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Additional file 2: Figure S2. Rarefaction curves for each sample: (a) Number of OTUs, (b) Chao 1 and (c) Shannon index

Background Wild boar has experienced several evolutionary trajectories from which domestic (under artificial selection) and the feral pig (under natural selection) originated. Strong adaptation deeply affects feral population’s morphology and physiology, including the microbiota community. The gut microbiota is generally recognized to play a crucial role in maintaining host health and metabolism. To date, it is unclear whether feral populations’ phylogeny, development stages or lifestyle have the greatest impact in shaping the gut microbiota, as well as how this can confer adaptability to new environments. Here, in order to deepen this point, we characterized the gut microbiota of feral population discriminating between juvenile and adult samples, and we compared it to the microbiota structure of wild boar and domestic pig as the references. Gut microbiota composition was estimated through the sequencing of the partial 16S rRNA gene by DNA metabarcoding and High Throughput Sequencing on DNA extracted from fecal samples. Results The comparison of microbiota communities among the three forms showed significant differences. The feral form seems to carry some bacteria of both domestic pigs, derived from its ancestral condition, and wild boars, probably as a sign of a recent re-adaptation strategy to the natural environment. In addition, interestingly, feral pigs show some exclusive bacterial taxa, also suggesting an innovative nature of the evolutionary trajectories and an ecological segregation in feral populations, as already observed for other traits. Conclusions The feral pig showed a significant change between juvenile and adult microbiota suggesting an influence of the wild environment in which these populations segregate. However, it is important to underline that we certainly cannot overlook that these variations in the structure of the microbiota also depended on the different development stages of the animal, which in fact influence the composition of the intestinal microbiota. Concluding, the feral pigs represent a new actor living in the same geographical space as the wild boars, in which its gut microbial structure suggests that it is mainly the result of environmental segregation, most different from its closest relative. This gives rise to interesting fields of exploration regarding the changed ecological complexity and the consequent evolutionary destiny of the animal communities involved in this phenomenon.

Microorganisms efficiently colonize the external and internal surfaces of the animal body establishing mutually beneficial interactions and forming site- and individual-specific microbiota. The degradation of complex polysaccharides in the animal gut, the production of useful compounds, protection against pathogenic microorganisms and contribution to the development of an efficient immune system are the main beneficial effects of a balanced microbiota. A dysbiosis, an imbalanced composition of the microbiota, has been associated with a large number of diseases from gastro-intestinal or urogenital disorders to allergies, cardiovascular and autoimmune diseases and even to the onset of certain cancers. A growing body of evidence has indicated that probiotic treatments, aimed at maintaining or rebalancing the microbiota, are useful to treat/prevent those illnesses. Lactic Acid Bacteria and Bifidobacteria are the most common microbes used in probiotic preparations; however, other bacteria and yeast cells are also widely used in commercial products. Here we focus on the use of bacterial spore formers as probiotics. Spore formers have been marketed as probiotics for over 50 years and are now extensively used for the treatment of intestinal disorders and as dietary supplements in humans, as growth promoters and competitive exclusion agents in animals.

Proteins and glycoproteins that form the surface layers of the Bacillus spore assemble into semipermeable arrays that surround and protect the spore cytoplasm. Such layers, acting like molecular sieves, exclude large molecules but allow small nutrients (germinants) to penetrate. We report that CotG, a modular and abundant component of the Bacillus subtilis spore coat, controls spore permeability through its central region, formed by positively charged tandem repeats. These repeats act as spacers between the N and C termini of the protein, which are responsible for the interaction of CotG with at least one other coat protein. The deletion but not the replacement of the central repeats with differently charged repeats affects the spore resistance to lysozyme and the efficiency of germination-probably by reducing the coat permeability to external molecules. The presence of central repeats is a common feature of the CotG-like proteins present in most Bacillus species, and such a wide distribution of this protein family is suggestive of a relevant role for the structure and function of the Bacillus spore. IMPORTANCE Bacterial spores are quiescent cells extremely resistant to a variety of unphysiological conditions, including the presence of lytic enzymes. Such resistance is also due to the limited permeability of the spore surface, which does not allow lytic enzymes to reach the spore interior. This article proposes that the spore permeability in B. subtilis is mediated by CotG, a modular protein formed by a central region of repeats of positively charged amino acid acting as a "spacer" between the N and C termini. These, in turn, interact with other coat proteins, generating a protein layer whose permeability to external molecules is controlled by the distance between the N and C termini of CotG. This working model is most likely expandable to most sporeformers of the Bacillus genus, since they all have CotG-like proteins, not homologous to CotG of B. subtilis but similarly characterized by central repeats.

Spore formers are ubiquitous microorganisms commonly isolated from most environments, including the gastro-intestinal tract (GIT) of insects and animals. Spores ingested as food and water contaminants safely transit the stomach and reach the intestine, where some of them germinate and temporarily colonize that niche. In the lower part of the GIT, they re-sporulate and leave the body as spores, therefore passing through their entire life cycle in the animal body. In the intestine, both un-germinated spores and germination-derived cells interact with intestinal and immune cells and have health-beneficial effects, which include the production of useful compounds, protection against pathogenic microorganisms, contribution to the development of an efficient immune system and modulation of the gut microbial composition. We report a genomic and physiological characterization of SF106 and SF174, two aerobic spore former strains previously isolated from ileal biopsies of healthy human volunteers. SF106 and SF174 belong respectively to the B. subtilis and Alkalihalobacillus clausii (formerly Bacillus clausii) species, are unable to produce toxins or other metabolites with cytotoxic activity against cultured human cells, efficiently bind mucin and human epithelial cells in vitro and produce molecules with antimicrobial and antibiofilm activities.

The Bacillus cereus group is a species complex of the Bacillus genus that includes several closely related species. Within this group, bacteria indicated as B. cereus sensu stricto (B. cereus) are the causative agent of two different types of gastrointestinal diseases associated with food poisoning. Outbreaks of this opportunistic pathogen are generally due to the resistance of its spores to heat, pH and desiccation that makes hard their complete inactivation from food products. B. cereus is commonly isolated from a variety of environments, including intestinal samples of infected and healthy people. We report the genomic and physiological characterization of MV19, a human intestinal strain closely related (ANI value of 98.81%) to the reference strain B. cereus ATCC 14579. MV19 cells were able to grow in a range of temperatures between 20 and 44°C. At the optimal temperature the sporulation process was induced very rapidly and mature spores efficiently released, however these appeared structurally and morphologically defective. At the sub-optimal growth temperature of 25°C sporulation was slow and less efficient but a high total number of fully functional spores was produced. Altogether, results reported here indicate that the reduced rapidity and efficiency of sporulation at 25°C are compensated by a high quality and quantity of released spores, suggesting the relevance of different performances at different growth conditions for the adaptation of this bacterium to diverse environmental niches.

To investigate whether short term fructose-rich diet induces changes in the gut microbiota as well as in skeletal muscle and adipose tissue physiology and verify whether they persist even after fructose withdrawal, young rats of 30 d of age were fed for 3 weeks a fructose-rich or control diet. At the end of the 3-weeks period, half of the rats from each group were maintained for further 3 weeks on a control diet. Metagenomic analysis of gut microbiota and short chain fatty acids levels (faeces and plasma) were investigated. Insulin response was evaluated at the whole-body level and both in skeletal muscle and epididymal adipose tissue, together with skeletal muscle mitochondrial function, oxidative stress, and lipid composition. In parallel, morphology and physiological status of epididymal adipose tissue was also evaluated. Reshaping of gut microbiota and increased content of short chain fatty acids was elicited by the fructose diet and abolished by switching back to control diet. On the other hand, most metabolic changes elicited by fructose-rich diet in skeletal muscle and epididymal adipose tissue persisted after switching to control diet. Increased dietary fructose intake even on a short-time basis elicits persistent changes in the physiology of metabolically relevant tissues, such as adipose tissue and skeletal muscle, through mechanisms that go well beyond the reshaping of gut microbiota. This picture delineates a harmful situation, in particular for the young populations, posed at risk of metabolic modifications that may persist in their adulthood.

The gut microbiota exerts a variety of positive effects on the intestinal homeostasis, including the production of beneficial molecules, control of the epithelial barrier integrity and the regulation of the balance between host’s cell death and proliferation. The interactions between commensal bacteria and intestinal cells are still under-investigated and is then of paramount importance to address such interactions at the molecular and cellular levels. We report an in vitro analysis of the effects of molecules secreted by Lactobacillus gasseri SF1183 on HCT116 cells, selected as a model of intestinal epithelial cells. SF1183 is a L. gasseri strain isolated from an ileal biopsy of a human healthy volunteer, able to prevent colitis symptoms in vivo. Expanding previous findings, we show that bioactive molecules secreted by SF1183 reduce the proliferation of HCT116 cells in a reversible manner determining a variation in cell cycle markers (p21WAF, p53, cyclin D1) and resulting in the protection of HCT116 cells from TNF-alfa induced apoptosis, an effect potentially relevant for the protection of the epithelial barrier integrity and reconstitution of tissue homeostasis. Consistently, SF1183 secreted molecules increase the recruitment of occludin, a major component of TJ, at the cell–cell contacts, suggesting a reinforcement of the barrier function.