Exploring different microbial communities through metagenomic approaches

Abstract

The human body harbors a complex and dynamic population of microorganisms residing in various compartments such as the gastrointestinal, genitourinary and respiratory tracts and the skin surfaces, all universally recognized as the human microbiota. Among the different human-related microbial communities, the gut microbiota is certainly one of the most studied due to the high complexity and the extreme heterogeneity of the microbial ecosystems it retains, which have co-evolved over the decades to form a mutually beneficial relationship with the host from which both parties take advantages. Recently, the scientific community has turned particular interest to study the bacterial component of the intestinal microbiota since bacteria are involved in a continuous dialogue with the host affecting its health. Indeed, the gut microbiota has been found to be crucial for immunologic, hormonal, and metabolic homeostasis of the host. However, several factors, like the environment and the host's lifestyles, may cause modifications in the microbiota composition, thus causing dysbiosis, which is often related to the onset of many diseases. Traditionally, the assessment of these bacterial communities has been based on conventional culture-dependent methods that do not allow an exhaustive characterization of the microbial biodiversity occurring in the human body. In the last decades, the advent and development of culture-independent approaches based on Next-Generation Sequencing (NGS) techniques, also known as metagenomics, allowed an accurate disentangling of the microbial populations inhabiting the human body. Specifically, metagenomics attempts offer the possibility to profile the bacterial taxonomy and predict the functional activities exploited by the microbial communities and thus underpinning the microbe-microbe and microbe-host interactions. Thanks to NGS approaches, the interest in studying the microbiome and its role in the establishment and maintenance of human health, also for diagnostic purposes has strikingly increased. Despite that, the study of microbiome composition in human biological specimens does not always come without challenges due to many procedural issues. The aim of this Ph.D. thesis is to explore the composition of different microbial communities by means of the most reliable metagenomics approach. Specifically, it aims to investigate the reliability of a widely used protocol for the depletion of eukaryotic DNA through the analysis of different human specimens rich in host DNA, focusing on the impact of this protocol on the detection of bacterial populations. Furthermore, through NGS techniques, the study of the microbiota, with a particular interest in the gut microbiota, also highlighted aspects concerning its modulation. In this context, there is growing scientific evidence on the correlation between the alteration of gut microbiota composition and disorders. Many potential factors such as diet, prebiotic compounds and probiotic products can modulate the human intestinal microbiota, trying to re-establish an altered bacterial composition. However, despite the wide commercial employment of probiotic formulations, very little is known about the molecular mechanisms of the action and the genetic features of probiotic bacteria. In this context, this Ph.D. thesis aims to unravel the microbiota composition of different probiotic products present on the Italian market by developing a powerful and reliable pipeline combining whole metagenome shotgun analyses and flow cytometry assays to verify the quality of probiotic formulations. Moreover, among the various factors that can influence the gut microbiota composition, there is solid and liquid diet. Concerning the gut modulation acted by food, in this Ph.D. thesis, we investigated the resident microbiome of many Italian raw milk cheeses being part of Protected Designation of Origin (PDO) denomination, revealing how the microbiota harbored by each cheese is mainly linked to the type of cheesemaking process together with local environmental factors rather than exclusively to the cheese type or the geographical origin. The study of food microbiota represents the first necessary step to get an overview of the possible influence these microbial communities could exploit on the consumer’s intestinal microbiota. Finally, water is in all respects considered as a food but also as a reservoir of microorganisms able to colonize and modulate the consumer’s intestinal microbiota. In this context, one of the purposes of this Ph.D. thesis is to evaluate the microbial composition inhabiting drinking water through a comprehensive shotgun metagenomics analysis of tap water microbiome, highlighting the occurrence of a deeply bacterial biodiversity and the presence of a conserved core tap water microbiota most represented by unknown microbial species, constituting the so-called microbial dark matter. Furthermore, genome reconstruction of the dominant bacterial genera of water microbiota allowed us to unveil their presence in the fecal microbiome of humans from various geographical locations, providing evidence of a potential novel route of horizontal microbial transmission.