VERS L IDENTIFICATION DE NOUVELLES CIBLES THÉRAPEUTIQUES : LES OUTILS D ANALYSE

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1 VERS L IDENTIFICATION DE NOUVELLES CIBLES THÉRAPEUTIQUES : LES OUTILS D ANALYSE 06/02/2018 1

2 Context 06/02/2018 2

3 The commensal microbiota: a growing field with many potential applications 06/02/2018 3

4 In human Over the past decade, numerous studies have found an association between the gut microbiota composition and many diseases. However, is it reality? Or is the truth hidden in the shadow of several thousand publications a year with inflated expectations in almost any disease?? Cani, P. D. (2017). "Gut microbiota - at the intersection of everything?" Nature Reviews. Gastroenterology and Hepatology. 06/02/2018 4

5 In animals Increase resistance to stress and infections Decrease feed conversion ratio Decrease methane emission «Classical probiotics» + prebiotics «SER-109 like» without ethanol selection «Nextgeneration»probiotics? Lactobacillus acidophilus, L. casei, L. plantarum, Bifidobacterium bifidum, Pediococcus pentosaceus, Undefined normal avian gut flora : more than 200 species Streptococcus spp. Bacterioides spp. Citrobacter spp. Clostridium spp. Enterococcus spp. Escherichia spp. Eubacterium spp. Fusobacterium spp. Lactobacillus spp. Propionibacterium spp. Ruminococcus spp. Polansky, O., et al. (2016). "Important Metabolic Pathways and Biological Processes Expressed by Chicken Cecal Microbiota." Applied and Environmental Microbiology 82(5): Based on data in this study, Anaerostipes, Anaerotruncus, and Subdoligranulum might therefore be suitable probiotic candidates for oral inoculation of newly hatched chickens. 06/02/2018 5

6 The race for «Next-generation probiotics» X 06/02/2018 6

7 Cocktails or single strains? VE-202 composition 06/02/2018 7

8 A quick, non-exhaustive overview of selected tools and technologies 06/02/2018 8

9 Next Generation Sequencing: a dramatic cost decrease Ion Torrent Sanger Technology SOLiD 5500 Illumina MiSeq Roche 454 Solexa Illumina HiSeq PacBio-RS Ion Proton Illumina NextSeq500 PacBio Sequel SOLiD 1.0 SOLiD 3.0 Date Cost per Mb Cost per Genome Sep-01 9 I $5 292,39 $ to10-6 cost reduction Wetterstrand KA. DNA Sequencing Costs: Data from the NHGRI Genome Sequencing Program (GSP) Available at: - Accessed Jan Date Cost per Mb Cost per Genome Oct-15 $0,014 $1 245 Jul-17 $0,012 $ /02/2018 9

10 Next Generation Sequencing: Are we comparing apples and oranges? As with all methods, the use of benchmarking controls has revealed critical differences among methods that impact sequencing results and later would impact study interpretation. We recommend that the community adopt PCR-free based approaches to reduce PCR bias that affects calculations of abundance and to improve assemblies for accurate taxonomic assignment. Furthermore, the inclusion of a known-input cell spike-in control provides accurate quantitation of organisms in clinical samples. M. B. Jones et al., Library preparation methodology can influence genomic and functional predictions in human microbiome research. Proc. Natl. Acad. Sci. U. S. A. 112, (2015). 06/02/

11 Next Generation Sequencing: Recently published standards Costea PI et al Towards standards for human fecal sample processing in metagenomic studies. Nat Biotechnol 35: The Microbiome Quality Control project Sinha R et al Assessment of variation in microbial community amplicon sequencing by the Microbiome Quality Control (MBQC) project consortium. Nat Biotechnol 35: ATCC Microbiome Standards quid virome, mycobiome, archeome??? 06/02/

12 Culturomics: playing with numbers Preselection Culture Identification Collection & storage Characterization 06/02/

13 Culturomics: Which culture conditions? Selection Preincubation Incubation n# 5µm active 80 C / filtration 20min Medium Atmosphere Medium Temp Atmosphere 1 N N anaerobic 37 C anaerobic Blood culture bottle 2 N Y anaerobic 37 C anaerobic 3 N N Blood culture bottle aerobic 37 C aerobic 4 N N + rumen anaerobic 37 C anaerobic 5 N N Blood culture bottle aerobic 37 C aerobic 6 N N + sheep blood anaerobic 37 C anaerobic 7 N N Blood culture bottle aerobic 37 C aerobic 8 N N + rumen + sheep anaerobic 37 C anaerobic 9 N N aerobic 37 C aerobic Marine broth 10 N N anaerobic 37 C anaerobic 11 N N Brain Heart Infusion aerobic 37 C aerobic 12 N N Trypticase soy broth aerobic 37 C aerobic 13 N N aerobic 37 C aerobic 14 N N anaerobic 37 C anaerobic 15 N N Columbia-like broth aerobic 28 C aerobic 16 N N + sheep blood anaerobic 28 C anaerobic 17 Y N aerobic 37 C aerobic 18 Y N anaerobic 37 C anaerobic 5% sheep blood agar? Sporulated bacteria? Adapted from Lagier et al Culture of previously uncultured members of the human gut microbiota by culturomics. Nat Microbiol 1: /02/

14 Targeted culturomics O 2 Phenotypic & genomic characterization O 2 Collections of isolated strains BD Influx cell sorter Gating & identification of regions of interest Extensive cultivation of regions of interest O 2 O 2 06/02/

15 Fluorescence Microfluidics: ultra-high throughput Microfluidic chamber Bright Field Imaging Fluorescence Imaging Flow Measurements Laser Lines A B C 06/02/2018 Time 15

16 «Finding the needle in a haystack» Vitamin B2- Secreting Lactic Acid Bacteria Taken from: Chen, J., M. Vestergaard, T. G. Jensen, J. Shen, M. Dufva, C. Solem and P. R. Jensen (2017). "Finding the Needle in the Haystack-the Use of Microfluidic Droplet Technology to Identify Vitamin-Secreting Lactic Acid Bacteria." MBio 8(3). 06/02/

17 Gnotobiotic pre-clinical models Wild-type mice with standard Microbiota Probiotic/bioactive to evaluate Specific tools for 3Din vivo monitoring Selected bacteria from mice microbiota, human microbiota Wild-type mice KO-mice Germ free mice Reproduction * * Image taken from Servick K Mouse microbes may make scientific studies harder to replicate. Science. 06/02/

18 Typical application Wild type or genetically engineered mice Wild type microbiota Non-responders µbiota Patients or healthy volunteers Non-responders Axenic mice FMT Gnotobiotic mice Responders µbiota Responders Culturomics Isolate and grow bacterial species/strains of interest * Image taken from Servick K Mouse microbes may make scientific studies harder to replicate. Science. 06/02/

19 Metabolomics and proteomics Integrated analytical approaches for microbiota metabolomics 06/02/2018 EXPANDING CREATIVITY, ADDING VALUE, TOGETHER. 19

20 Immunomonitoring: Recent developments Mass cytometry: up to 44 parameters 06/02/

21 Immunomonitoring: Discriminating circulating and tissue leukocytes 06/02/

22 Immunomonitoring: Discriminating circulating and tissue leukocytes Non-infected muscle Gated on CD45+ cells Inflammatory monocytes Non classical macrophages Neutrophils B cells Classical macrophages NK cells T cells Non inflammatory monocytes 06/02/2018

23 Immunomonitoring: Discriminating circulating and tissue leukocytes Non-infected Infected muscle muscle Inflammatory monocytes Gated on on CD45+ cells Non classical macrophages Neutrophils B cells Classical macrophages NK cells T cells Non inflammatory monocytes 06/02/2018

24 Conclusion 06/02/

25 High complexity! Choose your tools carefully Critical need for validation and standardization Sample prep is a major issue Bioinformatics and statistics are also critical 06/02/

26 Vincent THOMAS, PhD Head of Microbiota Program Cell.+33 (0) Campus de l Institut Pasteur, 28, rue du Docteur Roux PARIS, FRANCE EXPANDING CREATIVITY, ADDING VALUE, TOGETHER.