Bacillus subtilis SF106 and Bacillus clausii SF174 spores reduce the inflammation and modulate the gut microbiota in a colitis model.

Chronic intestinal inflammation is associated with strong alterations of the microbial composition of the gut. Probiotic treatments and microbiota-targeting approaches have been considered to reduce the inflammation, improve both gut barrier function as well as overall gastrointestinal health. Here, a murine model of experimental colitis was used to assess the beneficial health effects of Bacillus subtilis SF106 and Bacillus clausii (recently renamed Shouchella clausii) SF174, two spore-forming strains previously characterised in vitro as potential probiotics. Experimental colitis was induced in BALB/c mice by the oral administration of dextran sodium sulphate (DSS) and groups of animals treated with spores of either strain. Spores of both strains reduced the DSS-induced inflammation with spores of B. clausii SF174 more effective than B. subtilis SF106. Spores of both strains remodelled the mouse gut microbiota favouring the presence of beneficial microbes such as members of the Bacteroidetes and Akkermansia genera.

Carotenoids found in Bacillus.

AIMS: To identify the diversity of pigmented aerobic spore formers found in the environment and to characterize the chemical nature of this pigmentation. MATERIALS AND RESULTS: Sampling of heat-resistant bacterial counts from soil, sea water and the human gastrointestinal tract. Phylogenetic profiling using analysis of 16S rRNA sequences to define species. Pigment profiling using high-performance liquid chromatography-photo diode array analysis. CONCLUSIONS: The most commonly found pigments were yellow, orange and pink. Isolates were nearly always members of the Bacillus genus and in most cases were related with known species such as Bacillus marisflavi, Bacillus indicus, Bacillus firmus, Bacillus altitudinis and Bacillus safensis. Three types of carotenoids were found with absorption maxima at 455, 467 and 492 nm, corresponding to the visible colours yellow, orange and pink, respectively. Although the presence of other carotenoids cannot be ruled out, these three predominant carotenoids appear to account for the pigments obtained in most pigmented bacilli, and our analysis reveals the existence of a C30 biosynthetic pathway. Interestingly, we report the presence of a water-soluble pigment that may also be a carotenoid. The function of carotenoids is photoprotection, and carotenoid-containing spores exhibited significantly higher levels of resistance to UV radiation than non-carotenoid-containing Bacillus species. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that pigmented bacilli are ubiquitous and contain new carotenoid biosynthetic pathways that may have industrial importance.

Characterization of spore forming Bacilli isolated from the human gastrointestinal tract.

AIMS: To isolate and characterize spore-former bacteria able to colonize the human gastrointestinal tract (GIT). METHODS AND RESULTS: A total of 25 spore-formers was isolated from faeces and ileal biopsies of healthy human volunteers and identified at the species level. Physiological analysis was performed to evaluate the ability of the various isolates to form biofilms, to swarm, to produce surfactants and molecules that have antimicrobial activity against selected pathogens. To assess the potential probiotic activity of the isolates, we tested the resistance of cells and spores to simulated gastric conditions, the ability to grow and sporulate in anaerobic conditions and the presence of toxin-encoding genes in their genome. CONCLUSIONS: Spore-formers belonging to various bacterial species have been isolated from the gut of healthy human volunteers. These strains appear to be well adapted to the intestinal environment and we propose them as potential probiotic strains for human use and as oral vaccine vehicles. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge this is the first detailed characterization of spore-forming Bacilli from the human GIT. Our data suggest that the isolated species do not transit, but rather colonize this specific habitat and propose them as probiotic strains for human use.

Beneficial effects of carotenoid-producing cells of Bacillus indicus HU16 in a rat model of diet-induced metabolic syndrome.

A well-established rat model of diet-induced metabolic syndrome was used to evaluate the effects of the oral administration of spores or cells of HU16, a carotenoid-producing strain of Bacillus indicus. Symptoms of metabolic syndrome were induced in 90-days old, male Sprague-Dawley rats maintained for eight weeks on a high-fat diet, as previously reported. Parallel groups of animals under the same diet regimen also received a daily dose of 1×1010 cells or spores of B. indicus HU16. Cells of strain HU16 were able to reduce symptoms of metabolic syndrome, plasma markers of inflammation and oxidative markers in plasma and liver to levels similar to those observed in rats under a standard diet. HU16 cells did not affect obesity markers or the accumulation of triglycerides in the liver of treated animals. Denaturing gradient gel electrophoresis analysis showed that the oral administration of HU16 cells did not significantly affect the gut microbiota of high fat-fed rats, suggesting that the observed beneficial effects are not due to a reshaping of the gut microbiota but rather to metabolites produced by HU16 cells.

Bacillus megaterium SF185 induces stress pathways and affects the cell cycle distribution of human intestinal epithelial cells.

The interaction between the enteric microbiota and intestinal cells often involves signal molecules that affect both microbial behaviour and host responses. Examples of such signal molecules are the molecules secreted by bacteria that induce quorum sensing mechanisms in the producing microorganism and signal transduction pathways in the host cells. The pentapeptide competence and sporulation factor (CSF) of Bacillus subtilis is a well characterized quorum sensing factor that controls competence and spore formation in the producing bacterium and induces cytoprotective heat shock proteins in intestinal epithelial cells. We analysed several Bacillus strains isolated from human ileal biopsies of healthy volunteers and observed that some of them were unable to produce CSF but still able to act in a CSF-like fashion on model intestinal epithelial cells. One of those strains belonging to the Bacillus megaterium species secreted at least two factors with effects on intestinal HT29 cells: a peptide smaller than 3 kDa able to induce heat shock protein 27 (hsp27) and p38-MAPK, and a larger molecule able to induce protein kinase B (PKB/Akt) with a pro-proliferative effect.

Efficient insertional mutagenesis in Streptococcus thermophilus.

Bacteria have always been considered ideal organisms for genetic analysis. While this is true for some model organisms, like Escherichia coli, Bacillus subtilis and, more recently, Lactococcus lactis, genetic analysis of other organisms is often prevented by lack of valuable tools, like vectors, transposons and methods for transformation, gene inactivation and random insertional mutagenesis. This is the case of the moderately thermophilic bacterium Streptococcus thermophilus, an organism that, in spite of its widespread use for food fermentations, is only poorly characterized. We report here an insertional mutagenesis system that allows efficient random mutagenesis, easy characterization of the interrupted genes and construction of stable null mutations. This may become a powerful S. thermophilus tool for both genetic analysis and construction of 'food-grade' mutants of this biotechnologically relevant microorganism.

Control of ilvIH transcription during amino acid downshift in stringent and relaxed strains of Escherichia coli.

Transcription of the ilvIH operon was reduced during amino acid starvation of wild-type Escherichia coli. The effect was abolished by a relA mutation and was enhanced by a spoT mutation, thus suggesting a possible negative role of ppGpp accumulation on ilvIH transcription. No effect of amino acid downshift was observed on the synthesis of lrp mRNA, encoding the positive regulator (Lrp) of ilvIH transcription.

Bacillus subtilis spore coat assembly requires cotH gene expression.

Endospores of Bacillus subtilis are encased in a protein shell, known as the spore coat, composed of a lamella-like inner layer and an electron-dense outer layer. We report the identification and characterization of a gene, herein called cotH, located at 300 degrees on the B. subtilis genetic map between two divergent cot genes, cotB and cotG. The cotH open reading frame extended for 1,086 bp and corresponded to a polypeptide of 42.8 kDa. Spores of a cotH null mutant were normally heat, lysozyme, and chloroform resistant but were impaired in germination. The mutant spores were also pleiotropically deficient in several coat proteins, including the products of the previously cloned cotB, -C, and -G genes. On the basis of the analysis of a cotE cotH double mutant, we infer that CotH is probably localized in the inner coat and is involved in the assembly of several proteins in the outer layer of the coat.

Bacillus subtilis Vegetative Catalase Is an Extracellular Enzyme.

Strong catalase activity was secreted by Bacillus subtilis cells during stationary growth phase in rich medium but not in sporulation-inducing medium. N-terminal sequencing indicated that the secreted activity was due to the vegetative catalase KatA, previously considered an endocellular enzyme. Extracellular catalase protected B. subtilis cells from oxidative assault.

Assembly requirements and role of CotH during spore coat formation in Bacillus subtilis.

We report Western blot data showing that the 42.8-kDa product of the previously characterized cotH locus (8) is a structural component of the Bacillus subtilis spore coat. We show that the assembly of CotH requires both CotE and GerE. In agreement with these observations, the ultrastructural analysis of purified spores suggests that CotH is needed for proper formation of both inner and outer layers of the coat.

Fate and dissemination of Bacillus subtilis spores in a murine model.

Bacterial spores are being consumed as probiotics, although little is known about their efficacy or mode of action. As a first step in characterizing spore probiotics, we have studied the persistence and dissemination of Bacillus subtilis spores given orally to mice. Our results have shown that spores do not appear to disseminate across the mucosal surfaces. However, we found that the number of spores excreted in the feces of mice was, in some experiments, larger than the original inoculum. This was an intriguing result and might be explained by germination of a proportion of the spore inoculum in the intestinal tract, followed by limited rounds of cell growth and then sporulation again. This result raises the interesting question of whether it is the spore or the germinated spore that contributes to the probiotic effect of bacterial spores.

Characterization of two Bacillus probiotics.

Bacillus subtilis is currently used as an oral probiotic. We examined two commercial B. subtilis probiotic preparations, Enterogermina and Biosubtyl. Surprisingly, physiological and genetic characterization of the bacteria contained in each of these preparations has shown that neither contains B. subtilis.

Characterization of Bacillus species used for oral bacteriotherapy and bacterioprophylaxis of gastrointestinal disorders.

Bacillus subtilis spores are being used for oral bacteriotherapy and bacterioprophylaxis of gastrointestinal disorders in both humans and animals. Since B. subtilis is an aerobic saprophyte, how spores may benefit the gut microbiota is an intriguing question, since other probiotics such as Lactobacillus spp. which colonize the gut are anerobes. As a first step in understanding the potential effects of ingesting spores, we have characterized five commercial products. An extensive biochemical, physiological, and phylogenetic analysis has revealed that four of these products are mislabeled. Moreover, four of these products showed high levels of antibiotic resistance.