GUT MICROFLORA HAVE GUT MICROFLORA HAVE TO BE MODERNLY TO BE MODERNLY CONSIDERED A CONSIDERED A “MICROMIAL ORGAN” “MICROMIAL ORGAN” PLACED WITHIN A HOST PLACED WITHIN A HOST ORGAN.ORGAN.

•The intestinal habitat of an individual The intestinal habitat of an individual contains 300-500 different species of contains 300-500 different species of bacteria, and the number of microbial bacteria, and the number of microbial cells within the gut lumen is about 10 cells within the gut lumen is about 10 times larger than the number of times larger than the number of eukariotic cells in the human body. eukariotic cells in the human body. Bengmark S. Gut 1998Bengmark S. Gut 1998

•Gastric acid, bile and pancreatic Gastric acid, bile and pancreatic secretion kill most ingested secretion kill most ingested microorganisms.microorganisms.

•The large intestine contains a complex The large intestine contains a complex and dynamic microbial ecosystem with and dynamic microbial ecosystem with high densities of living bacteria which high densities of living bacteria which achieve concentrations of up to 10achieve concentrations of up to 101212or or 101013 13 cells/g of luminal contents. cells/g of luminal contents. Simon GL. Simon GL. Gastroenterology 1984Gastroenterology 1984

Czarniecki M. J Med Chem 2008

•A large proportion of the faecal mass A large proportion of the faecal mass consists of bacteria (around 60%). consists of bacteria (around 60%). Stephen AM, Cummings JH. J.Med.Microbiol.Stephen AM, Cummings JH. J.Med.Microbiol.

•Some of these bacteria are potential Some of these bacteria are potential pathogens. However, the presence of pathogens. However, the presence of bacterial competition and the bacterial competition and the constant interation between the host constant interation between the host and its microbial guests can infer and its microbial guests can infer important health benefits to the important health benefits to the human host. human host. Salminen S et al. Br J Nutr1998.Salminen S et al. Br J Nutr1998.

COMPOSITION OF THE FLORACOMPOSITION OF THE FLORA

-Colonization of the gastrointestinal tract starts -Colonization of the gastrointestinal tract starts immediately after birth, occurs within a few immediately after birth, occurs within a few days, and it is linked to the type of delivery, days, and it is linked to the type of delivery, the type of diet and environmental factors.the type of diet and environmental factors.

-Pioneer bacteria can modulate expression of -Pioneer bacteria can modulate expression of genes in host epithelial cells, thus creating a genes in host epithelial cells, thus creating a favourable habitat for themselves, and can favourable habitat for themselves, and can prevent growth of other bacteria in the prevent growth of other bacteria in the ecosystem. The initial colonisation is therefore ecosystem. The initial colonisation is therefore very relevant to the final composition of the very relevant to the final composition of the permanent flora. permanent flora. Hooper LV et al. Science 2001Hooper LV et al. Science 2001

-Resident bacteria deliver regulatory signals to -Resident bacteria deliver regulatory signals to the epithelium and instruct mucosal immune the epithelium and instruct mucosal immune response. response. O’hara AM. Sci World J 2007O’hara AM. Sci World J 2007

• Anaerobic bacteria outnumber aerobic Anaerobic bacteria outnumber aerobic bacteria by a factor of 100-1000. The genera bacteria by a factor of 100-1000. The genera bacterioides, bifidobacterium, eubacterium, bacterioides, bifidobacterium, eubacterium, clostridium, peptococcus,peptostreptococcus, clostridium, peptococcus,peptostreptococcus, and ruminococcus are predominant in human and ruminococcus are predominant in human beings, whereas aerobes(facultative beings, whereas aerobes(facultative anaerobe) such as escherichia, enterobacter, anaerobe) such as escherichia, enterobacter, enterococcus, klebsiella, lactobacillus, enterococcus, klebsiella, lactobacillus, proteus, etc are among the subdominant proteus, etc are among the subdominant genera. genera. Salminen S. Br J Nutr 1998Salminen S. Br J Nutr 1998

• Every individual presents a particular Every individual presents a particular combination of predominant species that is combination of predominant species that is distinct from that found in other individuals.distinct from that found in other individuals.

• Although bacterial composition can fluctuate Although bacterial composition can fluctuate under some circumstances (acute diarrhoeal under some circumstances (acute diarrhoeal illnesses, antibiotic treatment, dietary illnesses, antibiotic treatment, dietary interventions), individuals’ flora composition interventions), individuals’ flora composition pattern usually remain constant. pattern usually remain constant. Willson kh. Appl Willson kh. Appl Environ Microbiol 1996Environ Microbiol 1996

•Several bacteria that can be seen by Several bacteria that can be seen by direct microscopic examination of diluted direct microscopic examination of diluted faecal specimens cannot be grown in faecal specimens cannot be grown in culture media: 40-80% of total culture media: 40-80% of total microscopic counts are not recoverable by microscopic counts are not recoverable by culture, although estimates vary between culture, although estimates vary between individuals and between studies. individuals and between studies. Suau A et al. Suau A et al. Appl Environ Microbiol 1999Appl Environ Microbiol 1999

•Molecular biological procedures can now Molecular biological procedures can now be used to investigate the microbial be used to investigate the microbial ecology in the colon. Many DNA ecology in the colon. Many DNA sequences correspond to previously sequences correspond to previously undescribed bacteria and data suggest undescribed bacteria and data suggest that every individual has unique strains of that every individual has unique strains of bacteria. bacteria. Tannock GW. Am J Clin Nutr 2001Tannock GW. Am J Clin Nutr 2001

• LA METAGENOMICA E' UN APPROCCIO BASATO SULL'UTILIZZO DILA METAGENOMICA E' UN APPROCCIO BASATO SULL'UTILIZZO DI

TECNICHE GENOMICHE MODERNE PER LO STUDIO DI COMUNITA'TECNICHE GENOMICHE MODERNE PER LO STUDIO DI COMUNITA'

MICROBICHE DIRETTAMENTE NEL LORO AMBIENTE NATURALE,MICROBICHE DIRETTAMENTE NEL LORO AMBIENTE NATURALE,

EVITANDO IL PRELIEVO E LA COLTURA IN LABORATORIO.EVITANDO IL PRELIEVO E LA COLTURA IN LABORATORIO.

SI BASA SUL SEQUENZIAMENTO DEL GENOMA DI MICROORGANISMISI BASA SUL SEQUENZIAMENTO DEL GENOMA DI MICROORGANISMI

LA CUI ANALISI NEL LORO HABITAT NATURALE VIENE DEFINITA LA CUI ANALISI NEL LORO HABITAT NATURALE VIENE DEFINITA

METAGENOMA.METAGENOMA.

• LA METAGENOMICA HA MESSO IN EVIDENZA UNA DIVERSITA' LA METAGENOMICA HA MESSO IN EVIDENZA UNA DIVERSITA'

SENZA PRECEDENTI NELLA COMPOSIZIONE DELLE COMUNITA' SENZA PRECEDENTI NELLA COMPOSIZIONE DELLE COMUNITA' MICROBICHE CON UNA LARGHISSIMA PROPORZIONE DI NUOVIMICROBICHE CON UNA LARGHISSIMA PROPORZIONE DI NUOVI

GENI.GENI.

• THE HUMAN GUT HOSTS MORE THAN 100 THE HUMAN GUT HOSTS MORE THAN 100 TRILLIONTRILLION

BACTERIA, WHICH TOGETHER MAKE UP THE GUT BACTERIA, WHICH TOGETHER MAKE UP THE GUT MICROBIOTA. THE GUT MICROBIOTA CO-MICROBIOTA. THE GUT MICROBIOTA CO-EVOLVED WITH HUMANS TO ACHIEVE A EVOLVED WITH HUMANS TO ACHIEVE A SYMBIOTIC RELATIONSHIP LEADING TO SYMBIOTIC RELATIONSHIP LEADING TO PHYSIOLOGICAL HOMEOSTASIS. PHYSIOLOGICAL HOMEOSTASIS.

• THE HUMAN MICROBIOTA REMAINS GLOBALLY THE HUMAN MICROBIOTA REMAINS GLOBALLY STABLE OVER TIME IN HEALTHY CONDITIONS STABLE OVER TIME IN HEALTHY CONDITIONS AND ABSENCE OF PERTURBATION.AND ABSENCE OF PERTURBATION.

• APPLICATION OF METAGENOMICS TO THIS THIS APPLICATION OF METAGENOMICS TO THIS THIS ENVIRONMENT MAY HELP IN DECIPHERING KEY ENVIRONMENT MAY HELP IN DECIPHERING KEY FUNCTIONS AND KEY BACTERIA INVOLVED IN FUNCTIONS AND KEY BACTERIA INVOLVED IN HEALTH AND DISEASE.HEALTH AND DISEASE.

FAECAL BACTERIOTHERAPY: FAECAL BACTERIOTHERAPY:

AN UNTHINKABLE APPROACHAN UNTHINKABLE APPROACHFaecal bacteriotherapy has a longstanding history in animal Faecal bacteriotherapy has a longstanding history in animal

health. In human beings it has been used in selected cases, health. In human beings it has been used in selected cases, especially as a treatment of last resort for patients with severe especially as a treatment of last resort for patients with severe Clostridium difficile pseudomembranous colitis.Clostridium difficile pseudomembranous colitis.

Schwan A, Sjölin S, Trottestam U, Aronsson B.     Relapsing clostridium difficile enterocolitis cured by rectal infusion of homologous faeces.Lancet. 1983 Oct 8;2(8354):845. Borody TJ, Warren EF, Leis SM, Surace R, Ashman O, Siarakas S.     Bacteriotherapy using fecal flora: toying with human motions.J Clin Gastroenterol. 2004 Jul;38

Although it is unthinkable that this approach will gain the favour of the people, it could indicate that the rectal infusion represents an alternative physiological administration way of probiotics in selected clinical conditions.Ricci N, Caselli M. Rectal infusion of bacterial preparations for intestinal disorders. Lancet 1983; ii:1984-85

• IN THE ERA OF METAGENOMICS FECAL IN THE ERA OF METAGENOMICS FECAL TRANSPLANT AND SYNTHETIC TRANSPLANT AND SYNTHETIC MICROBIOME TRANSPLANT MIGHT BE MICROBIOME TRANSPLANT MIGHT BE VERY PROMISING THERAPIES FOR VERY PROMISING THERAPIES FOR DYSBIOSIS-ASSOCIATED DISEASES.DYSBIOSIS-ASSOCIATED DISEASES.

FUTURE OF MICROBIOME STUDIESFUTURE OF MICROBIOME STUDIES• NEED FOR STANDARDIZATIONNEED FOR STANDARDIZATION

• NEED FOR LARGE PROSPECTIVE COHORTSNEED FOR LARGE PROSPECTIVE COHORTS

• NEED FOR HOLISTIC APPROACHNEED FOR HOLISTIC APPROACH

• NEED FOR AN ECOLOGICAL UNDERSTANDINGNEED FOR AN ECOLOGICAL UNDERSTANDING

UNDERSTANDING CO-EVOLUTION OF THE UNDERSTANDING CO-EVOLUTION OF THE MICROBIAL GENOMES AND HOST MICROBIAL GENOMES AND HOST GENOME, FROM GEOGRAPHICALLY AND GENOME, FROM GEOGRAPHICALLY AND CULTURALLY DIVERSE POPULATIONS, CULTURALLY DIVERSE POPULATIONS, WILL BE KEY FOR FUTURE STRATEGIC WILL BE KEY FOR FUTURE STRATEGIC PREVENTIVE MEASURES THROUGHOUT PREVENTIVE MEASURES THROUGHOUT THE LIFE-SPAN.THE LIFE-SPAN.

METABOLOMIC VIEW METABOLOMIC VIEW

METABOLOMIC APPROACHES, IN WHICH A LARGE METABOLOMIC APPROACHES, IN WHICH A LARGE NUMBER OF SMALL MOLECULE METABOLITES CAN BE NUMBER OF SMALL MOLECULE METABOLITES CAN BE DEFINED IN A BIOLOGICAL SAMPLE, OFFER A VERY DEFINED IN A BIOLOGICAL SAMPLE, OFFER A VERY PROMISING AND POWERFUL AVENUE TO PROMISING AND POWERFUL AVENUE TO “FINGERPRINT” MICROBIOTA FUNCTIONAL STATUS, “FINGERPRINT” MICROBIOTA FUNCTIONAL STATUS, WITH FUNCTIONAL CHARACTERIZATION OF THE WITH FUNCTIONAL CHARACTERIZATION OF THE INTESTINAL MICROBIOTA AND ITS INTERACTIONS INTESTINAL MICROBIOTA AND ITS INTERACTIONS WITH THE HOST.WITH THE HOST.

WHAT IS MORE IMPORTANT IN INDUCING WHAT IS MORE IMPORTANT IN INDUCING INFLAMMATORY AND CANCEROUS INFLAMMATORY AND CANCEROUS CHANGES?CHANGES?

- A GENERAL DESTRUCTURATION OF THE ECOSYSTEM- A GENERAL DESTRUCTURATION OF THE ECOSYSTEM

- SOME SPECIFIC BACTERIA - SOME SPECIFIC BACTERIA

EXAMPLES OF EMERGIG SPECIFIC EXAMPLES OF EMERGIG SPECIFIC BACTERIA:BACTERIA:

-ENTEROTOXIGENIC BACTERIOIDES FRAGILIS-ENTEROTOXIGENIC BACTERIOIDES FRAGILISINVOLVED IN IBD AND COLORECTAL CANCERINVOLVED IN IBD AND COLORECTAL CANCER

-FUSOBACTERIA (NUCLEATUM, VARIUM, etc)-FUSOBACTERIA (NUCLEATUM, VARIUM, etc)INVOLVED IN IBD AND COLORECTAL CANCERINVOLVED IN IBD AND COLORECTAL CANCER

-SEGMENTED FILAMENTOUS BACTERIA (SFB)-SEGMENTED FILAMENTOUS BACTERIA (SFB)POSSIBLY INVOLVED IN AUTOIMMUNE DISEASESPOSSIBLY INVOLVED IN AUTOIMMUNE DISEASES

PER CONOSCERE BENE LE COSE,BISOGNA CONOSCERNE I PARTICOLARI; E SICCOMEQUESTI SONO INFINITI,LE NOSTRE CONOSCENZE SONO SEMPRE SUPERFICIALI EDIMPERFETTE. Francois de La Rochefoucauld, 1665