Created on 05 June 2013

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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