Novel Putative Transposable Element Associated with the Subtype E5 Botulinum Toxin Gene Cluster of Neurotoxigenic Clostridium butyricum Type E Strains from China

Previously, a whole-genome comparison of three Clostridium butyricum type E strains from Italy and the United States with different C. botulinum type E strains indicated that the bont/e gene might be transferred between the two clostridia species through transposition. However, transposable elements (TEs) have never been identified close to the bont/e gene. Herein, we report the whole genome sequences for four neurotoxigenic C. butyricum type E strains that originated in China. An analysis of the obtained genome sequences revealed the presence of a novel putative TE upstream of the bont/e gene in the genome of all four strains. Two strains of environmental origin possessed an additional copy of the putative TE in their megaplasmid. Similar putative TEs were found in the megaplasmids and, less frequently, in the chromosomes of several C. butyricum strains, of which two were neurotoxigenic C. butyricum type E strains, and in the chromosome of a single C. botulinum type E strain. We speculate that the putative TE might potentially transpose the bont/e gene at the intracellular and inter-cellular levels. However, the occasional TE occurrence in the clostridia genomes might reflect rare transposition events.

Effects of Clostridium butyricum on Growth Performance, Gut Microbiota and Intestinal Barrier Function of Broilers

This study was conducted to evaluate the effects of Clostridium butyricum dietary supplementation on the growth, antioxidant, immune response, gut microbiota, and intestinal barrier function of broilers under high stocking density (HSD) stress. A total of 324 1-day-old Arbor Acres male broilers were randomly assigned to three treatments with six replicates, each replicate including 18 chickens (18 birds/m2). The experiment lasted 6 weeks. The three treatments were basal diet (control, CON), basal diet supplemented with 1 × 109 colony forming units (cfu)/kg C. butyricum (CB), and basal diet supplemented with 10 mg/kg virginiamycin (antibiotic, ANT). The results showed that the body weight (BW) and average daily gain (ADG) of broilers in the CB group were significantly higher than those in the CON group in three periods (p < 0.05). The total antioxidant capacity (T-AOC) and the superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity in serum of the CB group were significantly increased compared with those in the CON and ANT groups at 42 days (p < 0.05). At 42 days, the serum immunoglobulin M (IgM) and immunoglobulin G (IgG) levels of the CB group were significantly higher than those of the CON group. Compared with the CON group, interleukin-1β (IL-1β) in the CB group was significantly decreased in the starter and grower stages (p < 0.05), but there was no significant difference between the two treatment groups (p > 0.05). C. butyricum significantly decreased the high stocking density-induced expression levels of IL-1β and tumor necrosis factor-α (TNF-α) in the ileum of broilers at different stages. Additionally, C. butyricum could increase the expressions of claudin-1 and zonula occludens-1 (ZO-1) in intestinal tissue. Moreover, C. butyricum significantly increased the Sobs and Shannon indices in the CB group compared with the ANT group (p < 0.05), while the Ace index in the CB group was significantly higher than that of the CON group (p < 0.05). Furthermore, by using 16S rRNA gene sequencing, the proportion of Bacteroides in the CB group was increased compared to those in the CON and ANT groups at the genus level. In conclusion, C. butyricum supplemented into feed could improve the growth performance and feed utilization of broilers by promoting immune and intestinal barrier function and benefiting the cecal microflora.

Guide-directed DNA cleavage by a prokaryotic Argonaute protein induces chromosome recombination in Escherichia coli

Emerging evidence supports the argument that some prokaryotic argonautes (pAgos) serve as a defensive system against invasion of viruses and plasmids through guide DNAs (gDNAs) directed DNA cleavage. This DNA-guided DNA interference motivates research to induce genomic mutations via pAgo mediated cleavage. Here we demonstrate that CbAgo, a pAgo from Clostridium butyricum, is able to induce chromosomal recombination between direct repeat sequences via its gDNA-directed cleavage in Escherichia coli chromosome. We also show that CbAgo targeting can assist Lambda-Red recombineering in RecA-deficient strain. Our study reveals that cleavage by CbAgo in E. coli chromosome can be mutagenic and suggests its broader application in genetic manipulation.

Clostridium butyricum Improves Rumen Fermentation and Growth Performance of Heat-Stressed Goats In Vitro and In Vivo

This study aimed to evaluate the effects of Clostridium butyricum on rumen fermentation and the growth performance of heat-stressed goats. The in vitro fermentation was carried out using Clostridium butyricum supplement at 0% (CG), 0.025% (CB1), 0.05% (CB2), 0.10% (CB3), and 0.20% (CB4) of the dry matter (DM) weight of basal diet. Results showed that ruminal pH and the concentrations of ammonia nitrogen, total volatile fatty acids, acetic acid, propionic acid, as well as the acetic acid to propionic acid ratio were significantly increased (p < 0.05) in CB2 and CB3 compared with the CG group. Additionally, significant increases (p < 0.05) in the degradability of DM, neutral detergent fiber, and acid detergent fiber were observed in CB2 and CB3 compared with the CG group. For the in vivo study, 12 heat-stressed goats were divided equally into three groups: the control (HS1) was fed the basal diet, and groups HS2 and HS3 were fed with 0.05% and 0.10% Clostridium butyricum added to the basal diet, respectively. The experiment was designed as a 3 × 3 Latin square. Similar effects on rumen fermentation and digestibility parameters were obtained with 0.05% of Clostridium butyricum supplement compared to the in vitro study. Moreover, the dry matter intake and average daily gain were significantly increased (p < 0.05) in HS2 compared with other groups. These results indicated that an effective dose of Clostridium butyricum supplement (0.05%) could improve the rumen fermentation and growth performance of heat-stressed goats.

Effects of Dietary Supplementation With Clostridium butyricum on the Amelioration of Growth Performance, Rumen Fermentation, and Rumen Microbiota of Holstein Heifers

In China, the use of antibiotics growth promoters as feed additives has been banned. The goal of raising dairy heifers is to gain a relatively high body weight on a high-fiber diet at first mating or calving, thus increasing economic benefits. The objective of this experiment was to explore the effects of supplemental Clostridium butyricum (C. butyricum) on growth performance, rumen fermentation and microbiota, and blood parameters in Holstein heifers. Twenty Holstein heifers [mean ± standard deviation (SD); age = 182 ± 4.20 d, body weight = 197.53 ± 5.94 kg, dry matter intake (DMI) = 6.10 ± 0.38 kg] were randomly assigned to one of two diets group for a 42-day feeding period: (1) basal diet (an untreated control group, i.e., the CON group) or (2) basal diet plus daily 2 × 108 (colony-forming unit, CFU) of C. butyricum per kg of DMI per heifer (the CB group). The results demonstrated that C. butyricum supplementation increased the average daily gain from d 21 to 42 and DMI compared to the control group. Supplementation with C. butyricum significantly decreased the molar proportion of acetate and the acetate to propionate ratio but increased the molar proportion of butyrate and propionate. Compared with the control group, the relative abundance of Butyrivibrio fibrisolvens, Ruminococcus albus, Ruminobacter amylophilus, Ruminococcus flavefaciens, and Streptococcus bovis increased during the trial period in the CB group. However, C. butyricum had no significant effect on the blood parameters in Holstein heifers. In conclusion, these results show that feeding C. butyricum can improve growth performance and rumen fermentation without any negative impact on blood parameters in Holstein heifers.

Clostridium butyricum Alleviates Enterotoxigenic Escherichia coli K88-Induced Oxidative Damage Through Regulating the p62-Keap1-Nrf2 Signaling Pathway and Remodeling the Cecal Microbial Community

Clostridium butyricum (CB) can enhance antioxidant capacity and alleviate oxidative damage, but the molecular mechanism by which this occurs remains unclear. This study used enterotoxigenic Escherichia coli (ETEC) K88 as a pathogenic model, and the p62-Keap1-Nrf2 signaling pathway and intestinal microbiota as the starting point to explore the mechanism through which CB alleviates oxidative damage. After pretreatment with CB for 15 d, mice were challenged with ETEC K88 for 24 h. The results suggest that CB pretreatment can dramatically reduce crypt depth (CD) and significantly increase villus height (VH) and VH/CD in the jejunum of ETEC K88-infected mice and relieve morphological lesions of the liver and jejunum. Additionally, compared with ETEC-infected group, pretreatment with 4.4×106 CFU/mL CB can significantly reduce malondialdehyde (MDA) level and dramatically increase superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels in the serum. This pretreatment can also greatly increase the mRNA expression levels of tight junction proteins and genes related to the p62-Keap1-Nrf2 signaling pathway in the liver and jejunum in ETEC K88-infected mice. Meanwhile, 16S rDNA amplicon sequencing revealed that Clostridium disporicum was significantly enriched after ETEC K88 challenge relative to the control group, while Lactobacillus was significantly enriched after 4.4×106 CFU/mL CB treatment. Furthermore, 4.4×106 CFU/mL CB pretreatment increased the short-chain fatty acid (SCFA) contents in the cecum of ETEC K88-infected mice. Moreover, we found that Lachnoclostridium, Roseburia, Lactobacillus, Terrisporobacter, Akkermansia, and Bacteroides are closely related to SCFA contents and oxidative indicators. Taken together, 4.4×106 CFU/mL CB pretreatment can alleviate ETEC K88-induced oxidative damage through activating the p62-Keap1-Nrf2 signaling pathway and remodeling the cecal microbiota community in mice.

Dietary Clostridium butyricum and Bacillus subtilis Promote Goose Growth by Improving Intestinal Structure and Function, Antioxidative Capacity and Microbial Composition

Probiotics are a substitute for antibiotics in the sense of intestinal health maintenance. Clostridium butyricum and Bacillus subtilis, as probiotic bacteria, have been widely used in animal production. The aim of this study was to investigate the effects of the two probiotic bacteria in geese. A total of 288 1-day old, healthy Yangzhou geese were randomly assigned into 4 groups (A, B, C and D) with 6 replicates of 12 birds each. Group A, as control, was fed a basal diet, and the treatment groups (B, C and D) were fed the basal diet supplemented with 250 mg/kg Clostridium butyricum (the viable count was 3.0 × 106 CFU/g), 250 mg/kg Bacillus subtilis (the viable count was 2.0 × 107 CFU/g), or a combination of the two probiotic bacteria for 70 days, respectively. The results indicated that: compared with the control group, dietary probiotics (1) promoted the growth and feed intake of the geese, (2) increased the absolute weight of duodenum, (3) increased the antioxidative capacity (total antioxidative capacity (T-AOC), total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-PX)) of intestinal mucosa, (4) improved intestinal morphology (the ratio of villus height to crypt depth), (5) but did not induce inflammation and changes of tight junction in the intestine, which was indicated by no induction of pro/inflammatory cytokines (IL-1β, IL-6, IL-10, TNFAIP3) and tight junction related genes (TJP1 and OCLN). Moreover, dietary probiotics increased the relative abundances of Firmicutes phylum and Lactobacillus genus and decreased the relative abundances of Proteobacteria phylum or Ralstonia genus in the intestinal content. In addition, the alpha diversity (observed species, Chao1, and estimate the number of OTUs in the community(ACE)) was reduced and the predicted functions of intestinal microflora, including peptidases, carbon fixation and metabolic function of starch and sugar, were enhanced by dietary probiotics. In conclusion, dietary probiotics promote the growth of geese by their positive effects on intestinal structure and function, the composition and functions of gut microflora, and intestinal antioxidative capacity.

Dietary Addition With Clostridium butyricum and Xylo-Oligosaccharides Improves Carcass Trait and Meat Quality of Huanjiang Mini-Pigs

This study was conducted to investigate the effects of dietary addition with Clostridium butyricum (CB) and xylo-oligosaccharides (XOS) on growth performance, carcass trait, and meat quality of pigs. A total of 128 Huanjiang mini-pigs with an initial body weight of 9.5 ± 0.1 kg were randomly assigned to one of four groups. The pigs in control (Con) group were fed a basal diet and those in the experimental groups were fed the basal diet supplemented with 0.05% CB (CB group), 0.02% XOS (XOS group), or 0.05% CB + 0.02% XOS (CB + XOS group). Eight replicate pens were used per group with four pigs per pen. On days 28, 56, and 84 of the trial, the growth performance, carcass trait, and meat quality were evaluated. The results showed that dietary CB addition decreased (p &lt; 0.05) the average daily gain and increased (p &lt; 0.05) the ratio of feed intake to body weight gain at day 28 of the trial; CB, XOS, and CB + XOS addition increased (p &lt; 0.05) the backfat thickness at day 84 of the trial compared with the Con group. Dietary CB, XOS, and CB + XOS addition increased (p &lt; 0.05) the pH45min, while decreased (p &lt; 0.05) the marbling score at day 28 of the trial compared with the Con group. Dietary CB + XOS addition increased (p &lt; 0.05) the contents of Ala, Arg, Asp, Gly, His, Leu, Lys, Met, Phe, Ser, Thr, Tyr, and Val in muscle at day 56 of the trial. At day 84 of the trial, dietary CB addition increased the contents of nonessential amino acid (NEAA), total amino acid (TAA), and monounsaturated fatty acid (MUFA), while decreased (p &lt; 0.05) the percentage of C20:1 in muscle compared with the Con group. Collectively, dietary addition with 0.05% CB and 0.02% XOS could not alter the growth performance, but increase carcass trait, meat quality, and muscular nutrient contents in Huanjiang mini-pigs.