Pangenome analyses of Clostridium butyricum provide insights into its genetic characteristics and industrial application.

Clostridium butyricum is a Gram-positive anaerobic bacterium known for its ability to produce butyate. In this study, we conducted whole-genome sequencing and assembly of 14C. butyricum industrial strains collected from various parts of China. We performed a pan-genome comparative analysis of the 14 assembled strains and 139 strains downloaded from NCBI. We found that the genes related to critical industrial production pathways were primarily present in the core and soft-core gene categories. The phylogenetic analysis revealed that strains from the same clade of the phylogenetic tree possessed similar antibiotic resistance and virulence factors, with most of these genes present in the shell and cloud gene categories. Finally, we predicted the genes producing bacteriocins and botulinum toxins as well as CRISPR systems responsible for host defense. In conclusion, our research provides a desirable pan-genome database for the industrial production, food application, and genetic research of C. butyricum.

Clostridium butyricum upregulates GPR109A/AMPK/PGC-1α and ameliorates acute pancreatitis-associated intestinal barrier injury in mice.

Acute pancreatitis frequently causes intestinal barrier damage, which aggravates pancreatitis. Although Clostridium butyricum exerts anti-inflammatory and protective effects on the intestinal barrier during acute pancreatitis, the underlying mechanism is unclear. The G protein-coupled receptors 109 A (GPR109A) and adenosine monophosphate-activated protein kinase (AMPK)/ peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) signaling pathways can potentially influence the integrity of the intestinal barrier. Our study generated acute pancreatitis mouse models via intraperitoneal injection of cerulein and lipopolysaccharides. After intervention with Clostridium butyricum, the model mice showed reduced small intestinal and colonic intestinal barrier damage, dysbiosis amelioration, and increased GPR109A/AMPK/PGC-1α expression. In conclusion, Clostridium butyricum could improve pancreatic and intestinal inflammation and pancreatic injury, and relieve acute pancreatitis-induced intestinal barrier damage in the small intestine and colon, which may be associated with GPR109A/AMPK/PGC-1α.

Multiplex genetic manipulations in Clostridium butyricum and Clostridium sporogenes to secrete recombinant antigen proteins for oral-spore vaccination.

BACKGROUND: Clostridium spp. has demonstrated therapeutic potential in cancer treatment through intravenous or intratumoral administration. This approach has expanded to include non-pathogenic clostridia for the treatment of various diseases, underscoring the innovative concept of oral-spore vaccination using clostridia. Recent advancements in the field of synthetic biology have significantly enhanced the development of Clostridium-based bio-therapeutics. These advancements are particularly notable in the areas of efficient protein overexpression and secretion, which are crucial for the feasibility of oral vaccination strategies. Here, we present two examples of genetically engineered Clostridium candidates: one as an oral cancer vaccine and the other as an antiviral oral vaccine against SARS-CoV-2. RESULTS: Using five validated promoters and a signal peptide derived from Clostridium sporogenes, a series of full-length NY-ESO-1/CTAG1, a promising cancer vaccine candidate, expression vectors were constructed and transformed into C. sporogenes and Clostridium butyricum. Western blotting analysis confirmed efficient expression and secretion of NY-ESO-1 in clostridia, with specific promoters leading to enhanced detection signals. Additionally, the fusion of a reported bacterial adjuvant to NY-ESO-1 for improved immune recognition led to the cloning difficulties in E. coli. The use of an AUU start codon successfully mitigated potential toxicity issues in E. coli, enabling the secretion of recombinant proteins in C. sporogenes and C. butyricum. We further demonstrate the successful replacement of PyrE loci with high-expression cassettes carrying NY-ESO-1 and adjuvant-fused NY-ESO-1, achieving plasmid-free clostridia capable of secreting the antigens. Lastly, the study successfully extends its multiplex genetic manipulations to engineer clostridia for the secretion of SARS-CoV-2-related Spike_S1 antigens. CONCLUSIONS: This study successfully demonstrated that C. butyricum and C. sporogenes can produce the two recombinant antigen proteins (NY-ESO-1 and SARS-CoV-2-related Spike_S1 antigens) through genetic manipulations, utilizing the AUU start codon. This approach overcomes challenges in cloning difficult proteins in E. coli. These findings underscore the feasibility of harnessing commensal clostridia for antigen protein secretion, emphasizing the applicability of non-canonical translation initiation across diverse species with broad implications for medical or industrial biotechnology.

Physiological and biochemical characteristics of the carbon ion beam irradiation-generated mutant strain Clostridium butyricum FZM 240 in vitro and in vivo.

Clostridium butyricum (C. butyricum) represents a new generation of probiotics, which is beneficial because of its good tolerance and ability to produce beneficial metabolites, such as short-chain fatty acids and enzymes; however, its low enzyme activity limits its probiotic efficacy. In this study, a mutant strain, C. butyricum FZM 240 was obtained using carbon ion beam irradiation, which exhibited greatly improved enzyme production and tolerance. The highest filter paper, endoglucanase, and amylase activities produced by C. butyricum FZM 240 were 125.69 U/mL, 225.82 U/ mL, and 252.28 U/mL, which were 2.58, 1.95, and 2.21-fold higher, respectively, than those of the original strain. The survival rate of the strain increased by 11.40 % and 5.60 % after incubation at 90 °C for 5 min and with simulated gastric fluid at pH 2.5 for 2 h, respectively, compared with that of the original strain. Whole-genome resequencing and quantitative real-time PCR(qRT-PCR) analysis showed that the expression of genes related to enzyme synthesis (GE000348, GE001963 and GE003123) and tolerance (GE001114) was significantly up-regulated, while that of genes related to acid metabolism (GE003450) was significantly down-regulated. On this basis, homology modeling and functional prediction of the proteins encoded by the mutated genes were performed. According to the results, the properties related to the efficacy of C. butyricum as a probiotic were significantly enhanced by carbon ion beam irradiation, which is a novel strategy for the application of Clostridium spp. as feed additives.

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.

Dietary inclusion of Clostridium butyricum cultures alleviated impacts of high-carbohydrate diets in largemouth bass (Micropterus salmoides).

A 60-d feeding trial was conducted to explore the potential regulatory effects of dietary Clostridium butyricum cultures (CBC) supplementation in high-carbohydrate diet (HCD) on carbohydrate utilisation, antioxidant capacity and intestinal microbiota of largemouth bass. Triplicate groups of largemouth bass (average weight 35·03 ± 0·04 g), with a destiny of twenty-eight individuals per tank, were fed low-carbohydrate diet and HCD supplemented with different concentration of CBC (0 %, 0·25 %, 0·50 % and 1·00 %). The results showed that dietary CBC inclusion alleviated the hepatic glycogen accumulation induced by HCD intake. Additionally, the expression of hepatic ampkα1 and insulin signaling pathway-related genes (ira, irb, irs, p13kr1 and akt1) increased linearly with dietary CBC inclusion, which might be associated with the activation of glycolysis-related genes (gk, pfkl and pk). Meanwhile, the expression of intestinal SCFA transport-related genes (ffar3 and mct1) was significantly increased with dietary CBC inclusion. In addition, the hepatic antioxidant capacity was improved with dietary CBC supplementation, as evidenced by linear decrease in malondialdehyde concentration and expression of keap1, and linear increase in antioxidant enzyme activities (total antioxidative capacity, total superoxide dismutase and catalase) and expression of antioxidant enzyme-related genes (nrf2, sod1, sod2 and cat). The analysis of bacterial 16S rRNA V3-4 region indicated that dietary CBC inclusion significantly reduced the enrichment of Firmicutes and potential pathogenic bacteria genus Mycoplasma but significantly elevated the relative abundance of Fusobacteria and Cetobacterium. In summary, dietary CBC inclusion improved carbohydrate utilization, antioxidant capacity and intestinal microbiota of largemouth bass fed HCD.

Enhancing and monitoring spore production in Clostridium butyricum using pH-based regulation strategy and a robust soft sensor based on back-propagation neural networks.

Clostridium butyricum is a probiotic that forms anaerobic spores and plays a crucial role in regulating gut microbiota. However, the total viable cell count and spore yield of C. butyricum in industrial production are comparatively low. To this end, we investigated the metabolic characteristics of the strain and proposed three distinct pH regulation strategies for enhancing spore production. In addition, precise measurement of fermentation parameters such as substrate concentration, total viable cell count, and spore concentration is crucial for successful industrial probiotics production. Nevertheless, online measurement of these intricate parameters in the fermentation of C. butyricum poses a considerable challenge owing to the complex, nonlinear, multivariate, and strongly coupled characteristics of the production process. Therefore, we analyzed the capacitance and conductivity acquired from a viable cell sensor as the core parameters for the fermentation process. Subsequently, a robust soft sensor was developed using a seven-input back-propagation neural network model with input variables of fermentation time, capacitance, conductivity, pH, initial total sugar concentration, ammonium ion concentration, and calcium ion concentration. The model enables the online monitoring of total viable biomass count, substrate concentrations, and spore yield, and can be extended to similar fermentation processes with pH changes as a characteristic feature.

Dynamic flux balance analysis of 1,3-propanediol production by clostridium butyricum fermentation.

To study the relationship between the yield of 1,3-propanediol (1,3-PDO) and the flux change of the Clostridium butyricum metabolic pathway, an optimized calculation method based on dynamic flux balance analysis was used by combining genome-scale flux balance analysis with a kinetic model. A more comprehensive and extensive metabolic pathway was obtained by optimization calculations. The primary extended branches include: the dihydroxyacetone node, which enters the pentose phosphate pathway; the α-oxoglutarate node, which has synthetic metabolic pathways for glutamic acid and amino acids; and the serine and homocysteine nodes, which produce cystathionine before homocysteine enters the methionine cycle pathway. According to the expanded metabolic network, the flux distribution of key nodes in the metabolic pathway and the relationship between the flux distribution ratio of nodes and the yield of 1,3-PDO were analyzed. At the dihydroxyacetone node, the flux of dihydroxyacetone converted to dihydroxyacetone phosphate was positively correlated with the yield of 1,3-PDO. As an important intermediate product, the flux change in the metabolic pathway of α-oxoglutarate reacting with amino acids to produce glutamic acid is positively correlated with the yield. When pyruvate was used as the central node to convert into lactic acid and α-oxoglutarate, the proportion of branch flux was negatively correlated with the yield of 1,3-PDO. These studies provide a theoretical basis for the optimization and further study of the metabolic pathway of C. butyricum.

Sensing of DNA modifications by pAgo proteins in vitro.

Many prokaryotic Argonaute (pAgo) proteins act as programmable nucleases that use small guide DNAs for recognition and cleavage of complementary target DNA. Recent studies suggested that pAgos participate in cell defense against invader DNA and may also be involved in other genetic processes, including DNA replication and repair. The ability of pAgos to recognize specific targets potentially make them an invaluable tool for DNA manipulations. Here, we demonstrate that DNA-guided DNA-targeting pAgo nucleases from three bacterial species, DloAgo from Dorea longicatena, CbAgo from Clostridium butyricum and KmAgo from Kurthia massiliensis, can sense site-specific modifications in the target DNA, including 8-oxoguanine, thymine glycol, ethenoadenine and pyrimidine dimers. The effects of DNA modifications on the activity of pAgos strongly depend on their positions relative to the site of cleavage and are comparable to or exceed the effects of guide-target mismatches at corresponding positions. For all tested pAgos, the strongest effects are observed when DNA lesions are located at the cleavage position. The results demonstrate that DNA cleavage by pAgos is strongly affected by DNA modifications, thus making possible their use as sensors of DNA damage.

Clostridium butyricum Prazmowski can degrade and utilize resistant starch via a set of synergistically acting enzymes.

Resistant starch is a prebiotic fiber that is best known for its ability to increase butyrate production by the gut microbiota. This butyrate then plays an important role in modulating the immune system and inflammation. However, the ability to use this resistant starch appears to be a rare trait within the gut microbiota, with only a few species such as Ruminococcus bromii and Bifidobacterium adolescentis having been demonstrated to possess this ability. Furthermore, these bacteria do not directly produce butyrate themselves, rather they rely on cross-feeding interactions with other gut bacteria for its production. Here, we demonstrate that the often-used probiotic organism Clostridium butyricum also possesses the ability to utilize resistant starch from a number of sources, with direct production of butyrate. We further explore the enzymes responsible for this trait, demonstrating that they exhibit significant synergy, though with different enzymes exhibiting more or less importance depending on the source of the resistant starch. Thus, the co-administration of Clostridium butyricum may have the ability to improve the beneficial effects of resistant starch.IMPORTANCEClostridium butyricum is seeing increased use as a probiotic, due to potential health benefits tied to its ability to produce butyrate. Here, we demonstrate that this organism can use a variety of resistant starch sources and characterize the enzymes it uses to accomplish this. Given the relative rarity of resistant starch utilizing ability within the gut and the health benefits tied to resistant starch, the combined use of this organism with resistant starch in synbiotic formulations may prove beneficial.

Clostridium butyricum MIYAIRI 588 alleviates periodontal bone loss in mice with diabetes mellitus.

The gut microbiota is a bridge linking periodontitis and systemic diseases, such as diabetes mellitus (DM). The probiotic Clostridium butyricum MIYAIRI 588 (CBM588) is reportedly an effective therapeutic approach for gut dysbiosis. Here, in a mouse model, we explored the therapeutic effect of CBM588 on periodontal bone destruction in DM and DM-associated periodontitis (DMP), as well as the underlying mechanism. Micro-computed tomography revealed that DM and DMP both aggravated periodontal bone destruction, which was alleviated by intragastric supplementation with CBM588. Moreover, 16S rRNA sequencing and untargeted metabolite analysis indicated that CBM588 ameliorated DMP-triggered dysbiosis and led to reduced oxidative stress associated with elevated 4-hydroxybenzenemethanol (4-HBA) in serum. Furthermore, in vitro and in vivo experiments found that the metabolite 4-HBA promoted nuclear factor erythroid 2-related factor 2 (Nrf2) signaling activation and modulated the polarization of macrophages, thus ameliorating inflammatory bone destruction in DMP. Our study demonstrates the protective effects of CBM588 in DM-induced mice, with and without ligature-induced periodontitis. The mechanism involves regulation of the gut microbiota and restoration of the integrity of the gut barrier to alleviate oxidative damage by elevating serum 4-HBA. This study suggests the possibility of CBM588 as a therapeutic adjuvant for periodontal treatment in diabetes patients.

[Effects of Peroxisome Proliferator-Activated Receptor γ on Modulating Angiopoietin-Like Protein 4 Synthesis in Caco-2 Cells Exposed to Clostridium butyricum].

Angiopoietin-like protein 4 (ANGPTL4) is considered to be one of the important circulating mediators linking intestinal microorganisms and host lipid metabolism. The objective of this study was to assess the effects of peroxisome proliferator-activated receptor у (PPARγ) on modulating ANGPTL4 synthesis in Caco-2 cells exposed to Clostridium butyricum. The viability of Caco-2 cells and the expression of PPARγ and ANGPTL4 in Caco-2 cells were detected after the Caco-2 cells were co-cultured with C. butyricum at the concentration of 1 x 10^(6), 1 x 10^(7) and 1 x 10^(8) CFU/mL. The results showed that cell viability was enhanced by C. butyricum. Besides, PPARγ and ANGPTL4 expression and secretion in Caco-2 cells was significantly increased by 1 x 10^(7) and 1 x 10^(8) CFU/mL of C. butyricum. Furthermore, the effects of PPARγ on modulating ANGPTL4 synthesis in Caco-2 cells regulated by 1 x 10^(8) CFU/mL of C. butyricum was also be expounded in PPARγ activation/inhibition model based on Caco-2 cells and via ChIP technique. It was found that C. butyricum promoted the binding of PPARγ to the PPAR binding site (chr19: 8362157-8362357, located upstream of the transcriptional start site of angptl4) of the angptl4 gene in Caco-2 cells. However, the PPARγ was not the only way for C. butyricum to stimulate ANGPTL4 production. Taken together, PPARγ played a role in the regulation of ANGPTL4 synthesis by C. butyricum in Caco-2 cells.

Clostridium butyricum and its metabolite butyrate promote ferroptosis susceptibility in pancreatic ductal adenocarcinoma.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with limited therapeutic options. The diversity and composition of the intratumoral microbiota are associated with PDAC outcomes, and modulating the tumor microbiota has the potential to influence tumor growth and the host immune response. Here, we explore whether intervention with butyrate-producing probiotics can limit PDAC progression. METHODS: Based on the TCGA (PAAD) database, we analyzed the differential communities of intratumoral microbiota in PDAC patients with long survival and short survival and explored the relevant mechanisms of Clostridium butyricum and its metabolite butyrate in the treatment of PDAC. Treatment with Clostridium butyricum or butyrate in combination with the ferroptosis inducer RSL3 in a PDAC mouse model has an inhibitory effect on PDAC progression. The potential molecular mechanisms were verified by flow cytometry, RNA-seq, Western blotting, qRT‒PCR and immunofluorescence. RESULTS: We found that the tumoral butyrate-producing microbiota was linked to a better prognosis and less aggressive features of PDAC. Intervention with Clostridium butyricum or its metabolite butyrate triggered superoxidative stress and intracellular lipid accumulation, which enhanced ferroptosis susceptibility in PDAC. CONCLUSION: Our study reveals a novel antitumor mechanism of butyrate and suggests the therapeutic potential of butyrate-producing probiotics in PDAC.

Clostridium butyricum improves cognitive dysfunction in ICV-STZ-induced Alzheimer's disease mice via suppressing TLR4 signaling pathway through the gut-brain axis.

In recent years, the relationship between gut-brain axis and Alzheimer's disease (AD) attracted increasing attention. The aim of this study is to investigate the therapeutic effect of Clostridium butyricum (CB) on intraventricular injection of streptozotocin (ICV-STZ)-induced mice and the potential mechanisms. ICV-STZ mice were treated with CB by gavage for 21 consecutive days. The pharmacological effect of CB was assessed by behavior test, brain tissue H&E staining and tau protein phosphorylation levels of hippocampus tissues. The expression levels of TLR4, MYD88, NF-κB p65, TNF-α, iNOS, Occludin and ZO-1 in hippocampal and colonic tissues were detected by Western-blot method. 16S rRNA gene sequencing analysis was used to analyze the intestinal microbiota of mice. The results showed that CB improved the cognitive dysfunction of ICV-STZ mice, restored the structure and cell number of hippocampal and cortical neurons, decreased the protein levels of pSer404-tau protein in hippocampal tissues and TLR4, MYD88, NF-κB p65 and iNOS in hippocampal and colonic tissues, and increased the protein levels of Occludin and ZO-1 in colonic tissues. Meanwhile, CB reversed the changes of intestinal microbiota in AD mice. Therefore, the mechanisms of cognitive function and brain pathological changes in AD mice improved by CB may be related to the regulation of TLR4 signaling pathway and intestinal microbiota. This study supports the potential anti-AD effect of CB and initially revealed its pharmacological mechanism of CB, providing a theoretical basis for further clinical application of CB.

Structure, antioxidant properties, and protective effects on DNA damage of exopolysaccharides from Clostridium butyricum.

Exopolysaccharides (EPSs) of probiotics are naturally nontoxic antioxidants with some interesting biological activities. This research aims to explore the structural and antioxidant properties of the EPS from Clostridium butyricum, a probiotics widely existed in human and animal intestines. EPS of C. butyricum RO-07 was purified through a combination of anion-exchange column chromatography and gel chromatography and determined to be composed of glucosamine, arabinose, galactosamine, galactose, glucose, and xylose in a molar ratio of 1:1:1:2:1:1 with a molecular weight 1.23 × 104  Da. It exhibited a stronger antioxidant activity than ascorbic acid, with scavenging activities up to 75.2% and 95.0% against hydroxyl radical (•OH) and superoxide radical (O2 - •), respectively. It also performed protective effects on DNA against radiation destruction by ultraviolet and reactive oxygen species generated oxidation stress. With these superior advantages in oxidants and radiation resistance, the EPS from C. butyricum RO-07 therefore has great potential to be applied in food and cosmetic industry.

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.

Engineered probiotics Clostridium butyricum-pMTL007-GLP-1 improves blood pressure via producing GLP-1 and modulating gut microbiota in spontaneous hypertension rat models.

Hypertension is a significant risk factor of cardiovascular diseases (CVDs) with high prevalence worldwide, the current treatment has multiple adverse effects and requires continuous administration. The glucagon-like peptide-1 receptor (GLP-1R) agonists have shown great potential in treating diabetes mellitus, neurodegenerative diseases, obesity and hypertension. Butyric acid is a potential target in treating hypertension. Yet, the application of GLP-1 analogue and butyric acid in reducing blood pressure and reversing ventricular hypertrophy remains untapped. In this study, we combined the therapeutic capability of GLP-1 and butyric acid by transforming Clostridium butyricum (CB) with recombinant plasmid pMTL007 encoded with hGLP gene to construct the engineered probiotics Clostridium butyricum-pMTL007-GLP-1 (CB-GLP-1). We used spontaneous hypertensive rat (SHR) models to evaluate the positive effect of this strain in treating hypertension. The results revealed that the intragastric administration of CB-GLP-1 had markedly reduced blood pressure and improved cardiac marker ACE2, AT2R, AT1R, ANP, BNP, ß-MHC, α-SMA and activating AMPK/mTOR/p70S6K/4EBP1 signalling pathway. The high-throughput sequencing further demonstrated that CB-GLP-1 treatments significantly improved the dysbiosis in the SHR rats via downregulating the relative abundance of Porphyromonadaceae at the family level and upregulating Lactobacillus at the genus level. Hence, we concluded that the CB-GLP-1 greatly improves blood pressure and cardiomegaly by restoring the gut microbiome and reducing ventricular hypertrophy in rat models. This is the first time using engineered CB in treating hypertension, which provides a new idea for the clinical treatment of hypertension.

Whole cell of pure Clostridium butyricum CBT-1 from anaerobic bioreactor effectively hydrolyzes agro-food waste into biohydrogen.

Recycling organic waste and converting them into renewable energy are a promising route for environment protection and effective biochemical reactions suitable for industrial hydrogen synthesis. This study targeted to isolate a pure anaerobic culture with potential to hydrolyze different biomass and production of biohydrogen. For this, a sample of full-scale anaerobic digester, fed with a multicomponent solid, was inoculated on Reinforced Clostridial Medium (RCM) in strict anaerobic conditions. An anaerobic Clostridium butyricum CBT-1 strain was isolated, identified from morphological and 16S rRNA sequence. The CBT-1 culture expressed amylase, cellulase and peroxidases activities. The strain exhibited visual decolorization of both Azure B and crystal violet dyes. In batch fermentation experiment, the CBT-1 produced highest of 3.06, 2.67 and 2.46 mol/mol H2 yield from glucose, starch and cellulose respectively, whereas, the CBT-1 showed low 0.43 mol H2/mol of substrate from untreated rice straw due to lignin in compact structure and comparatively high H2 yield of 1.91 and 2.01 mol H2/mol of substrate rice straw hydrolysate and kitchen food waste (KFWS) respectively. The cumulative volumetric yield of H2 was 358.15, 300.8 and 294.5NmL/gSub from glucose, starch and cellulose respectively. Similarly, the cumulative H2 volume was 76.7, 184.4, 237.2 NmL/gVS from untreated rice straw, rice straw hydrolysate and kitchen food waste. This study emphasizes the prospects to find similar robust anaerobic culture for hydrolyzing complex biomass. Such strains could be used as standard co-inoculum for biohydrogen obtaining and as the biocatalyst for commercial scale applications.

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.

Clostridium butyricum and Bifidobacterium pseudolongum Attenuate the Development of Cardiac Fibrosis in Mice.

Cardiac fibrosis is an integral aspect of every form of cardiovascular diseases, which is one of the leading causes of death worldwide. It is urgent to explore new effective drugs and treatments. In this paper, transverse aortic constriction (TAC)-induced cardiac fibrosis was significantly alleviated by a cocktail of antibiotics to clear the intestinal flora, indicating that the gut microbiota was associated with the disease process of cardiac fibrosis. We transplanted feces from sham-operated and TAC-treated mice to mice treated with a cocktail of antibiotics. We found that TAC-treated gut microbiota dysbiosis cannot cause cardiac fibrosis on its own. Interestingly, healthy fecal microbiota transplantation could alleviate cardiac fibrosis, indicating that targeted probiotics and related metabolite intervention may restore a normal microenvironment for the treatment or prevention of cardiac fibrosis. We used 16S rRNA sequencing of fecal samples and discovered that butyric acid-producing bacteria and Bifidobacterium pseudolongum were the dominant bacteria in the group with the lowest degree of cardiac fibrosis. Moreover, we demonstrated that sodium butyrate prevented the development of cardiac fibrosis. The effect of Clostridium butyricum (butyric acid-producing bacteria) was better than that of B. pseudolongum on cardiac fibrosis. Surprisingly, the cocktail of two probiotics had a stronger ability than a single probiotic. In conclusion, therapies targeting the gut microbiota and metabolites such as probiotics present new strategies for treating cardiovascular disease. IMPORTANCE Cardiac fibrosis is a basic process in cardiac remodeling. It is related to almost all types of cardiovascular diseases (CVD) and has become an important global health problem. Basic research and a number of clinical studies have shown that myocardial fibrosis can be prevented and reversed to a certain extent. It is urgent to explore new effective drugs and treatments. We indicated a causal relationship between cardiac fibrosis and gut microbiota. Gut microbiota dysbiosis cannot cause cardiac fibrosis on its own. Interestingly, healthy fecal microbiota transplantation could alleviate cardiac fibrosis. According to our findings, the combined use of butyric acid-producing bacteria and B. pseudolongum can help prevent cardiac fibrosis. Therapies targeting the gut microbiota and metabolites, such as probiotics, represent new strategies for treating cardiovascular disease.