes of arginine into ornithine and citrulline in the bacteria. Net glutamine utilization increased in pure bacterial strains with increased concentrations of arginine. With the addition of arginine, net utilization of threonine, glycine, phenylalanine and branched-chain AA increased (P<0.05) in Streptococcus sp. and Klebsiella sp., but decreased in E. coli. Net utilization of lysine, threonine, isoleucine, leucine, glycine and alanine by jejunal or ileal mixed bacteria decreased (P<0.05) with the addition of arginine. Complete utilization of asparagine, aspartate and serine were observed in pig small-intestinal bacteria after 3 h of incubation. Overall, the addition of arginine affected the metabolism of the arginine-family of AA and the serine- and aspartate-family of AA in small-intestinal bacteria and reduced the utilization of most AA in ileal mixed bacteria. These novel findings indicate that arginine exerts its beneficial effects on swine nutrition partially by regulating AA utilization and metabolism in the small-intestinal microbiota.

Efficacy and Safety of Ceftaroline Fosamil in Hospitalized Patients with Community-Acquired Pneumonia in China: Subset Analysis of an International Phase 3 Randomized Controlled Trial.

BACKGROUND: Ceftaroline fosamil has demonstrated superior clinical efficacy versus ceftriaxone for hospitalized adults with moderate-to-severe community-acquired pneumonia (CAP) in a Phase 3 trial in Asia and in a meta-analysis of three trials in Asia, North America, and Europe. Efficacy and safety outcomes for the subset of patients in China in the ASIA CAP trial were analyzed to determine if the same conclusions hold in the China subpopulation. METHODS: Hospitalized adults with Pneumonia Outcomes Research Team risk class III-IV CAP were randomized (1:1) to receive either intravenous ceftaroline fosamil 600 mg every 12 h or ceftriaxone 2 g every 24 h for 5-7 days. The primary efficacy variable was clinical response at test-of-cure (TOC) in the clinically evaluable (CE) population. Secondary endpoints included microbiological responses and safety. RESULTS: Of 302 patients randomized in China, 205 were included in the CE population. Clinical cure rates at TOC were 80/105 (76.2%) for ceftaroline fosamil and 61/100 (61.0%) for ceftriaxone (difference 15.2%, 95% CI 2.5, 27.6), thereby meeting predefined non-inferiority and superiority criteria for the overall study. Subgroup analyses of the primary endpoint demonstrated consistency of favourable efficacy of ceftaroline fosamil across age groups, Pneumonia Outcomes Research Team risk classes and CURB-65 scores. Microbiological responses were presumed from clinical outcomes. Adverse events were consistent with the study treatments' known safety profiles. CONCLUSION: The China subset results are consistent with the overall study population, despite the smaller sample size. Ceftaroline fosamil was both non-inferior and superior to ceftriaxone for empiric treatment of Chinese patients with moderate-to-severe CAP. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT01371838.