IMPACT OF DIET ON MICROBIOTA COMPOSITION AND FUNCTION IN THE SMALL INTESTINE Microbiome Drug Development Summit 2017 29-06-2017 Els van Hoffen Senior scientist and project manager Nutrition & Health els.vanhoffen@nizo.com
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MICROBIOTA IN HEALTH AND DISEASE IDENTIFICATION OF MICROBIOME MODULATORS OF THE FUTURE Importance of microbiota in health and disease is wellrecognized (GI tract, skin, brain, female health) NIZO pipeline: expertise-driven study design, mechanistic understanding, microbiome modulator discovery & validation. Fields of application Human and animal health, food safety and fermentation
NIZO MICROBIOMICS PLATFORM FROM UNDERSTANDING TO MODULATOR DISCOVERY Data-analytics & Expert Interpretation In silico Sequencing DNA/RNA extraction Sample collection & logistics MicrobiOMICs Identification Microbiome Markers Microbiome markers of health/disease Human POC, Challenge Field trial Discovery Platform Microbiome Modulators In vitro strains In vitro community Upscaling & production microbiome modulators Expert consultation design In vitro Host-microbe interactomics
PROFILING THE GASTROINTESTINAL, SKIN, AND VAGINAL MICROBIOTA TOWARDS CAUSALITY AND INTERVENTION STUDIES GI-tract Skin Women s health Forehead Armpit Left Inner Elbow Left Lowerback 8/9 Lowerback 3 inner - elbow 1 Fronthead inner - elbow Armpit Fronthead Fronthead ~ P. acnes Microbiota management in GI health - Dietary interventions studies - Microbial challenge experiments - Microbiota & Immunity - Microbiota & GI comfort - Infant formula studies - In vitro high throughput screening Microbiota in skin health & disease: Psoriasis, AD Acne, dandruff, (mal)odour Microskin 3D skin models: host-microbe interactions Manage or modulate vaginal microbiota: - Clinical study design and sampling - (Longitudinal) microbiota studies - In silico prediction of modulators - In vitro assays to test/validate modulators - Markers for monitoring women s health
SAMPLING MICROBES IN THE SMALL INTESTINE RATIONALE Largely unexplored, mainly due to inaccessibility & invasive procedures Small intestine is first site of interaction between intestinal microbiota & diet/drugs NUMBERS! Lowly abundant but relevant species in the small intestine can be diffused/undetectable within the colon microbiota community Impact on Health: Major immunomodulation site, metabolic control, gut-brain, etc. Impact on Disease: Dysbiotic signatures identified in the ileum could not be identified in feces (Gevers et al 2014; Bajaj et al 2012)
INTELLICAP DISPOSABLE CAPSULE Microfluidic Container Harbors Reservoir (~ 300 µl) Microelectronic Body Batteries Motor for controlled sampling Subject monitoring sensors (ph, temperature) µ-computer Real time communication unit
SAMPLE QUENCHING IN VITRO QUENCHING FOR METAGENOMIC ANALYSIS Quencher development by NIZO Requirements quencher: Qualitative and quantitative preservation of the microbiota Safety Sufficient sampling volume Combination of IntelliCap system and the quencher: Remotely controlled intelligent capsule for small intestinal sampling of the microbiome
THE STUDY: USING A NON-INVASIVE CAPSULE TO STUDY THE SMALL INTESTINAL MICROBIOME IN HUMANS Main research questions: Is the IntelliCap a suitable tool for sampling of small intestinal (ileal) microbiota? Is it possible to detect dietary changes in small intestinal microbiota composition by using the IntelliCap? Fully controlled diets for the IntelliCap validation study: High protein versus high carbohydrate target species with different metabolic profiles WUR - Metabolism & Genomics Group Division of Human Nutrition
HUMAN VALIDATION STUDY CROSS-OVER DESIGN
STUDY OUTCOMES SAMPLING TIMEPOINTS # Values not calibrated
WORKFLOW MICROBIOME ANALYSIS Study design, sample collection (feces, capsules) Generation of DNA (dedicated DNA isolation protocols; purification) Amplification of the library by PCR (16S rrna gene V3-V4 region & shotgun metagenomics) (Barcoded) primer design and PCR optimization Data generation via NGS sequencing (Illumina) Bioinformatics data analysis & expert interpretation QIIME, classification, visualization & statistical analysis
SMALL INTESTINAL VS FECAL SAMPLE COMPOSITION SAMPLES CLUSTER ON ORIGIN (SI VS FAECES) AND ON SUBJECT
BACTERIAL COMPOSITION IN SI VS FAECES SPECIFIC GROUPS CONTRIBUTE TO DISTINCTION BETWEEN SI AND FAECAL SAMPLES 10-fold higher Streptococcus in SI Bilophila only in SI Small intestine: Streptococcus Veillonella Gemella Rothia Bilophyla Granulicatella Haemophilus Feces: Bifidobacterium Dorea Adlercreutzia
PREDICTION OF FUNCTIONAL COMPOSITION OF THE MICROBIOTA DISTINCT ENRICHMENT OF CARBOHYDRATE TRANSPORT FUNCTIONS IN HIGH VS LOW CARB DIET PICRUSt: module focussing on microbial functions associated with metabolism Difference in PTS-functionality: phosphotransferase system carbohydrate uptake control of carbon metabolism Low-carb/high-protein diet: Advantage for bacteria that can import simple sugars (e.g. glucose, mannose, lactose) High-carb/low-protein diet: Advantage for bacteria that can import linear polyols that are not absorbed in the small intestine and remain available in the colon (e.g. sorbitol, mannitol)
FUNCTIONAL COMPOSITION OF THE MICROBIOTA: SHOTGUN METAGENOMICS DISTINCT ENRICHMENT OF BACTERIAL GROUPS AND FUNCTIONS IN HIGH VS LOW CARB DIET High Carb Low Carb Coprococcus Dorea Streptococcus Pathways more abundant in Low Carb/High Protein: L-isoleucine degradation I L-leucine degradation I superpathway of L-phenylalanine biosynthesis superpathway of L-alanine biosynthesis L-histidine degradation I L-lysine biosynthesis II L-histidine degradation III L-ornithine biosynthesis Bacterial groups with selective advantage: confirms 16S amplicon data Identifies additional functionality compared to PICRUSt
NIZO MICROBIOMICS PLATFORM - SMALL INTESTINAL MICROBIOTA COMPOSITION AND FUNCTION - Data-analytics & Expert Interpretation In silico Sequencing DNA/RNA extraction Sample collection & logistics MicrobiOMICs Identification Microbiome Markers Microbiome markers of health/disease Human POC, Challenge Field trial Discovery Platform Microbiome Modulators In vitro strains In vitro community Upscaling & production microbiome modulators Expert consultation design In vitro Host-microbe interactomics
THE ROLE OF DATA & EXPERT KNOWLEDGE 18 1990 2020 D A T A Colony typing One organism (q)pcr One gene Amplicon sequencing (16S) All organisms Shotgun Metagenomics All organisms, all genes Metagenomics: new standard for studying the microbiome. Crucial: combination of data analysis and expert knowledge. Expert knowledge
CONCLUSIONS The IntelliCap validation study demonstrates the NIZO approach to understand the impact of the microbiota community in health and disease Creative design of innovative strategies to study microbiota composition and function Dedicated tools and protocols to build evidence for the impact of the microbiota community in health and disease (feces, small intestine, skin, vagina, etc.) Benefits of understanding functional pathways: Establish causality Claim substantiation: health benefits Design microbiome modulators
ACKNOWLEDGMENTS NIZO: Harro Timmerman Jos Boekhorst Michiel Kleerebezem Alwine Kardinaal Elly Lucas Sabina Lukovac Saskia van Schalkwijk Iris van Swam Michiel Wels Mariëlle Voortman WUR - Metabolism & Genomics Group Division of Human Nutrition: Diederik Esser Guido Hooiveld Lydia Afman Medimetrics: Christoph Wanke Suzanne Tibosch SPONSORS: