The Human Microbiome: The Undiscovered Country<br />Walter J. Coyle, MD, FACP,FACG<br />
Movement of the Talk<br />Describe the Microbiome<br />Microbiology 101<br />Review the diversity of human gut flora<br />...
The Human Microbiome<br />Definitions: <br />Microbiome:  Aggregate of all gut species<br />Microbiota: Individual bacteri...
Anatomic Regions of the Gut<br />Upper GI tract: 102 – 104 cells/ml	<br />Lactobacilli, streptococci, H pylori<br />Ileum:...
Microbes and Humans<br />Dethlefsen Nature 2007; 449:812-818<br />
Micro 101<br />Pace in Science 1997  276:735-740<br />
The Human Gut Flora<br />DiBiase, et al. Mayo Clin Proc 2008;83:460-469 <br />
Mouse intestine, mucus gel layer<br />Home to billions of organisms<br />Sonnenburg J, et al. in Nature<br />Immunology 20...
The Microbiome: Who’s there?<br />Early gut colonization has four phases<br />Phase 1: Sterile gut <br />Phase 2: Initial ...
The Microbiome: Who’s there?<br />Adult Microbiome:<br />Increasing diversity of flora as we age<br />In some newer PCR (1...
The Microbiome: Who’s there?<br />Multiple species: eukaryotic, bacterial, archael<br />Firmicutes (Gram +/ motile) and Ba...
Ley R, et al in Cell 2006:124:837-848<br />
Gut Flora and Metabolism<br />
Metabolomics<br />Study of the metabolites and small molecules that the body and gut bacteria produce.<br />New area of sc...
Gut Flora and Metabolism<br />Microbial genomes enhance our metabolic activity<br />May indirectly or directly effect our ...
Gut Flora and Metabolism<br />Energy salvage: esp via the short-chain fatty acids<br />Acetate, butyrate, propionate (SCFA...
Gut Flora Metabolic Reactions<br />Metabolic reactions<br />Goldin BR in Gastrointestinal Microbiology  138-154, 2006<br />
Gut Flora and the Brain<br />Collins and Bercik in GASTRO 2009;136:2003–2014<br />
Obesity and the Gut Flora<br />
Ley, et al.  PNAS. 2005, 102: 11070-75<br />
The Microbiome: Changes in flora<br />Mice and humans have different gut flora but the two largest divisions are shared in...
Ley, et al.  PNAS. 2005, 102: 11070-75 <br />
The Microbiome: Changes in flora<br />Obesity changes the relative proportions of divisions. <br />Obese mice AND obese pe...
Gut Flora and Obesity<br />Germ free mice studied by Gordon, et al<br />Control mice (remaining germ free) were changed fr...
Gut Flora and Obesity<br />Mice then studied for change in weight and fat pad size<br />Assessed response to fatty meal lo...
Gut Flora and Obesity<br />Gordon J, et al, PNAS 2007;104:983<br />
Gut Flora and Obesity<br />GF mice are “resistant” to the Western diet<br />Gut flora are responsible for increasing energ...
“Let food be thy medicine and medicine be thy food.”<br />Hippocrates<br />
The Microbiome: transplant experiments<br />Ob Ob mouse<br />Wild type mouse<br />Turnbaugh, et al.  Nature 2006; 444:1027...
The Microbiome: transplant experiments<br />Germ-free mice given ob/ob or wild-type flora<br />Chow consumption and exerci...
Ley R, et al in Nature 2006:444<br />Human Flora and Obesity<br />Mice data links gut flora with obesity and diet<br />Stu...
Human Flora and Obesity<br />Ley R, et al in Nature 2006:444<br />
Ley R, et al in Nature 2006:444<br />Human Flora and Obesity<br />Firmicutes dominate in obese subjects<br />All subjects ...
Proposed Mechanisms in Obesity<br />DiBiase, et al. Mayo Clin Proc 2008;83:460-469 <br />
Microbiome: regulators<br />Archae:  1-2 % of mouse and human flora<br />Represent a major microbial group in gut flora<br...
The Gut and Obesity: Options<br />Diet may manipulate flora<br />Low carbs, high fat<br />High carbs, low fat<br />Probiot...
Coyle’s Corollary<br />It is better to be a stool donor than a recipient.<br />
Probiotics<br />
Probiotics<br />Definition: Live microorganisms which when ingested in adequate amounts confer a health benefit on the hos...
Common Probiotics<br />Khedkar and Ouwehand in GastrointestMicrobiology  315-334, 2006<br />
Probiotics<br />VSL #3<br />4 lactobacilli<br />L. plantarum, casei, acidopholus, delbrueckiispp<br />3 bidifobacteria<br ...
Probiotics<br />Digestive Advantage<br />Ganeden BC30<br />Bacillus coagulans<br />Erythritol<br />Cellulose<br />Other mi...
Probiotics<br />Bifidobacteriuminfantis 35624  aka Bifantis<br />“Patented” strain of probiotic in Align<br />Decreased sy...
Probiotics<br />Saccharomyces boulardii<br />Other minor ingredients<br />Shown in Rand / PC trials to help prevent recurr...
Probiotics in Food (Actimel)<br />L. caseiImmunitas™<br />Claim it is scientifically proven to be effective<br />“Each bot...
Prebiotics<br />
Prebiotics<br />Ingested substances that selectively stimulate the proliferation and/or activity of desirable bacterial po...
Prebiotics<br />Inulin: plant polymers mainly comprising fructose units, use have a terminal glucose<br />Indigestable fib...
Prebiotics<br />Is is possible to design a food, sugar, protein, or fat that would alter your gut flora to promote weight ...
Designing Probiotics: The Future?<br />GASTROENTEROLOGY 2009;136:2015–2031<br />
Conclusions<br />The human microbiome and the Microbiome Project:  research just beginning…<br />Gut flora by their genes,...
Conclusions<br />Future studies must focus on the mechanisms of influence of our gut flora.<br />Studies must be placebo c...
The Future	<br />Define who is there:  in healthy subjects and those with disease<br />Study at Scripps in subjects with d...
Questions<br />
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The Human Microbiome: The Undiscovered Country

  1. 1. The Human Microbiome: The Undiscovered Country<br />Walter J. Coyle, MD, FACP,FACG<br />
  2. 2. Movement of the Talk<br />Describe the Microbiome<br />Microbiology 101<br />Review the diversity of human gut flora<br />Understand how our bacteria influence our metabolism and obesity<br />Review the plethora of probiotics<br />Describe prebiotics and their potential uses<br />Conclusions and the Future<br />
  3. 3. The Human Microbiome<br />Definitions: <br />Microbiome: Aggregate of all gut species<br />Microbiota: Individual bacterial species in the biome<br />Over 100 trillion organisms (1014)<br />Passengers in the mobile colonic petri dish<br />Over 500 species identified so far (70 divisions)<br />90% of the cells in our body our microbial!<br />100 fold more genes in our gut then in us<br />Our flora are an integral part of our genetic landscape and evolution<br />
  4. 4. Anatomic Regions of the Gut<br />Upper GI tract: 102 – 104 cells/ml <br />Lactobacilli, streptococci, H pylori<br />Ileum: 106-1012 cells /ml, upper bacteria plus<br />Faculative anaerobes: Enterobacteriaceae<br />Obligate anaerobes: Bacteroides, Veillonella, FusobacteriumandClostridium species<br />Colon: distal human colon is the most biodense natural ecosystem known (1010-1012 cells/ml)<br />Complex and diverse<br />Comprise most of our bacterial biomass<br />
  5. 5. Microbes and Humans<br />Dethlefsen Nature 2007; 449:812-818<br />
  6. 6. Micro 101<br />Pace in Science 1997 276:735-740<br />
  7. 7. The Human Gut Flora<br />DiBiase, et al. Mayo Clin Proc 2008;83:460-469 <br />
  8. 8. Mouse intestine, mucus gel layer<br />Home to billions of organisms<br />Sonnenburg J, et al. in Nature<br />Immunology 2004:5<br />
  9. 9.
  10. 10. The Microbiome: Who’s there?<br />Early gut colonization has four phases<br />Phase 1: Sterile gut <br />Phase 2: Initial acquisition: vagina, feces, hospital<br />Phase 3: Breast feeding or bottle-feeding (different)<br />Breast fed more bifidobacteria (up to 90% of flora)<br />Bottle fed more diverse; more Bacteroides , and Clostridial species<br />Phase 4: Start of solids; move to adult flora <br />Bifidobacteria remain key flora into adulthood <br />Ley, Peterson, Gordon. Cell 2006 ;124:837<br />Ley, et al. PNAS. 2005, 102: 11070 <br />Edwards, et al. Br J Nutr. 2002<br />
  11. 11. The Microbiome: Who’s there?<br />Adult Microbiome:<br />Increasing diversity of flora as we age<br />In some newer PCR (16S rRNA) studies, up to 92% of the flora in adults were “novel” species<br />Serial stool collections show remarkable stability by an individual<br />Greatest concordance with twins<br />Less concordance with households<br />Host genetic influence unexplored.<br />McCartney and Gibson in Gastrointestinal Microbiology, 51-73, 2006<br />
  12. 12. The Microbiome: Who’s there?<br />Multiple species: eukaryotic, bacterial, archael<br />Firmicutes (Gram +/ motile) and Bacteroidetes (Gram -) : Majority<br />These two groups comprise > 90% <br />Anaerobes / aerobes: 1000:1<br />Far fewer species than the environment<br />High levels of interpersonal diversity<br />My stool is not your stool<br />Ley, Peterson, Gordon. Cell 2006 ;124:837<br />Ley, et al. Science 2005; 307: 1915 <br />
  13. 13. Ley R, et al in Cell 2006:124:837-848<br />
  14. 14. Gut Flora and Metabolism<br />
  15. 15. Metabolomics<br />Study of the metabolites and small molecules that the body and gut bacteria produce.<br />New area of science <br />Broader than proteonomics<br />Includes bacteria products with our own genetic products<br />Pioneered by Jeremy Nicholson and Jeff Gordon<br />
  16. 16. Gut Flora and Metabolism<br />Microbial genomes enhance our metabolic activity<br />May indirectly or directly effect our metabolism<br />The colon is very active metabolically<br />20-70 gms of carbos and 5-20 gms of protein/day<br />Over 100 kcal per day!<br />Mass of colonic microbiome = single kidney<br />Metabolically as active as the liver<br />Hooper, et al. Annu Rev Nutr, 2002<br />
  17. 17. Gut Flora and Metabolism<br />Energy salvage: esp via the short-chain fatty acids<br />Acetate, butyrate, propionate (SCFAs)<br />Absorbed into body and used by liver and others organs<br />Acetate and propionate modulate glucose metabolism in the liver and adipocytes (glycemic index)<br />50-70% of colonic cell energy derived from butyrate<br />Number and diversity of organisms key<br />Do we acquire flora that program us for obesity or normal weight?<br />Mackowiak, NEJM, 1982; 307:83-93 Hooper, et al. Annu Rev Nutr, 2002<br />
  18. 18. Gut Flora Metabolic Reactions<br />Metabolic reactions<br />Goldin BR in Gastrointestinal Microbiology 138-154, 2006<br />
  19. 19. Gut Flora and the Brain<br />Collins and Bercik in GASTRO 2009;136:2003–2014<br />
  20. 20. Obesity and the Gut Flora<br />
  21. 21. Ley, et al. PNAS. 2005, 102: 11070-75<br />
  22. 22. The Microbiome: Changes in flora<br />Mice and humans have different gut flora but the two largest divisions are shared in common<br />Bacteroidetes (Gram -)<br />Firmicutes (Gram +)<br />These flora change in response to diet and obesity of host<br />Ley, et al. PNAS. 2005, 102: 11070-75<br />
  23. 23. Ley, et al. PNAS. 2005, 102: 11070-75 <br />
  24. 24. The Microbiome: Changes in flora<br />Obesity changes the relative proportions of divisions. <br />Obese mice AND obese people have more Firmicutes than Bacteroidetes: dieting and weight gain shifts these proportions.<br />What are the implications of the change in our colonic metabolic machine<br />Chicken or the egg?<br />Ley, et al. PNAS. 2005, 102: 11070-75 <br />
  25. 25. Gut Flora and Obesity<br />Germ free mice studied by Gordon, et al<br />Control mice (remaining germ free) were changed from low fat diet to “Western” diet<br />Mice given normal gut flora (conventialized mice) also had same diet change.<br />Low fat diet: 5% lipids<br />“Western” diet: 41% lipids, 41% carbos (with simple sugars), 18% protein<br />Gordon J, et al, PNAS 2007;104:983<br />
  26. 26. Gut Flora and Obesity<br />Mice then studied for change in weight and fat pad size<br />Assessed response to fatty meal loading<br />Assessed locomotion of mice via gastrocnemius muscle activity.<br />Results: See Figures<br />Gordon J, et al, PNAS 2007;104:983<br />
  27. 27. Gut Flora and Obesity<br />Gordon J, et al, PNAS 2007;104:983<br />
  28. 28. Gut Flora and Obesity<br />GF mice are “resistant” to the Western diet<br />Gut flora are responsible for increasing energy production from food<br />“Doctor, I don’t eat” May be partially true! <br />Gut flora also influence lipid production, adipose cell storage, and appear to influence mouse locomotor activity<br />“Couch potato” gut flora<br /> My gut flora made me do it!<br />Gordon J, et al, PNAS 2007;104:983<br />
  29. 29. “Let food be thy medicine and medicine be thy food.”<br />Hippocrates<br />
  30. 30. The Microbiome: transplant experiments<br />Ob Ob mouse<br />Wild type mouse<br />Turnbaugh, et al. Nature 2006; 444:1027<br />
  31. 31. The Microbiome: transplant experiments<br />Germ-free mice given ob/ob or wild-type flora<br />Chow consumption and exercise the same for both groups<br />Both sets had similar starting weight and % BF.<br />The ob/ob microbiota promote host adiposity<br />Turnbaugh, et al. Nature 2006; 444:1027<br />
  32. 32. Ley R, et al in Nature 2006:444<br />Human Flora and Obesity<br />Mice data links gut flora with obesity and diet<br />Studied 12 obese human subjects; low calorie diet for one year<br />Fat-restricted (FAT-R)<br />Carbohydrate restricted (CARB-R)<br />Microbiota studied for one year (16s rRNA)<br />Firmicutes and bacteroidetes dominated (92.6%)<br />
  33. 33. Human Flora and Obesity<br />Ley R, et al in Nature 2006:444<br />
  34. 34. Ley R, et al in Nature 2006:444<br />Human Flora and Obesity<br />Firmicutes dominate in obese subjects<br />All subjects (no diff with diet) had bloom of all Bacteroidetes with decline in Firmicutes<br />CARB-R: Change began at 2% wgt loss<br />FAT-R: Change began at 6% wgt loss<br />Dynamic link between obesity and gut flora exists in humans (proven 1st in mice)<br />
  35. 35. Proposed Mechanisms in Obesity<br />DiBiase, et al. Mayo Clin Proc 2008;83:460-469 <br />
  36. 36. Microbiome: regulators<br />Archae: 1-2 % of mouse and human flora<br />Represent a major microbial group in gut flora<br />Increased in obese mice<br />Many are methanogenic : Methanobactersmithii<br />Converts CO2 and H2 gas to methane<br />By decreasing the partial pressure of H2 gas these bacteria can drive bacterial metabolism<br />The flora of obese mice are more efficient at extracting energy: “The Energy Harvest”<br />
  37. 37. The Gut and Obesity: Options<br />Diet may manipulate flora<br />Low carbs, high fat<br />High carbs, low fat<br />Probiotics<br />Prebiotics<br />Stool transplants<br />“Guarantee weight loss with thin people’s feces!”<br />Could this be the future?<br />
  38. 38. Coyle’s Corollary<br />It is better to be a stool donor than a recipient.<br />
  39. 39. Probiotics<br />
  40. 40. Probiotics<br />Definition: Live microorganisms which when ingested in adequate amounts confer a health benefit on the host. <br />Majority of probiotics are Gram +, lactic acid producers<br />Bifidobacterial species and Lactobacillus species<br />Survive transit through stomach and duodenum<br />Others include: non-pathogenic streptococci, enterococci, E coli Nissle 1917, Saccharomyces boulardii (yeast)<br />Fooks, et al. Int Dairy J, 1999 <br />Sheil, et al. In Gastrointestinal Microbiology, 2006<br />
  41. 41. Common Probiotics<br />Khedkar and Ouwehand in GastrointestMicrobiology 315-334, 2006<br />
  42. 42. Probiotics<br />VSL #3<br />4 lactobacilli<br />L. plantarum, casei, acidopholus, delbrueckiispp<br />3 bidifobacteria<br />B. infantis, breve, longum<br />1 streptococcus<br />Streptococcus salivarius ssp. thermophilus<br />Rand, PC studies have shown efficacy <br />in pouchitis and IBS<br />
  43. 43. Probiotics<br />Digestive Advantage<br />Ganeden BC30<br />Bacillus coagulans<br />Erythritol<br />Cellulose<br />Other minor ingredients <br />
  44. 44. Probiotics<br />Bifidobacteriuminfantis 35624 aka Bifantis<br />“Patented” strain of probiotic in Align<br />Decreased symptoms in two large trials in subjects with IBS*<br />*Whorwell P, et al. Am J Gastro 2006; 101<br />O’Mahoney L, et al. Gastro 2005;128<br />
  45. 45. Probiotics<br />Saccharomyces boulardii<br />Other minor ingredients<br />Shown in Rand / PC trials to help prevent recurrent C. difficile infection<br />
  46. 46. Probiotics in Food (Actimel)<br />L. caseiImmunitas™<br />Claim it is scientifically proven to be effective<br />“Each bottle contains 10 billion live” bacteria “that survive and remain active in the<br /> digestive tract.”<br />
  47. 47. Prebiotics<br />
  48. 48. Prebiotics<br />Ingested substances that selectively stimulate the proliferation and/or activity of desirable bacterial populations present in the host intestinal tract.<br />Usually target bifidobacteria and lactobacilli<br />Bifidogenic or bifidus factors explored in the 50s<br />Usually are non-digestible oligosaccharides (NDOs)<br />Lactulose, galacto-oligosaccharides, lactosucrose…<br />Crittenden and Playne. In Gastrointestinal Microbiology, 2006, pg 285-314.<br />
  49. 49. Prebiotics<br />Inulin: plant polymers mainly comprising fructose units, use have a terminal glucose<br />Indigestable fiber<br />Gut flora produce H2, CO2, methane gas from inulin<br />
  50. 50. Prebiotics<br />Is is possible to design a food, sugar, protein, or fat that would alter your gut flora to promote weight loss?<br />More likely possibility is to give a prebiotic that decreases your “Energy Harvest” of colonic bacteria<br />ie. lose weight by making your gut flora less efficient at digesting your left over food <br />
  51. 51. Designing Probiotics: The Future?<br />GASTROENTEROLOGY 2009;136:2015–2031<br />
  52. 52. Conclusions<br />The human microbiome and the Microbiome Project: research just beginning…<br />Gut flora by their genes, by-products, and metabolic activity influence our metabolism, weight, activity, immunity, health and disease.<br />Manipulation of gut flora may be an integral part of weight loss programs and different disease treatments in the future.<br />
  53. 53. Conclusions<br />Future studies must focus on the mechanisms of influence of our gut flora.<br />Studies must be placebo controlled and high quality.<br />Truly need translational science to work at the levels of the petri dish, genomics, and clinical outcomes.<br />Much more to come!<br />
  54. 54. The Future <br />Define who is there: in healthy subjects and those with disease<br />Study at Scripps in subjects with diet change, NASH, and IBD<br />Elucidate the gut flora host interaction<br />Design trials that truly assess the potential for probiotics and prebiotics to make a difference in health and disease.<br />
  55. 55. Questions<br />
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