Application of metagenomic approaches in discovering the gut microbiota functionality

Abstract

In the last years, the advent of the Next-Generation Sequencing (NGS) technologies allowed to unveil the composition and functionality of the microbial populations inhabiting humans and other animals, i.e. microbiota. In the human context, it has been estimated that 1014 microorganisms reside in various compartments of the body such as the gastrointestinal, genitourinary and respiratory tracts and the skin surfaces. Among these, the human intestine retains a complex microbial ecosystem, that has co-evolved through the decades to form a mutually beneficial relationship with the host. In this regard, the range of studies focusing on this topic suggested that the gut microbiota exploits crucial functions largely responsible for the healthy status of the host. The gut microbiota can promote, through various mechanisms, intestinal metabolism and immune system modulation. Conversely, several factors, such as the environment and the lifestyle, may cause an upset in the composition of the microbiota, and thus causing dysbiosis. Gut dysbiosis is typically related to a negative impact on host health, and it is associated with a large array of human disorders and disease, such as Inflammatory Bowel Disease (IBD), metabolic syndrome, neurological disorders and colorectal cancer (CRC). The aim of this Ph.D. thesis is to explore the role of intestinal bacteria in human and animal health by means of several metagenomics approaches. In particular, it aims to investigate bifidobacterial compositional differences in various mammalian species (including humans), through a metagenomic analysis, based on the bioinformatics sequencing and analysis of the Internal Transcribed Spacer (ITS) sequences for bifidobacterial profiling (Bifidobacterial-ITS profiling). In addition, it discusses the role of bifidobacteria in the gastrointestinal tract of mammals and how these microorganisms can persist and colonize the human gut. Furthermore, it provides an in depth overview of the mucosa microbiota composition associated with gastrointestinal polyps. This PhD thesis also evaluates the correlation between adenomatous polyps and publicly available metagenomics datasets of CRC, in order to identify possible microbial biomarkers associated with an early detection of CRC. Despite the extensive commercial exploitation of probiotic formulations, there are still major knowledge gaps regarding the precise molecular mechanism of action and corresponding genetic/genomic properties of probiotic bacteria. In this context, this PhD thesis aims to unveil microbiota composition of different probiotic products through the development of an industrial pipeline encompassing 16S rRNA-microbial analysis, combined with a shotgun metagenomics approach. Thus, in order to confirm the obtained in silico metagenomic results, a culturomic approach was used to perform a census of the bacterial populations of all assayed probiotics products. Finally, the purpose of this PhD thesis is also to provide a new metagenomic tool for species-level profiling of complex lactobacilli communities, which complements phylogenetic group assignments that can be obtained from 16S rRNA gene profiling data.