The human gut microbiota

Transcription

1 The human gut microbiota Marco Ventura, PhD. Laboratory of Probiogenomics, Department of Life Sciences University of Parma, Italy 1

2 The Gastrointestinal Tract (GIT) The gastrointestinal tract begins with the mouth, leads to the oesophagus and extends through the stomach, small and large intestine, ending at the distal intestine. 2

3 Human GIT microbiota The gastrointestinal tract (GIT) of human beings is a complex ecosystem whose bacterial populations (microbiota) show the highest densities in natural ecosystems. The human GIT microbiota acts as a multifunctional organ that drives essential human functions. The composition of the human GIT microbiota is widely unknown. Bifidobacteria represent one of the first bacterial colonizers of the GIT. 3 Turroni et al., 2008; Antonie van Leeuwenhoek

4 The GIT microbiota II The gut contains our largest collection of resident microorganisms. Up to bacteria/g in the large intestine of adult. Adult humans are composed of an estimated 10 times more resident microbial than human cells. The GIT microbiota includes both an indigenous microflora (autochthons) and a foreign microflora (contaminant). The composition of autochthonous microbiota is largely unknown. 4

5 Microbial diversity in the human gut The adult GIT contains all three domains of life, Bacteria, Archaea and Eukarya. The divisions that predominate are: Bacteroides (e.g. Bacteroides) Actinobacteria (e.g. Bifidobacterium) Firmicutes (e.g. Lactobacillus) Flavobacterium 5

6 How microbes and humans have coevolved in the intestine Human intestine is lined with an epithelium that turns over rapidly and continuously throughout life. The gut epithelium is overlaid by a mucous-polysaccharide gel layer that constitute a biofilm-like community. Emerging evidences suggests that a dynamic interplay between the microbiota, the mucosal immune system and mucous gel layer may affect microbial community structure in the intestine. 6

7 Bacterial colonisation of the GIT At the birth stage, the human GIT is sterile. The first bacterial colonisation occurs during childbearing. The composition of intestinal microbiota is affected by the different weaning system, i.e. breast-feeding or bottle-feeding. -Breast-feeding: bifidobacteria and LAB -Bottle-feeding: streptococcae and enterobacteria. With ageing the GIT microbiota changes and becoming more complex. 7

8 Human GIT microbiota Up to 1000 species 70% unculturable Closely co-evolved microbial partners Interactions with; Diet Drugs Immune system Gut physiology Systemic metabolism 8 Turroni et al., 2008; Antonie van Leeuwenhoek

9 Relevance of the GIT microbiota Ulcers, gastric cancer Intestinal putrefaction Colon cancer IBD IBS Obesity? Diarrhoea, infections, toxin production Inhibition harmful bacteria H. pylori Digestion/ absorption of food Staphylococci ingredients & minerals C. difficile (SCFA) C. perfringens Immunomodulation Veillonellae Phytochemical Streptocci/Enterococci conversions E. coli Sulphate Reducers Bacteroides Bifidobacteria Lactobacilli C. leptum; C. coccoides; Eubacterium sp. Health status 11 Vitamin Synthesis 9

10 Microbial markers and metabolic disorders Specific microbial taxa are often associated with metabolic disorders such as obesity and diabetes. In obese as well as diabetes patients there is a considerably decline in the population of bifidobacteria Turnbaugh et al., 2009, Nature; Ley et al., 2005, PNAS; Cani et al., 2007, Diabetologia.

11 The human GIT microbiota and bifidobacteria The GIT microbiota is originated at the time of birth from mother s microflora. Bifidobacteria represent one of the first bacterial colonizers of the GIT 11

12 Bifidobacterial ecology Bifidobacteria are commensals of the gastro intestinal tracts (GIT) of humans and animals. Bifidobacterium breve Bifidobacterium bifidum Bifidobacterium pseudocatenulatum Bifidobacterium longum subsp. infantis Bifidobacterium longum subsp. longum Bifidobacterium adolescentis Bifidobacterium catenulatum Bifidobacterium angulatum Bifidobacterium gallicum Bifidobacterium dentium Bifidobacterium tsurumiense Bifidobacterium scardovii Bifidobacterium coryneforme Bifidobacterium indicum Bifidobacterium asteroides Human gut Oral cavity Human blood Insect Bifidobacterium pseudolongum subsp. pseudolongum Bifidobacterium thermacidophilum subsp. porcinum Bifidobacterium psychraerophilum Bifidobacterium thermophilum Bifidobacterium longum subsp. suis Bifidobacterium choerinum Bifidobacterium animalis subsp animalis/lactis Bifidobacterium cuniculi Bifidobacterium magnum Bifidobacterium saeculare Bifidobacterium ruminantium Bifidobacterium merycicum Bifidobacterium pseudolongum subsp. globosum Bifidobacterium boum Bifidobacterium gallinarum Bifidobacterium pullorum Animal gut Bifidobacterium thermacidophilum subsp. thermacidophilum Bifidobacterium minimum Sewage Few bifidobacterial species possess a cosmopolitan lifestyle 12 Ventura et al., 2004; Antonie van Leeuwenhoek

13 Turroni et al, 2009, AEM. Bifidobacterial ecology of the GIT A polyphasic approach involving culture-based methods followed by bacterial identification through the sequencing of 16S-ITS rrna allowed to assess the bifidobacterial diversity of the human GIT microbiota. Bifidobacteria Isolation Seven main taxa forms the intestinal bifidobacterial microbiota Sampling 34 mucosal samples 34 fecal samples 900 isolates S-ITS sequences Bifidobacteria Identification Bifidobacteria s rrna sequence database 13

14 Bifidobacterial diversity in the GIT Turroni et al, 2009 AEM highly variable at inter-subject level; Bifidobacterial populations are: Very different between fecal and mucosal microbiota. stable between different mucosal sites within the same subject; Authocthous vs. allocthonous microbiota are different Few bifidobacterial isolates are widely distributed in the GIT A higher ecological fitness of these bacteria? 14

15 Ventura et al., 2009, FBS Novel approaches to study gut biodiversity: microbiomics Culture independent approaches, such as metagenomics, allowed to get a high level of resolution of the biodiversity of complex microbial communities Gut microbiota Culturable bacteria Unculturable bacteria 15

16 Diversity of the human gut microbiota Eckburg et al Science 16

17 Microbiomic studies and bifidobacteria Environmental sampling Chromosomal DNA extraction PCR amplification of 16S rrna gene & gene cloning Sequencing and taxa identification Metagenomics analyses: a modest contribution of bifidobacteria on the biodiversity of the human intestinal microbiota Commercial kit Enzymatic & mechanical Eckburg et al., PNAS 2005 Turroni et al., 2009 AEM Palmer et al., 2007 Plos Biol. Wang et al., 2005 FEMS Ecol. 17

18 Microbiomic analysis of bifidobacterial population in the human GIT Chromosomal DNA extraction PCR amplification of 16S rrna gene amplicon cloning Next generation sequencing and taxa identification Environmental sampling Enzymatic and mechanical lysis PCR amplification using LM3-LM26 (bifido Genus specific) 16S ITS 23S P0 P6 LM26 LM3

19 New evidences of the bifidobacterial Human mucosa biopsies; impact on the human microbiota: Chr. DNA extraction with enzymatic & mechanical lysis; microbiomics analyses 16S rrna gene PCR amplification with bifidobacterial genus specific primers; 5 mucosal samples 1312 clone 16rRNA sequences 1216 bifidobacterial clones 16S rrna gene cloning and sequening Taxa analysis 19 Turroni et al, 2009 ISME J

20 Do we know the whole human gut bifidobacterial diversity? Phylogenetic groups % B.longum group 17 B.adolescentis group 8.5 B.pseudolongum group 50 uncultured bacteria 11.5 new phylotypes 13 Novel 75 phylotypes: they could be new species? High inter-subject variability 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 32F FS29 M MS51 R All libraries B. longum group B. adolescentis group B. pseudolongum group uncultured bacteria new species 20 Turroni et al, 2009 ISME J

21 Bifidobacterial biodiversity in the infant gut Environmental sampling Chromosomal DNA extraction Enzymatic and mechanical lysis 16S PCR amplification of 16S rrna gene Primers Puni6-P6 ITS Next generation sequencing and taxa identification 23S Puni6 P6 11 infant faecal samples (collected from 3 different geographical origin) and 4 mothers. We obtained a total of reads.

22 Interindividual variability detected in the infant gut microbiota Phylum Family Bifidobacterium genus PCoA highlighted that there is no correlation between any of the classification parameters considered. Turroni et al, 2012, PLoS One, in press

23 Similarities and differences among population patterns OTU corresponding to B. longum OTU corresponding to B. pseudocatenulatum OTU corresponding to Str. thermophilus OTUs corresponding to Ruminococcaceae and uncultured Clostridiales There are common OTUs between infant- and adult- samples. Turroni et al, 2012, PLoS One, in press

24 Analysis of the core microbiota of infant/mother subjects Mothers Phylum Family Bifidobacterium genus Infants Taxonomical classification of the two datasets revealed large differences between infants and adults. With respect to the genus Bifidobacterium, no bifidobacterial species are unique of infants or adults. Turroni et al, 2012, PLoS One, in press

25 A novel approach to understand genetic biodiversity of complex environments: metagenomics Gut microbes outnumbered human cells by a factor of 10; The overall gene set of gut microbes is 150-times higher than the human genome; Although certain microbial species appear to be shared between individuals, considerable variation occurs both in types of microbes and in diversity of microbial genes at inter-individual level; Isolation of DNA from environmental samples followed by direct DNA sequencing allows to identify the microbial gene content of that environment; Metagenomics and Microbiome

26 The relevance of the microbiome

27 Dissection of the human microbiome Human microbiome project (NHI) The European Human gut microbiome project Human elderly microbiome project

28 Translation RNA proc. Transcription Replication Cell cycle contr. Defense mech. Signal transd. Cell wall membr. Cell motility Intra-traffick. Posttransl. mod. Energy product. Carbohydrate met. Amino acid met. Nucleotide met. Coenzyme met. Lipid met. Inorganic ion met. Secondary bios. General function Function unknown The Human gut microbiome Analyses of the human gut microbiome might provide evidences about functionality exerted by the human gut microbiota. Clusters of Orthologous Groups (COG) Bifidobacteria Actinobacteria Bacteria Metagenomics studies allow the identification of gene biomarkers 28

29 Phages and human gut microbiota Phage diversity unknown; What are their roles in the human gut microbiota? Knowledge gap about modulation of gut microbial populations; And about their contribution of genetic variability to bacterial populations? What about contribution of genes that increase the ecological adaptation (survival) of bacteria to a specific niche? Identification of the human virome 29

30 Edwards et al., 2005, Nat. Microbiol. Rev. Environmental microbiology and phages Human microbiome project What is the contribution of phage sequences to metagenomic libraries? What is the impact of phage on bacterial biodiversity? 30

31 Edwards et al., 2005, Nat. Microbiol. Rev. Phage community structure An example of phage community distribution Are there new phages? A very different number of phage genotypes have been detected in the various environmental samples. 31

32 Progresses of scientific knowledge and gut microorganisms Microbiomic Metagenomics 32

33 Conclusions The composition of the human gut microbiota is still largely unknown. Preliminary data demonstrated that phylogenetic changes are associated to various diseases and metabolic dysfunction. Gut microbial profiling data suggest that the count of specific bacteria (e.g., microbial biomarkers) is inversely related to fat mass development, inflammation and diabetes. Possible intervention with prebiotic and probiotic therapy could represent a valuable strategy in counteract the drop in target bacteria. Understanding the parameters that influence colonization, development and composition of the microbiota from a very early stage following birth, may be crucial for the development of strategies that guide formation of healthsustaining or -promoting microbiota into subsequent life stages. 33

34 Acknowledgments University of Parma, Italy Probiogenomics Lab. Francesca Turroni Elena Foroni Francesca Bottacini Fausta Serafini Alice Viappiani Christian Milani Sabrina Duranti Francesco Strati University of Verona, Italy GenProbio srl., Italy University of Milan, Italy Prof. Diego Mora Dr. Simone Guglielmetti University of Cork, Irelad Prof. Douwe van Sinderen IPLA, Asturias, Spain Dr. Abelardo Margolles Dr. Borja Sanchez Prof. Massimo Delledonne Dr. Alberto Ferrarini University of California, Davis, USA Prof. David Mills Dr. Jae Kim University of Aberdeen, UK Prof. Denise Kelly Dr. Imke Mulder