Molecular characterization of the human gut microbiota: the effect of aging

Age-related physiological changes in the gastrointestinal tract, as well as modification in lifestyle, nutritional behaviour, and functionality of the host immune system, inevitably affect the gut microbiota. The study presented here is focused on the application and comparison of two different microarray approaches for the characterization of the human gut microbiota, the HITChip and the HTF-Microb.Array, with particular attention to the effects of the aging process on the composition of this ecosystem. By using the Human Intestinal Tract Chip (HITChip), recently developed at the Wageningen University, The Netherland, we explored the age-related changes of gut microbiota during the whole adult lifespan, from young adults, through elderly to centenarians. We observed that the microbial composition and diversity of the gut ecosystem of young adults and seventy-years old people is highly similar but differs significantly from that of the centenarians. After 100 years of symbiotic association with the human host, the microbiota is characterized by a rearrangement in the Firmicutes population and an enrichment of facultative anaerobes. The presence of such a compromised microbiota in the centenarians is associated with an increased inflammation status, also known as inflamm-aging, as determined by a range of peripheral blood inflammatory markers. In parallel, we overtook the development of our own phylogenetic microarray with a lower number of targets, aiming the description of the human gut microbiota structure at high taxonomic level. The resulting chip was called High Taxonomic level Fingerprinting Microbiota Array (HTF-Microb.Array), and was based on the Ligase Detection Reaction (LDR) technology, which allowed us to develop a fast and sensitive tool for the fingerprint of the human gut microbiota in terms of presence/absence of the principal groups. The validation on artificial DNA mixes, as well as the pilot study involving eight healthy young adults, demonstrated that the HTF-Microb.Array can be used to successfully characterize the human gut microbiota, allowing us to obtain results which are in approximate accordance with the most recent characterizations. Conversely, the evaluation of the relative abundance of the target groups on the bases of the relative fluorescence intensity probes response still has some hindrances, as demonstrated by comparing the HTF.Microb.Array and HITChip high taxonomic level fingerprints of the same centenarians.

Dietary strategies to minimize the detrimental impact of weaning on gut morphology, physiology and microbiota of piglets

Weaning is a crucial period in the management of piglets. In modern piggeries economic interest make weaning age decrease more and more and the detrimental consequences of weaning have as much importance as earlier the weaning occurs. The risk of development of post-weaning diarrhea (PWD) in piglets is high and PWD is the cause of serious economic losses in pig herds. In the past the supplementation of the feed given after weaning with growth promoters antibiotics, in order to keep PWD under control, used to be a common practice, but their usage has been banned in EU since 2006. This measure led to the investigation of alternative suitable feed supplements that would be reasonably efficient in protecting and sustaining animal health and performance. Aim of this thesis was to evaluate the effect of some different alternatives to growth-promoters antibiotics on weaning piglets and to assess if some of them could be considered as valuables options to replace auxinic in animal feeding. The study is composed by four experimental trials. The first one aims to identify mechanisms involved in the auxinic effects of antibiotics in the diets; the following three evaluate the addition butyric acid, tryptophan, and nitrate as alternative to in-feed antimicrobials. Although some results are controversial, it appears from the data presented that the alternatives to in-feed antibiotics considered may exert positive effects on some zootechnical and health parameters on piglet in the post-weaning period. Anyway, the mechanism of action and the interaction with microbiota of such additives should be investigated inside out because many effects remains poorly understood.

Taste receptors in the gut: a chemosensitive mechanism from fish to human

The ingestion of a meal evokes a series of digestive processes, which consist of the essential functions of the digestive system: food transport, secretory activity, absorption of nutrients and the expulsion of undigested residues do not absorbed. The gastrointestinal chemosensitivity is characterized by cellular elements of the endocrine gastrointestinal mucosa and nerve fibers, in particular of vagal nature. A wide range of mediators endocrine and/or paracrine can be released from various endocrine cells in response to nutrients in the diet. These hormones, in addition to their direct activity, act through specific receptors activating some of the most important functions in the control of energy intake and energy homeostasis in the body. For integration of this complex system of control of gastrointestinal chemosensitivity, recent evidence demonstrates the presence of taste receptors (TR) belonging to the family of G proteins coupled receptor expressed in the mucosa of the gastrointestinal tract of different mammals and human. This thesis is divided into several research projects that have been conceived in order to clarify the relationship between TR and nutrients. To define this relationship I have used various scientific approaches, which have gone on to evaluate changes in signal molecules of TR, in particular of the α-transducin in the fasting state and after refeeding with standard diet in the gastrointestinal tract of the pig, the mapping of the same molecule signal in the gastrointestinal tract of fish (Dicentrarchus labrax), the signaling pathway of bitter TR in the STC-1 endocrine cell line and finally the involvement of bitter TR in particular of T2R38 in patients with an excessive caloric intake. The results showed how there is a close correlation between nutrients, TR and hormonal release and how they are useful both in taste perception but also likely to be involved in chronic diseases such as obesity.

Metagenomic trajectory of gut microbiome in the human lifespan

Co-evolving with the human host, gut microbiota establishes configurations, which vary under the pressure of inflammation, disease, ageing, diet and lifestyle. In order to describe the multi-stability of the microbiome-host relationship, we studied specific tracts of the bacterial trajectory during the human lifespan and we characterized peculiar deviations from the hypothetical development, caused by disease, using molecular techniques, such as phylogenetic microarray and next-generation sequencing. Firstly, we characterized the enterocyte-associated microbiota in breast-fed infants and adults, describing remarkable differences between the two groups of subjects. Successively, we investigated the impact of atopy on the development of the microbiome in Italian childrens, highlithing conspicuous deviations from the child-type microbiota of the Italian controls. To explore variation in the gut microbiota depending on geographical origins, which reflect different lifestyles, we compared the phylogenetic diversity of the intestinal microbiota of the Hadza hunter-gatherers of Tanzania and Italian adults. Additionally, we characterized the aged-type microbiome, describing the changes occurred in the metabolic potential of the gut microbiota of centenarians with respect to younger individuals, as a part of the pathophysiolology of the ageing process. Finally, we evaluated the impact of a probiotics intervention on the intestinal microbiota of elderly people, showing the repair of some age-related dysbioses. These studies contribute to elucidate several aspects of the intestinal microbiome development during the human lifespan, depicting the microbiota as an extremely plastic entity, capable of being reconfigured in response to different environmental factors and/or stressors of endogenous origin.

Interactions between the gut microbiota, short-chain fatty acids and the immune system in pediatric patients undergoing hematopoietic stem cell transplantation

The gut microbiota (GM) is essential for human health and contributes to several diseases; indeed it can be considered an extension of the self and, together with the genetic makeup, determines the physiology of an organism. In this thesis has been studied the peripheral immune system reconstitution in pediatric patients undergoing allogeneic hematopoietic stem cell transplantation (aHSCT) in the early phase; in parallel, have been also explored the gut microbiota variations as one of the of primary factors in governing the fate of the immunological recovery, predisposing or protecting from complications such as the onset of acute graft-versus-host disease (GvHD). Has been demonstrated, to our knowledge for the first time, that aHSCT in pediatric patients is associated to a profound modification of the GM ecosystem with a disruption of its mutualistic asset. aGvHD and non-aGvHD subjects showed differences in the process of GM recovery, in members abundance of the phylum Bacteroidetes, and in propionate fecal concentration; the latter are higher in the pre-HSCT composition of non-GvHD subjects than GvHD ones. Short-chain fatty acids (SCFAs), such as acetate, butyrate and propionate, are end-products of microbial fermentation of macronutrients and distribute systemically from the gut to blood. For this reason, has been studied their effect in vitro on human DCs, the key regulators of our immune system and the main player of aGvHD onset. Has been observed that propionate and, particularly, butyrate show a strong and direct immunomodulatory activity on DCs reducing inflammatory markers such as chemokines and interleukins. This study, with the needed caution, suggests that the pre-existing GM structure can be protective against aGvHD onset, exerting its protective role through SCFAs. They, indeed, may regulate cell traffic within secondary lymphoid tissues, influence T cell development during antigen recognition, and, thus, directly shape the immune system.

Relevance of the pig stomach for the detection of dietary factors and gut maturation and control

In this thesis two approaches were applied to achieve a double general objective. The first chapter was dedicated to the study of the distribution of the expression of genes of several bitter and fat receptor in several gastrointestinal tracts. A set of 7 genes for bitter taste and for 3 genes for fat taste was amplified with real-time PCR from mRNA extracted from 5 gastrointestinal segments of weaned pigs. The presence of gene expression for several chemosensing receptors for bitter and fat taste in different compartments of the stomach confirms that this organ should be considered a player for the early detection of bolus composition. In the second chapter we investigated in young pigs the distribution of butyrate-sensing olfactory receptor (OR51E1) receptor along the GIT, its relation with some endocrine markers, its variation with age, and after interventions affecting the gut environment and intestinal microbiota in piglets and in different tissues. Our results indicate that OR51E1 is strictly related to the normal GIT enteroendocrine activity. In the third chapter we investigated the differential gene expression between oxyntic and pyloric mucosa in seven starter pigs. The obtained data indicate that there is significant differential gene exression between oxintic of the young pig and pyloric mucosa and further functional studies are needed to confirm their physiological importance. In the last chapter, thymol, that has been proposed as an oral alternative to antibiotics in the feed of pigs and broilers, was introduced directly into the stomach of 8 weaned pigs and sampled for gastric oxyntic and pyloric mucosa. The analysis of the whole transcript expression shoes that the stimulation of gastric proliferative activity and the control of digestive activity by thymol can influence positively gastric maturation and function in the weaned pigs.