Istituto di Microbiologia. Università Cattolica del Sacro Cuore, Roma. Gut Microbiota assessment and the Meta-HIT program.

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1 Istituto di Microbiologia Università Cattolica del Sacro Cuore, Roma Gut Microbiota assessment and the Meta-HIT program Giovanni Delogu 1

2 Most of the bacteria species living in the gut cannot be cultivated in common media 2 main phylum: Firmicutes (Gram-positive) > 200 genera (Lactobacillus, Mycoplasma, Bacillus and Clostridium) Bacteroidetes (Gram-negative) ~20 genera

3 Data and analysis workflow for microbiome analysis; Weinstock G.M. (2012) Genomic approaches to studying the human microbiota. Nature 489,

4 Weinstock G.M. (2012) Genomic approaches to studying the human microbiota. Nature 489,

5 Giving the diversity among individuals, only studies involving a significant number of subjects provide relevant and valuable data; Illumina-based metagenomic sequencing; 124 european individuals; Qin J. Et al. (2010) A human gut microbial gene catalogue established by metagenomic sequencing. Nature 464,

6 Sanger sequenced 22 metagenomes from Danish, French, Italian and Spanish individuals. Functional and phylogenetic profiles of human gut microbiome Arumugam M. et al & MetaHIT consortium (2011) Enterotypes of the human gut microbiome. Nature 473,

7 Bacteroides is the most abundant but also the most variable genus across samples. Sanger sequenced 22 metagenomes from Danish, French, Italian and Spanish individuals. Arumugam M. et al & MetaHIT consortium (2011) Enterotypes of the human gut microbiome. Nature 473,

8 33 metagenomes (Sanger) 85 metagenomes (Illumina) Multidimensional cluster analysis and principal components analysis (PCA) revealed the forming of three distint clusters that have been designated as «enteroptypes». 154 metagenomes (16S pyroseq.) Each enterotype is identifiable by the variation of one of three genera: Bacteroides, Prevotella and Ruminococcus. Enterotypes are driven by groups of species that together contribute to the preferred community compositions. Arumugam M. et al & MetaHIT consortium (2011) Enterotypes of the human gut 8 microbiome. Nature 473,

9 ENTEROTYPE 1 Drivers of this enterotype seem to derive energy primarily from carbohydrates and proteins through fermentation, as these closely related genera have a very broad saccharolytic potential. Arumugam M. et al & MetaHIT consortium (2011) Enterotypes of the human gut microbiome. Nature 473,

10 ENTEROTYPE 2 Degrade mucin glycoproteins Arumugam M. et al & MetaHIT consortium (2011) Enterotypes of the human gut microbiome. Nature 473,

11 ENTEROTYPE 3 It is the most frequent and comprises species capable of degrading mucin. Arumugam M. et al & MetaHIT consortium (2011) Enterotypes of the human gut microbiome. Nature 473,

12 Functional differences between enterotypes These phylogenetic and functional differences among enetrotypes thus reflect different combinations of microbial trophic chains with a probable impact on synergistic interrelations with the human hosts. Arumugam M. et al & MetaHIT consortium (2011) Enterotypes of the human gut 12 microbiome. Nature 473,

13 Assessment of the gut microbiota Culture techniques DNA amplification Not reliable; Limited information; 16S rrnarealtime PCR; Not cultivable bacteria; (semi) quantitative analysis; 13

14 Analysis of predominant bacteria in colon biopsies and faeces Lyra A et al, World J Gastroenterol 2012

15 Analysis of predominant bacteria in feces Quantità DNA batterico (rdna 16S) 1.40E E E E E E E E+00 CTR DSS DSS ta 1X DSS ta 10X Lattobacilli Enterococchi Enterobatteri Clostridi Bacteroides % DNA batterico (rdna 16S) 100% 80% 60% 40% 20% 0% CTR DSS DSS ta 1X DSS ta 10X Lattobacilli Enterococchi Enterobatteri Clostridi Bacteroides Scaldaferri F., Bilotta M, Delogu G., Gasbarrini A. et al preliminary results

16 Metametrix, USA 16

17 Does the gut microbiota assessment, based on these methodologies, provide reliable information? 17

18 Assessment of the gut microbiota Metagenomic approach (Sanger, 16S rrna pyroseq, Illumina) Data analysis Genera and species identification; Phylogenetic grouping; Metagenome; Gene composition and function; 18

19 Illumina GA short-read sequencing on total faecal DNA; 124 individuals of European origin; The catalogue of non-redundant human intestinal microbial genes contains 3,3 million genes (150-fold more than the human gene complement); 99,1% are of bacterial origin, 0,1% eukaryotic and viral origin and the remaining archael; The cohort harbours 1150 prevalent bacterial species and each individual harbours at least 160 bacterial species; 75 species common to >50% of individuals and 57 species common to >90%; 19

20 242 adults (4788 specimens); Microbial taxas varies; Metabolic pathways remain stable; Functional redundancy; The HMP consortium. Structure, function and diversity of the healthy human microbiome. Nature 486 (June 2011)

21 Qin J. Et al. (2010) A human gut microbial gene catalogue established by metagenomic seqeuncing. Nature 464,

22 242 adults (4788 specimens); Microbial taxas varies; The HMP consortium. Structure, function and diversity of the healthy human microbiome. Nature 486 (June 2011)

23 Phylogentic three based on whole genome orthomcl clusters The genus Bacteroides comprises more that sixty species which are morphologically, biochemically and physiologically very heterogeneous; Taxonomy has been continuously revised in the last few decades though recent data emerging from genomic studies is providing a clearer picture; Karlsson FH, et al. (2011) A closer look at Bacteroides: phylogenetic relationship and genomic implications of a life in the human gut. Microb. Ecol. EAGEN Rome 2012 B. caccae B. vulgatus 23

24 Bacteroides play an important role in the human gut CPS Contributing to gut homeostasis by interacting with the host immune response; PULs Degrading polysaccharide components; 70% energy 10% energy EAGEN Rome

25 PULs in B. thetaiotaomicron genome 88 PULs 866 genes 18% total genome B. thetaiotaomicron s ability to opportunistically use many glycan sources likely makes it an important generalist EAGEN Rome 2012 among intestinal Bacteroidetes. 25

26 PULs in B. ovatus genome 112 PULs encompassing 1129 ORFs EAGEN Rome Martens EC et al, PLoS Biology 2011

27 EAGEN Rome

28 B. ovatus Plant cell wall, hemicellulose B. distasonis B. vulgatus Stronger in plant degradation, no carbon degradation; pre-digest products Target pectin, fruit-associated glycans B. thetaiotaomicron B. fragilis More soluble andaccessible components of plant cell wals. O- glycans mucus layer EAGEN Rome 2012 Adapted from Koropatkin NM, et al (2012) Nat Rev Microb

29 Deterioration of global Synteny in Bacteroides Synteny measure the conserved gene order along the chromosomes of different species or strains of the same species. Xu J., et al. (2007) Evolution of symbiotic bacteria in the distal human intestine. Plos Biology, 5 (7) EAGEN Rome

30 DIET ANTIBIOTICS PREBIOTICS INFLAMMATION PATHOGENS B. ovatus B. distasonis B. vulgatus B. thetaiotaomicron B. fragilis Bacterial genetic variability (interspecies, intra-species); Competion/mutualism EAGEN Rome 2012 Adapted from 30 Koropatkin NM, et al (2012) Nat Rev Microb

31 Challenge: Implementing a reliable and feasible methodology to assess gut microbiota in clinical practice. LAB CLINIC Weinstock G.M. (2012) Genomic approaches to studying the human microbiota. Nature 489,

32 Tools to assess the gut microbiome: Different parameters; Economic e relatively simple procedures; Easy to interpret data; Addressing specific and tailored points; MULTIPARAMETRI C PROFILE Weinstock G.M. (2012) Genomic approaches to studying the human microbiota. Nature 489,

33 33