Gut microbiota dynamics and fecal SCFAs after colonoscopy: accelerating microbiome stabilization by Clostridium butyricum.

BACKGROUND: Colonoscopy is a classic diagnostic method with possible complications including abdominal pain and diarrhoea. In this study, gut microbiota dynamics and related metabolic products during and after colonoscopy were explored to accelerate gut microbiome balance through probiotics. METHODS: The gut microbiota and fecal short-chain fatty acids (SCFAs) were analyzed in four healthy subjects before and after colonoscopy, along with seven individuals supplemented with Clostridium butyricum. We employed 16S rRNA sequencing and GC-MS to investigate these changes. We also conducted bioinformatic analysis to explore the buk gene, encoding butyrate kinase, across C. butyricum strains from the human gut. RESULTS: The gut microbiota and fecal short-chain fatty acids (SCFAs) of four healthy subjects were recovered on the 7th day after colonoscopy. We found that Clostridium and other bacteria might have efficient butyric acid production through bioinformatic analysis of the buk and assessment of the transcriptional level of the buk. Supplementation of seven healthy subjects with Clostridium butyricum after colonoscopy resulted in a quicker recovery and stabilization of gut microbiota and fecal SCFAs on the third day. CONCLUSION: We suggest that supplementation of Clostridium butyricum after colonoscopy should be considered in future routine clinical practice.

Effect of Clostridium butyricum on High-Fat Diet-Induced Intestinal Inflammation and Production of Short-Chain Fatty Acids.

BACKGROUND/AIMS: A high-fat diet (HFD) can cause intestinal inflammation and alter the gut microbiota; probiotics, however, are known to have anti-inflammatory effects. This study aimed to investigate the response of rat colon to HFD and the effect of Clostridium butyricum on HFD-induced intestinal inflammation and production of short-chain fatty acids (SCFAs) according to sex. METHODS: Male and female 6-week-old Fischer-344 rats were fed a chow diet or HFD for 8 weeks, and Biovita or three different concentrations of C. butyricum were orally gavaged. The levels of tight junction proteins (TJPs), inflammatory markers in the ascending colonic mucosa, and bile acids (BAs) and SCFAs in stool were measured. RESULTS: HFD significantly increased the histological inflammation scores and fat proportions. Fecal BA levels were higher in the HFD group than in the control group, with a more prominent increase in deoxycholic acid/cholic acid after probiotics administration in females; however, no statistically significant differences were observed. TJPs showed an opposite response to HFD depending on sex, and tended to increase and decrease after HFD in males and females, respectively. The HFD-reduced TJPs were recovered by probiotics, with some statistical significance in females. HFD-decreased butyric acid in stools appeared to be recovered by probiotics in males, but not in females. The expression of inflammatory markers (TNF-α) was increased by HFD in males and decreased with medium-concentration probiotic supplementation. The opposite was observed in females. MPO was increased by HFD in both sexes and decreased by probiotic supplementation. CONCLUSIONS: The probiotic C. butyricum improved indicators of HFD-induced colonic inflammation such as levels of inflammatory markers and increased the production of SCFAs and the expression of TJPs. These effects tended to be more pronounced in male rats, showing sex difference.

Clostridium butyricum-induced ω-3 fatty acid 18-HEPE elicits anti-influenza virus pneumonia effects through interferon-λ upregulation.

The precise mechanism by which butyrate-producing bacteria in the gut contribute to resistance to respiratory viral infections remains to be elucidated. Here, we describe a gut-lung axis mechanism and report that orally administered Clostridium butyricum (CB) enhances influenza virus infection resistance through upregulation of interferon (IFN)-λ in lung epithelial cells. Gut microbiome-induced ω-3 fatty acid 18-hydroxy eicosapentaenoic acid (18-HEPE) promotes IFN-λ production through the G protein-coupled receptor (GPR)120 and IFN regulatory factor (IRF)-1/-7 activations. CB promotes 18-HEPE production in the gut and enhances ω-3 fatty acid sensitivity in the lungs by promoting GPR120 expression. This study finds a gut-lung axis mechanism and provides insights into the treatments and prophylaxis for viral respiratory infections.

Acceleration of lactate-utilizing pathway for enhancing biohydrogen production by magnetite supplementation in Clostridium butyricum.

A conductive metal compound can be used as a catalyst for enhancing hydrogen production by dark fermentation. This study aimed to identify mechanisms of enhanced hydrogen production by magnetite supplementation. Experiments were performed with lactate and/or magnetite supplementation to confirm that the lactate-utilizing pathway is the key cause of enhanced hydrogen production. Also, ribonucleic acid sample was collected for monitoring gene regulation under each condition. Hydrogen production was significantly enhanced by approximately 25.6% and 58.9%, respectively, via magnetite alone and with lactate. Moreover, the expression of genes involved in hydrogen production, including pyruvate ferredoxin oxidoreductase, hydrogenase, and ferredoxin, via magnetite alone and with lactate was upregulated by 0.26, 0.71, and 3.50 and 1.06, 2.14, and 1.94 times, respectively.

Butyrate-producing human gut symbiont, Clostridium butyricum, and its role in health and disease.

Clostridium butyricum is a butyrate-producing human gut symbiont that has been safely used as a probiotic for decades. C. butyricum strains have been investigated for potential protective or ameliorative effects in a wide range of human diseases, including gut-acquired infection, intestinal injury, irritable bowel syndrome, inflammatory bowel disease, neurodegenerative disease, metabolic disease, and colorectal cancer. In this review we summarize the studies on C. butyricum supplementation with special attention to proposed mechanisms for the associated health benefits and the supporting experimental evidence. These mechanisms center on molecular signals (especially butyrate) as well as immunological signals in the digestive system that cascade well beyond the gut to the liver, adipose tissue, brain, and more. The safety of probiotic C. butyricum strains appears well-established. We identify areas where additional human randomized controlled trials would provide valuable further data related to the strains' utility as an intervention.

Bacteria producing antimicrobials against Clostridium difficile isolated from human stool.

Clostridium difficile infection (CDI) is a common cause of morbidity and mortality in hospitalized patients worldwide. The major problem facing current treatment is multiple recurrences, prompting the need for alternative therapies. In this study we isolated bacterial species, from Egyptian individuals' stool, with antimicrobial activity against clinical isolates of C. difficile and tried to examine the nature of the produced antimicrobials. In vitro antibacterial activity against C. difficile was initially screened in 123 fecal samples cultures using an agar overlay method. The isolates with antimicrobial activity against C. difficile in addition to Clostridium isolates were identified using partial 16S rDNA gene sequencing analysis. The isolates acting against C. difficile belonged to Lactobacillus, Enterococcus and Clostridium genera. The concentrated cell-free supernatants (CFSs) from these bacterial isolates were examined for antimicrobial activity against C. difficile growth by broth dilution method. 10 x concentrated CFSs of five isolates showed inhibition for C. difficile growth which was significantly different (p < 0.001) from control. Lactobacillus agilis T99A and Clostridium butyricum T58A isolates were selected for further evaluation of the produced antimicrobials. The antimicrobial activity of 10x CFSs of the two isolates was stable after enzymatic treatment with proteinase K or heating treatments up to 90 °C or neutralizing pH. The spectrum of activity of the two isolates was evaluated using different gram-positive and gram-negative bacterial species and did not show antimicrobial activity against these species. Our results showed two unconventional bacterial isolates: L. agilis T99A and C. butyricum T58A producing extracellular thermo stable antimicrobial agents against C. difficile clinical isolates.

The effect of dietary supplementation with Clostridium butyricum on the growth performance, immunity, intestinal microbiota and disease resistance of tilapia (Oreochromis niloticus).

This study was conducted to assess the effects of dietary Clostridium butyricum on the growth, immunity, intestinal microbiota and disease resistance of tilapia (Oreochromis niloticus). Three hundreds of tilapia (56.21 ± 0.81 g) were divided into 5 groups and fed a diet supplemented with C. butyricum at 0, 1 x 104, 1 x 105, 1 x 106 or 1 x 107 CFU g-1 diet (denoted as CG, CB1, CB2, CB3 and CB4, respectively) for 56 days. Then 45 fish from each group were intraperitoneally injected with Streptococcus agalactiae, and the mortality was recorded for 14 days. The results showed that dietary C. butyricum significantly improved the specific growth rate (SGR) and feed intake in the CB2 group and decreased the cumulative mortality post-challenge with S. agalactiae in the CB2, CB3 and CB4 groups. The serum total antioxidant capacity and intestinal interleukin receptor-associated kinase-4 gene expression were significantly increased, and serum malondialdehyde content and diamine oxidase activity were significantly decreased in the CB1, CB2, CB3 and CB4 groups. Serum complement 3 and complement 4 concentrations and intestinal gene expression of tumour necrosis factor α, interleukin 8, and myeloid differentiation factor 88 were significantly higher in the CB2, CB3 and CB4 groups. Intestinal toll-like receptor 2 gene expression was significantly upregulated in the CB3 and CB4 groups. Dietary C. butyricum increased the diversity of the intestinal microbiota and the relative abundance of beneficial bacteria (such as Bacillus), and decreased the relative abundance of opportunistic pathogenic bacteria (such as Aeromonas) in the CB2 group. These results revealed that dietary C. butyricum at a suitable dose enhanced growth performance, elevated humoral and intestinal immunity, regulated the intestinal microbial components, and improved disease resistance in tilapia. The optimal dose was 1 x 105 CFU g-1 diet.

RNA-Seq transcriptome analysis of breast muscle in Pekin ducks supplemented with the dietary probiotic Clostridium butyricum.

BACKGROUND: Increased attention is being paid to breast muscle yield and meat quality in the duck breeding industry. Our previous report has demonstrated that dietary Clostridium butyricum (C. butyricum) can improve meat quality of Pekin ducks. However, the potential biological processes and molecular mechanisms that are modulated by dietary C. butyricum in the breast muscle of Pekin ducks remain unknown. RESULTS: Supplementation with C. butyricum increased growth performance and meat yield. Therefore, we utilized de novo assembly methods to analyze the RNA-Seq transcriptome profiles in breast muscle to explore the differentially expressed genes between C. butyricum-treated and control Pekin ducks. A total of 1119 differentially expressed candidate genes were found of which 403 genes were significantly up-regulated and 716 genes were significantly down-regulated significantly. qRT-PCR analysis was used to confirm the accuracy of the of RNA-Seq results. GO annotations revealed potential genes, processes and pathways that may participate in meat quality and muscle development. KEGG pathway analysis showed that the differentially expressed genes participated in numerous pathways related to muscle development, including ECM-receptor interaction, the MAPK signaling pathway and the TNF signaling pathway. CONCLUSIONS: This study suggests that long-time dietary supplementation with C. butyricum can modulate muscle development and meat quality via altering the expression patterns of genes involved in crucial metabolic pathways. The findings presented here provide unique insights into the molecular mechanisms of muscle development in Pekin ducks in response to dietary C. butyricum.

Computational Tale of Two Enzymes: Glycerol Dehydration With or Without B12.

We present a series of QM/MM calculations aimed at understanding the mechanism of the biological dehydration of glycerol. Strikingly and unusually, this process is catalyzed by two different radical enzymes, one of which is a coenzyme-B12-dependent enzyme and the other which is a coenzyme-B12-independent enzyme. We show that glycerol dehydration in the presence of the coenzyme-B12-dependent enzyme proceeds via a 1,2-OH shift, which benefits from a significant catalytic reduction in the barrier. In contrast, the same reaction in the presence of the coenzyme-B12-independent enzyme is unlikely to involve the 1,2-OH shift; instead, a strong preference for direct loss of water from a radical intermediate is indicated. We show that this preference, and ultimately the evolution of such enzymes, is strongly linked with the reactivities of the species responsible for abstracting a hydrogen atom from the substrate. It appears that the hydrogen-reabstraction step involving the product-related radical is fundamental to the mechanistic preference. The unconventional 1,2-OH shift seems to be required to generate a product-related radical of sufficient reactivity to cleave the relatively inactive C-H bond arising from the B12 cofactor. In the absence of B12, it is the relatively weak S-H bond of a cysteine residue that must be homolyzed. Such a transformation is much less demanding, and its inclusion apparently enables a simpler overall dehydration mechanism.

Effects of the butyric acid-producing strain Clostridium butyricum MIYAIRI 588 on broiler and piglet zootechnical performance and prevention of necrotic enteritis.

The objective of this study was to assess the effects of a probiotic strain Clostridium butyricumMIYAIRI 588 (CBM588) on broiler and weaned piglet health and zootechnical performance. Five field studies were carried out in broilers and five in weaned piglets under European feed additive guidelines. Each study followed a randomized blocked design with two treatments: Control (basal diet) and CBM588 supplemented groups. The zootechnical performance parameters selected were body weight, daily gain, feed intake and feed efficiency (feed:gain). Broilers fed diets with CBM588 gained significantly more weight (+2%, p < .001) and exhibited significantly better feed efficiency (-1.6%, p < .001) in comparison with Controls. Similarly, analysis of pooled data of weaned piglet trials showed that CBM588-fed piglets were significantly heavier than Controls (+2.6%, p = .014), exhibited significantly higher mean daily gain (+4.7%; p = .004), and significantly improved feed efficiency (-4.2%, p = .001). In addition to the zootechnical efficacy studies, the preventive effect of CBM588 on necrotic enteritis (NE) was assessed in a natural challenge model in broilers where CBM588 reduced the incidence and severity of NE lesions. These data indicate the potential of CBM588 to improve broiler and weaned piglet zootechnical performance, and to make a positive contribution to animal health.

Anti-diabetic Effects of Clostridium butyricum CGMCC0313.1 through Promoting the Growth of Gut Butyrate-producing Bacteria in Type 2 Diabetic Mice.

Patients with type 2 diabetes (T2D) have decreased butyrate-producing bacteria. We hypothesized that supplementation with butyrate-producing bacteria may exert beneficial effects on T2D. The current study investigated the effects of well-characterized butyrate-producing bacteria Clostridium butyricum CGMCC0313.1 (CB0313.1) on hyperglycemia and associated metabolic dysfunction in two diabetic mouse models. CB0313.1 was administered daily by oral gavage to leptin db/db mice for 5 weeks starting from 3 weeks of age, and to HF diabetic mice induced by high fat diet (HFD) plus streptozotocin (STZ) in C57BL/6J mice for 13 weeks starting from 4 weeks of age. CB0313.1 improved diabetic markers (fasting glucose, glucose tolerance, insulin tolerance, GLP-1 and insulin secretion), and decreased blood lipids and inflammatory tone. Furthermore, CB0313.1 reversed hypohepatias and reduced glucose output. We also found that CB0313.1 modulated gut microbiota composition, characterized by a decreased ratio of Firmicutes to Bacteroidetes, reduced Allobaculum bacteria that were abundant in HF diabetic mice and increased butyrate-producing bacteria. Changes in gut microbiota following CB0313.1 treatment were associated with enhanced peroxisome proliferator-activated receptor-γ (PPARγ), insulin signaling molecules and mitochondrial function markers. Together, our study suggests that CB0313.1 may act as a beneficial probiotic for the prevention and treatment of hyperglycemia and associated metabolic dysfunction.

Biorefinery development through utilization of biodiesel industry by-products as sole fermentation feedstock for 1,3-propanediol production.

Rapeseed meal (RSM) hydrolysate was evaluated as substitute for commercial nutrient supplements in 1,3-propanediol (PDO) fermentation using the strain Clostridium butyricum VPI 1718. RSM was enzymatically converted into a generic fermentation feedstock, enriched in amino acids, peptides and various micro-nutrients, using crude enzyme consortia produced via solid state fermentation by a fungal strain of Aspergillus oryzae. Initial free amino nitrogen concentration influenced PDO production in batch cultures. RSM hydrolysates were compared with commercial nutrient supplements regarding PDO production in fed-batch cultures carried out in a bench-scale bioreactor. The utilization of RSM hydrolysates in repeated batch cultivation resulted in a PDO concentration of 65.5 g/L with an overall productivity of 1.15 g/L/h that was almost 2 times higher than the productivity achieved when yeast extract was used as nutrient supplement.

An improved screening method for microorganisms able to convert crude glycerol to 1,3-propanediol and to tolerate high product concentrations.

A new screening method was developed and established to find high-performance bacteria for the conversion of crude glycerol to 1,3-propanediol. Three soil samples from palm oil-rich habitats were investigated using crude glycerol of a German biodiesel plant. Nine promising 1,3-propanediol producers could be found. Because of a special pH buffer system, a fast evaluation on microscale and high 1,3-propanediol concentrations up to 40 g L⁻¹ could be achieved. Three strains demonstrated very high product tolerance and were identified as Clostridium butyricum. Two strains, AKR91b and AKR102a, grew and produced 1,3-propanediol in the presence of 60 g L⁻¹ initial 1,3-propanediol, the strain AKR92a even in the presence of 77 g L⁻¹ 1,3-propanediol. The strains AKR91b and AKR102a tolerated up to 150 g L⁻¹ crude glycerol and produced 80% of the 1,3-propanediol attained from pure glycerol of the same concentration. Further criteria for the choice of a production strain were the pathogenicity (risk class), ability to grow on low-cost media, e.g., with less yeast extract, and robustness, e.g., process stability after several bioconversions. Overall, the strain C. butyricum AKR102a was chosen for further process optimization and scale-up due to its high productivity and high final concentration in a pH-regulated bioreactor.