Human beings are reconsidered as “super-organisms” in co-evolution with their own indigenous microbial community (1). The vast majority of these microbes (10 to 100 trillions) inhabits the gastrointestinal tract, and constitutes the human intestinal microbiota. Their collective genome, defined as microbiome, is estimated to contain >=100 times as many genes as our 2.85-billion base pair human genome (2). Thus, the human “super-organism” possesses a “meta-genome”, which is consisting of human genes and the genes present in the genomes of the trillions of symbiotic microbes that mainly colonize the intestine. Throughout an extensive microbial-mammalian co-metabolism, the intestinal microbiome evolved to exert a marked influence on the human metabolic phenotype (3). Indeed, the intestinal ecosystem is partially responsible for maintaining human health. It is involved in the protection against pathogens, education of the immune system and modulation of gastrointestinal development. Besides, intestinal microbes play a pivotal role or contribute to cause many diseases. Alterations of the composition of the intestinal microbiota are associated with inflammatory bowel diseases, irritable bowel syndrome and allergic diseases (4, 5). Differences in the composition of the intestinal microbiota are also linked to Type 1 and Type 2 diabetes (6), and celiac sprue (7).
Recently, the microbiota associated to saliva was also a studied issue because of the importance of the oral microorganisms for oral hygiene and development of oral diseases. Seven major phyla were found in all relevant studies on salivary microbiota (8). Even though the microbiota is rather stable over time, intra- and inter-individual variations were found (9). The salivary microbiota is also recognized as associated to or as specific marker for diseases, being such diseases not necessarily oral pathologies. Indeed, specific microbial consortia that populate the saliva ecosystem were associated to obesity (10), cancer (11) and HIV (12). Interestingly, the similarity between salivary and fecal microbiota was investigated (13). Identical lactobacilli were able to inhabit both ends of the orogastrointestinal tract, whereas the composition of the other bacterial groups studied varied between the two sites.
The composition of the oral and intestinal microbiota is influenced by genetic factors (14), age (15) and diet (16, 17). Diet is the main reservoir of microbes and, especially, is the nutrient source for the host, and related oral and intestinal microbiota. Excluding obvious geographic differences, three main dietary habits are worldwide diffused: omnivore, vegetarian and vegan. No literature data (ISI Web of Knowledge) are available on the number of microorganisms that are daily ingested as influenced by dietary habits. Further, no literature data describe the composition of the oral and intestinal microbiota as comparatively affected by omnivore, vegetarian and vegan diets. Previously, the effect of the vegetarian diet only on fecal microbiota was investigated through culture-dependent and -independent methods (17-23). Only one study described such effect using high-throughput of the 16S rRNA gene sequencing and biochemical analyses (24). Based on this lack of information, two major aspects are worthy of investigation: (i) the presumptive microbial load, which is daily ingested by humans depending on the main types of diet; and (ii) the high-throughput and integrated “-omics” characterization of the oral and intestinal microbiota, as comparatively affected by dietary habits. Both these topics are consistent with aim of the present project.
Foods eaten are rarely sterile. In most of the cases, they contain associations of autochthonous, contaminant and/or deliberately added microorganisms that gain access, grow, survive and interact in the food matrix over the time. Fermented foods are those, which contain the highest number of microorganisms (25). Depending on dietary habits, the consumption of fermented products may correspond up to ca. 1/3 of the human daily intake of foods (26). Milk beverages, cheeses, sausages and fermented vegetables are the most common fermented products. The main features of fermented foods, including the number of microorganisms harboured, may markedly vary depending on numerous factors such as the technology of manufacture, the type (artisanal or industrial) of production, and the conditions of storage. Besides, a number of foods, specifically eaten by vegetarian and vegan consumers (e.g., soy sprouts, tofu, edible algae), are poorly or not at all investigated. When exhaustive information on dietary habits are provided by a statistically significant number of individuals, the presumptive intake of microorganisms should be estimable. The combined use of culture-dependent and -independent methods, including next generation sequencing, is the most appropriate approach to describe the microbial diversity. To get an overall picture, the metabolome characterization of foods should necessarily complete the relationship between foods and humans before beginning the physiology studies. Foods contain thousands of chemical compounds, which upon digestion and metabolism give rise to complex physiological reactions and possible changes in the intestinal microbiota. These changes result in a number of metabolites that are detectable in saliva, feces and urine. It is estimated that the omnivore diet exposes humans to more than 15,000 components, 8,000 of which are non-nutrients. Some of these components directly affect the composition of the oral and intestinal microbiota, and the related metabolic output, which may harbour physiologically active compounds for the host (27).
Depending on dietary habits, nutrients from foods affect the diversity of microorganisms that inhabit the saliva and intestine, in different ways. Within saliva and intestinal microbiota, an important distinction can be made between individual “core” and common “core”. The former represents a group of microorganisms that are stably present in the microbiota of an individual, while the latter indicates populations that are common to the majority of the individuals (28). The studies focused on complex microbial ecosystems have progressively entered into the era of “mega-sequencing projects”, which for instance include the Genomic Encyclopaedia of Bacteria and Archaea project (http://www.jgi.doe.gov/programs/GEBA), the Microbial Earth Project (http://genome.jgi.doe.gov/programs/bacteria-archaea/MEP/index.jsf), the Human Microbiome Project (http://nihroadmap.nih.gov/hmp), the Metagenomics of the Human Intestinal Tract consortium (http://www.metahit.eu),the Terragenome Initiative (http://www.terragenome.org) and the Earth Microbiome Project (http://www.earthmicrobiome.org) (29). The current development of high-throughput “-omics” methods allow for unique insight in the functions of complex communities (e.g., intestinal microbiota) and make possible to have a look at the mixed microbial communities as one “meta-organism” (30). Only coupling meta-genomics, meta-transcriptomics, meta-proteomics and meta-metabolomics the entire genetic and metabolic potential of the human oral and intestinal microbiota may be unravelled to answer not only the “who's there” question, but also “what can they do together?” Such approach was never used to study the effect of the dietary habits on the oral and fecal microbiota. Overall, the study of the effect of diet on the composition of the intestinal microbiota has an intrinsic limitation. For obvious ethical reasons, biopsies are not available. Although the composition of the intestinal microbiota partly differs from that of the fecal microbiota, numerous studies profitably used fecal samples to describe such complex ecosystem (18-24). Especially when the population of individuals to be studied is numerous and when the project aims to compare various dietary habits, the flow chart of activities and the experimental plan need to make use of the most suitable combination of techniques. For this purposes, the essential value of traditional techniques (e.g., culture-dependent) and well established molecular methods in ecological studies (e.g., PCR-DGGE) should not be overlooked, but used in combination with high-throughput “-omics” techniques (4).
To trace the most complete picture, further functional features of the “meta-organism” should be depicted. One of these aspects concerns the role of the faecal microbiota on the modulation of genotoxic and mutagenic risks at the level of intestine (31). Depending on dietary habits (e.g., intake of fat, proteins, fibre), the risks of carcinogenicity and chronic degenerative diseases may vary between individuals (32). Several compounds, either directly deriving from foods (e.g., mycotoxins, polycyclic aromatic hydrocarbons) or originating from the microbial metabolism (e.g., secondary bile acids, nitrosamines), have genotoxic properties (33). Microbial communities that inhabit the intestine possess antigenotoxic properties, which may cause the significant decrease of the biological activity of such chemical compounds (34). The development of the antibiotic resistance is another concrete risk for human health. As highly adaptable organisms, especially bacteria (e.g., enterococci and lactobacilli) are becoming more and more resistant to conventional antibiotics, thereby reducing the number of available antimicrobial agents. The phenomenon of acquired resistance in lactic acid bacteria is particularly risky since they may act as the reservoir of antibiotic resistant genes, which are transferred to foodborne or enteric intestinal and oral pathogens, when localized on mobile genetic elements (e.g., plasmids and transposons) (35). Since dietary intake represents one of the main route for the entrance of antibiotic resistant bacteria into the oral and intestinal tracts, the estimation of the impact of different dietary habits should provide new insights to control the mechanisms of transmission. Diseases caused by dysfunctional interactions between foods and human body are as numerous as they are broad, ranging from gastrointestinal to respiratory syndromes. The immune system is at the cornerstone of this interaction (36). It may be argued that there are four dominant ways for interaction between immune system and diet: over-nutrition (e.g., obesity), under-nutrition (e.g., anorexia and scurvy), dysfunctional nutrition (e.g., food allergy), and via genetic disease (37). More and more often evidences on the intestinal microbiota as the master regulator of the immune equilibrium, which confers protection to the host against inflammatory, autoimmune, and allergic diseases, are reported (38).
Based on this description of the state of the art, the response of the organism to the diet and the relative alteration of the susceptibility to disease may be investigated in order to comprehend the role of the microbiota to maintain the state of well being in humans. If the composition of the human microbiota is considered as an indicator of the state of health, it may constitute the basis for further exploration of the different types of diet as a key health determinant and open new horizons for disease prevention. Accordingly, the present project aims at determining the number and diversity of microorganisms carried by the omnivore, vegetarian and vegan diets, and at describing the diversity of the saliva and fecal microbiota of individuals subjected to different dietary habits. Next generation sequencing and high-throughput meta-genomic, meta-transcriptomic, meta-proteomic and meta-metabolomic techniques will be used to look at the mixed microbial communities as a whole meta-organism.
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