Cultivating Changing Gut Microbial Communities Lauri O. Byerley, RDN, LDN, FAND Associate Professor Louisiana State University Health Sciences Center Department of Physiology Disclaimer Research Funding Through the Years: • NCI • NIAAA • US Army • American Institute of Cancer Research • California Walnut Commission • American Public University • Louisiana State University Health Sciences Center Hippocrates….”All Diseases begin in the gut” Collaborators: DAVID WELSH CHRISTOPHER TAYLOR BRITTANY LORENZO SHELIA BANKS MENG LUO MONICA PONDER (VIRGINIA TECH) EUGENE BLANCHARD Seminar Layout • Learning objectives • Definition of terms • Introduction • Microbe view of the gut • Why cultivating microbes is important • Detecting gut microbes • Feeding our gut microbes • Proof of concept – Can we cultivate specific microbes? • Summary Learning Objectives • Diagram the GI tract from a microbe’s perspective • Describe why we should cultivate gut microbes • Explain the process by which microbes are detected • Produce a list of several different fiber types and several foods/ingredients associated with each fiber type • Describe alterations in gut microbiota following dietary changes • List the most appropriate foods to cultivate our gut microbiome Definition of Terms • Microbiota – a collection or community of microbes. Includes bacteria, fungi, archaea and viruses • Microbiome – refers to the genomes of all the microbes in a community • Metagenomics ‐ a technique that reveals biological functions of an entire community • Metabolomics – measurement of metabolites https://media.npr.org/assets/img/2016/08 /09/e‐coli_wide‐ b2909f1ef2e257a4c5fa01112174ab941c24 7871‐s800‐c85.jpg Introduction • We are more bacteria than human… • More bacteria in and on our body than human cells • Healthy human has approximately 40 trillion bacteria in gut • 500 to 1,000 species encompassing 10,000 strains • Humans – 23,000 genes • Microbiome – estimated 3,300,000 genes (150 fold more) • Mutual relationship http://www.global‐engage.com/wp‐content/uploads/2017/04/human‐microbiome‐NGS.jpg Microbiome – “Human Organ” • Weight ‐ approximately 1‐2 kg • No distinct structure ‐ organized system of cells like the immune system • Each person has a unique microbiome • So unique we may be able to track forensically http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1500832/ Microbes view of our gut • Major barriers for microbes entering the gut: • low pH • Saliva • Bile • Immune system • Finding a place to attach to intestinal wall • Surviving a widely varied diet https://elearning.kullabs.com/img/note_images/b7nZNAW6sMPqqMTg.jpg Keep these things in mind when cultivating our gut microbiome Microbial Biomass & pH Figure 1(A) Small intestine mucosal immune system landscape. M Zaeem Cader, and Arthur Kaser Gut 2013;62:1653-1664 Figure 1(B) Colon (large intestine) mucosal immune system. M Zaeem Cader, and Arthur Kaser Gut 2013;62:1653-1664 What is happening in the gut? For those microbes that manage to colonize the gut: • gut flora perform regular tasks of digestion, vitamin production, many others • Gene transfer between the myriad of species in the gut can generate new combinations including ones resistant to drugs “superbugs” https://i2.wp.com/thescientificparent.or g/wp‐content/uploads/2017/03/GI‐ Tract‐Small.png?fit=816%2C971&ssl=1 Why is Cultivating Important? Many Functions of Our Gut Bacteria https://www.researchgate.net/profile/Brigitta_Brinkman/publication/281518789/figure/fig1/AS:313058971930624@1451650483890/Main‐functions‐of‐bacteria‐in‐the‐gut‐ Bacteria‐benefit‐the‐host‐in‐many‐ways‐Besides.png Who Are the Players? Scientific Classification or Taxonomic Rank • Don’t get hung up on this – just know it exists • King Play Chess On Fine Green Silk • 3 Kingdoms: • Bacteria • Archaea King Play • Eukaryota Chess Kingdom Bacteria Bacteria On Fine Phylum Bacteriodetes Firmicutes Green Class Bacteriodetes Clostridia Silk Order Bacteriodales Clostridiales Family Bacteroidaceae Ruminococcaceae Genus Bacteroides Anaerotruncus Species B. fragilis A. colihominis Methods • We cannot grow most bacteria in the lab • We must detect DNA • Fecal samples collected from the descending colon • DNA isolated • V3 hypervariable region of 16S rRNA gene deep sequenced on an Illumina MiSeq. • Data analyzed using the UPARSE and QIIME pipeline and SAS. Species Name LOB1NTBMCA LOB1PFW LOB2PFW None;Other;Other;Other;Other;Other;Other 00 0 k__Bacteria;Other;Other;Other;Other;Other;Other 0.004396 0.001905 0.003637 k__Bacteria;p__Actinobacteria;Other;Other;Other;Other;Other 7.14E‐05 0 0.000222 k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Actinomycetales;Other;Other;Other 0 0 0 k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Actinomycetales;f__Actinomycetaceae;g__Actinomyces;Other 0 0 0 k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Actinomycetales;f__Actinomycetaceae;g__Arcanobacterium;s__ 0 0.000159 0 • Excel spreadsheet with numbers • Use these numbers to determine significant differences http://learn.genetics.utah.edu/content/microbiome/study/image How to analyze data? s/microbesdoing.jpg Fig. 4 Alpha diversity: species richness (number of taxa) within a single microbial ecosystem. How many different microbial species could be detected in one sample? Beta diversity: diversity in microbial community between different environments (difference in taxonomic abundance profiles from different samples). How different is the microbial composition in one environment compared to another? https://www.researchgate.net/profile/Patrice_Cani/publication/2Atherosclerosis 2018 268, 117-126DOI:81288066/figure/fig5/AS:271086130561031@1441643378758/Mice‐Fed‐ (10.1016/j.atherosclerosis.2017.11.023) a‐Lard‐Diet‐Have‐Increased‐Adiposity‐ and‐Distinct‐Gut‐Microbiota‐Composition.png Microbe Communities • Microbes act in communities • Microbes react to their surroundings • Shift when the environment changes • Change quickly http://learn.genetics.utah.edu/conte nt/microbiome/intro/images/microb es.jpg Factors Affect Human Gut Microbiome • Age • Activity/Exercise • Emotional State/Stress • Diet • Gender • Climate • Occupation • Hygiene • Antibiotics • Medications • Living with animals • Where you live • Diurnal Variation http://learn.genetics.utah.edu/content/microbiome/changing/images/change6.jpg Diet is a big player for the gut microbiome Diet Influences the Gut Microbiome • We acquire microbiome at birth • Long‐term diet effects: Bacteroides (Genus) Prevotella (Genus) Diet (Phyla ‐ Bacteriodetes) (Phyla – Baceriodetes) High animal protein, high fat, low High Low carbohydrate (Atkins, Paleo) High carbohydrates, low animal Low High protein, low fat • Japanese harbor organisms that aids in seaweed digestion • African children – high Bacteroidetes and deplete of Firmicutes to maximize energy uptake from fiber‐rich diet • vegetarians have gut flora that are better equipped to break down plant roughage, making otherwise indigestible molecules such as cellulose available for humans Scientific Evidence – Diet Influences Gut Microbiome Carnivore Omnivore Hervifore Meugge BD et al. Science. 2011 May 20; 332(6032): 970–974. Partitioning of People Into Enterotypes • Difficult studies because increases in one macronutrient typically associated with decrease in another • Higher complex carbohydrate diet – more Prevotella • Higher fat/protein diet – more Bacteroides Wu GD, et al. Science 334:105, 2011 Macronutrient composition rapidly impacts microbiome Changes within 24 hours Wu GD et al. Science. 2011 Oct 7; 334(6052): 105–108. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3368382/ Macronutrient composition rapidly impacts microbiome Plant‐based Animal‐based Diet Diet Similarity of each individual’s gut microbiota to their baseline communities (β‐diversity, Jensen‐Shannon distance) decreased on the animal‐based diet (dates with q<0.05 identified with asterisks; Bonferroni‐corrected, two‐sided Mann‐Whitney U test). David, LA Et al Nature. 2014 Jan 23; 505(7484): Community differences were apparent one day after a tracing dye 559–563. showed the animal‐based diet reached the gut (blue arrows depict https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3957 appearance of food dyes added to first and last diet day meals 428/bin/nihms536070f1.jpg Scientific Evidence ‐ Diet Influences Gut Microbiome 16S rRNA gene surveys reveal a clear separation of two children populations investigated. BF = Children from a rural African village of Burkina Faso (BF) Diet is • low in fat and animal protein • rich in starch, fiber, and plant polysaccharides • predominantly vegetarian EU = European children Diet is • typical western diet high in animal protein, sugar, starch, and fat • low in fiber. Carlotta De Filippo et al. PNAS 2010;107:33:14691-14696 ©2010 by National Academy of Sciences Diet Influences Microbiome • Changes occur rapidly ‐ within 24 hours of eating a high fat/low fiber or low fat/high fiber diet • Changes are significant • Changes are modest • Changes only last for at least 10 days The adult gut microbiome is characterized as existing in a steady state that requires a major disturbance to permanently alter that Microbiome is strongly associated state. Short-term diet interventions may transiently alter the gut microbiome community with long‐term diet. structure, but long-term diet changes are required to shift to a new steady-state. Voreades N., Kozil, A, Weir TL. Front Microbiol. 2014; 5: 494. What is our microbes favorite food? Fiber Dietary Fiber: Non-digestible soluble and insoluble carbohydrates ( > 3 monomeric units), and lignin