Our Gut Flora: The Internist’s Guide
Walter J. Coyle, MD, FACP,FACG Scripps Clinic Grand Rounds 2012 Disclosures
Speaker’s Bureau: Takeda Pharmaceuticals Consultant: CSA Medical
Specifically, I have no financial relationships with any probiotic or prebiotic company Movement of the Talk
Describe the Microbiome Microbiology 101 Review the diversity of human gut flora Understand how our bacteria influence our metabolism, obesity, and selected diseases Review the plethora of probiotics Describe prebiotics and synbiotics and their potential uses Conclusions and the Future The Human Microbiome
Definitions: Microbiome: Aggregate of all gut species Microbiota: Individual bacterial species in the biome Over 100 trillion organisms (1014) Passengers in the mobile colonic petri dish Over 500 species identified so far (70 divisions) 90% of the cells in our body our microbial! 100 fold more genes in our gut then in us Our flora are an integral part of our genetic landscape and evolution Anatomic Regions of the Gut
Upper GI tract: 102 –104 cells/ml Lactobacilli, streptococci, H pylori Ileum: 106-1012 cells /ml, upper bacteria plus FaculativeFusobacterium anaerobes: Enterobacteriaceae Obligate anaerobes: Bacteroides, Veillonella, and Clostridium species Colon: distal human colon is the most biodense natural ecosystem known (1010-1012 cells/ml) Complex and diverse Comprise most of our bacterial biomass Microbes and Humans
Dethlefsen Nature 2007; 449:812-818 Micro 101
Pace in Science 1997 276:735-740 The Human Gut Flora
DiBiase, et al. Mayo Clin Proc 2008;83:460-469 Mouse intestine, mucus gel layer Home to billions of organisms
Sonnenburg J, et al. in Nature Immunology 2004:5
The Microbiome: Who’s there?
Early gut colonization has four phases Phase 1: Sterile gut Phase 2: Initial acquisition: vagina, feces, hospital Phase 3: Breast feeding or bottle-feeding (different) Breast fed more bifidobacteria (up to 90% of flora) Bottle fed more diverse; more Bacteroides , and Clostridial species Phase 4: Start of solids; move to adult flora Bifidobacteria remain key flora into adulthood
Ley, Peterson, Gordon. Cell 2006 ;124:837 Ley, et al. PNAS. 2005, 102: 11070 Edwards, et al. Br J Nutr. 2002 The Microbiome: Who’s there?
Adult Microbiome: Increasing diversity of flora as we age In some newer PCR (16S rRNA) studies, up to 92% of the flora in adults were “novel” species Serial stool collections show remarkable stability by an individual Greatest concordance with twins Less concordance with households Host genetic influence unexplored.
McCartney and Gibson in Gastrointestinal Microbiology, 51-73, 2006 16s ribosomal RNA
Important tool for bacterial identification 1500 bp Unique to bacteria and conserved through time Over 8000 species can be identified by 16s rRNA The Microbiome: Who’s there?
Multiple species: eukaryotic, bacterial, archael Firmicutes (Gram +/ motile) and Bacteroidetes (Gram -) : Majority These two groups comprise > 90% Anaerobes / aerobes: 1000:1 Far fewer species than the environment High levels of interpersonal diversity My stool is not your stool
Ley, Peterson, Gordon. Cell 2006 ;124:837 Ley, et al. Science 2005; 307: 1915 Ley R, et al in Cell 2006:124:837-848 Gut Flora and Metabolism Metabolomics
Study of the metabolites and small molecules that the body and gut bacteria produce. New area of science Broader than proteonomics Includes bacteria products with our own genetic products Pioneered by Jeremy Nicholson and Jeff Gordon Gut Flora and Metabolism
Microbial genomes enhance our metabolic activity May indirectly or directly effect our metabolism The colon is very active metabolically 20-70 gms of carbos and 5-20 gms of protein/day Over 100 kcal per day! Mass of colonic microbiome = single kidney Metabolically as active as the liver
Hooper, et al. Annu Rev Nutr, 2002 Gut Flora and Metabolism
Energy salvage: esp via the short-chain fatty acids Acetate, butyrate, propionate (SCFAs) Absorbed into body and used by liver and others organs Acetate and propionate modulate glucose metabolism in the liver and adipocytes (glycemic index) 50-70% of colonic cell energy derived from butyrate Number and diversity of organisms key Do we acquire flora that program us for obesity or normal weight?
Mackowiak, NEJM, 1982; 307:83-93 Hooper, et al. Annu Rev Nutr, 2002 Gut Flora Metabolic Reactions
Metabolic reactions
Goldin BR in Gastrointestinal Microbiology 138-154, 2006 Gut Flora in Health and Disease
Koch’s postulates: old view of microbes New view: microecologic disease or “pathogenic microbial community” Disease not due to one organism but changes in mix Dysbiosis: Abnormal state of the microbial ecosystem in the host Harmful and protective species or families Gut Enterotypes: New Concept
Recent evidence suggests 3 distinct gut types exist May be independent of geography May be programmed early Can we change the gut type No clear link in this study to BMI or metabolic syndrome
Nature 2011; 473:174-180 The Human Microbiome
Immunologically active environment Protection: 2 levels Colonization resistance: Competition, pH, gases, bacteriocins, etc… Stimulate immune function: Indigenous bacteria stimulate mucosal defense and immune development GALT: Gut-associated lymphoid tissue J Allergy Clin Immunol 2011;127:1087-94
J Allergy Clin Immunol 2011;128:646-52.) Gut Flora and Immunity
Neonates by vaginal delivery have more diverse microbiota more bifidobacteria, lactobacilli, and Bacteroides vs C-section neonates Atopic children have less bifidobacteria, lactobacilli, and Bacteroides Loss of diversity in children associated with more atopy, eczema, and obesity Studies with probiotics have not been able to reliably influence atopy, eczema, or asthma
J Allergy Clin Immunol 2011;127:1087-94 J Allergy Clin Immunol 2011;128:646-52 Gut Flora and Immunity
Gut flora differ from industrialized society and 3rd world nations Possible connection Data supports vitamin D being important in the immune reaction to gut flora Possible role in allergy and obesity Convincing data that manipulation of the gut flora can help is limited.
J Allergy Clin Immunol 2011;127:1087-94 Obesity and the Gut Flora Ley, et al. PNAS. 2005, 102: 11070-75 The Microbiome: Changes in flora
Mice and humans have different gut flora but the two largest divisions are shared in common Bacteroidetes (Gram -) Firmicutes (Gram +) These flora change in response to diet and obesity of host
Ley, et al. PNAS. 2005, 102: 11070-75 Ley, et al. PNAS. 2005, 102: 11070-75 The Microbiome: Changes in flora
Obesity changes the relative proportions of divisions. Obese mice AND obese people have more Firmicutes than Bacteroidetes: dieting and weight gain shifts these proportions. What are the implications of the change in our colonic metabolic machine Chicken or the egg?
Ley, et al. PNAS. 2005, 102: 11070-75 Gut Flora and Obesity
Germ free mice studied by Gordon, et al Control mice (remaining germ free) were changed from low fat diet to “Western” diet Mice given normal gut flora (conventialized mice) also had same diet change. Low fat diet: 5% lipids “Western” diet: 41% lipids, 41% carbos (with simple sugars), 18% protein
Gordon J, et al, PNAS 2007;104:983 Gut Flora and Obesity
Mice then studied for change in weight and fat pad size Assessed response to fatty meal loading Assessed locomotion of mice via gastrocnemius muscle activity. Results: See Figures
Gordon J, et al, PNAS 2007;104:983 Gut Flora and Obesity
Gordon J, et al, PNAS 2007;104:983 Gut Flora and Obesity
GF mice are “resistant” to the Western diet Gut flora are responsible for increasing energy production from food “Doctor, I don’t eat” May be partially true! Gut flora also influence lipid production, adipose cell storage, and appear to influence mouse locomotor activity “Couch potato” gut flora My gut flora made me do it!
Gordon J, et al, PNAS 2007;104:983 “Let food be thy medicine and medicine be thy food.”
Hippocrates The Microbiome: transplant experiments
Wild type mouse Ob Ob mouse
Turnbaugh, et al. Nature 2006; 444:1027 The Microbiome: transplant experiments
Germ-free mice given ob/ob or wild-type flora Chow consumption and exercise the same for both groups Both sets had similar starting weight and % BF. The ob/ob microbiota promote host adiposity
Turnbaugh, et al. Nature 2006; 444:1027 Human Flora and Obesity
Mice data links gut flora with obesity and diet Studied 12 obese human subjects; low calorie diet for one year Fat-restricted (FAT-R) Carbohydrate restricted (CARB-R) Microbiota studied for one year (16s rRNA) Firmicutes and bacteroidetes dominated (92.6%)
Ley R, et al in Nature 2006:444 Human Flora and Obesity
Ley R, et al in Nature 2006:444 Human Flora and Obesity
Firmicutes dominate in obese subjects All subjects (no diff with diet) had bloom of all Bacteroidetes with decline in Firmicutes CARB-R: Change began at 2% wgt loss FAT-R: Change began at 6% wgt loss Dynamic link between obesity and gut flora exists in humans (proven 1st in mice)
Ley R, et al in Nature 2006:444 Murine model Given various antibiotics early in life Found changes in fat composition Metabolic changes also occurred The mix of flora changed Firmacutes >> Bacteroidetes
Blaser, et al. Nature 2012 Antibiotic exposure in mice
Blaser, et al. Nature 2012 Studied 11,532 children in Avon, UK (91-93) Mapped antibiotic use (<6m,6-14m,15-23m) Body mass documented at 6wks, 10m, 20m, 38m and 7 yrs Multivariate analysis: role of tob, mother’s BMI, other medications, etc…
International J of Obesity; 2012: 1-8 Antibiotics in children and obesity
Blaser, et al. Nature 2012 Proposed Mechanisms in Obesity
DiBiase, et al. Mayo Clin Proc 2008;83:460-469 Microbiome: regulators
Archae: 1-2 % of mouse and human flora Represent a major microbial group in gut flora Increased in obese mice Many are methanogenic : Methanobacter smithii Converts CO2 and H2 gas to methane By decreasing the partial pressure of H2 gas these bacteria can drive bacterial metabolism The flora of obese mice are more efficient at extracting energy: “The Energy Harvest” The Gut and Obesity: Options
Diet may manipulate flora Low carbs, high fat High carbs, low fat Probiotics Prebiotics Stool transplants “Guarantee weight loss with thin people’s feces!” Could this be the future? Fecal Transplant Workgroup Good review and provides “cookbook” recipe for performing fecal transplant
CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2011;9:1044–1049 Stool Transplants: How To
Stool transplants: “prepared” feces by NGT or enema or colonoscopy Usually family member; 30-50 g fresh stool Stool homogenized for delivery No infectious complications to date Screen for Hepatitis, HIV, etc… 73-100% response reported in C Diff
Gastro 2006;130 Clin Infect Dis 2003;36 Stool Transplant: Evidence
2003 case series of refractory C diff patients Stool via NG from healthy family member 15 of 18 became recurrence-free 2009 case series of refractory C diff patients 11 of 15 became recurrence-free 2010 case series of refractory C diff patients Stool via colonoscopy 12 of 12 with immediate and sustained response Clin Infect Dis 2003;36: 540-544 QJM 2009;102:781-784 Yoon, J of Clin Gastro 2010, 44:562-66 Colonoscopy Stool Transplants Coyle’s Corollary
It is better to be a stool donor than a recipient. Stool donor cards will be made available after this lecture. Stool Donor Card
Share your stool; stop obesity CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2010;8:471–473 Coming to your neighborhood soon….
Stool transplants done here.
Donations accepted. Probiotics Probiotics
Definition: Live microorganisms which when ingested in adequate amounts confer a health benefit on the host. Majority of probiotics are Gram +, lactic acid producers (ie. Firmacutes) boulardiiBifidobacterial species and Lactobacillus species Survive transit through stomach and duodenum Others include: non-pathogenic streptococci, enterococci, E coli Nissle 1917, Saccharomyces (yeast)
Fooks, et al. Int Dairy J, 1999 Sheil, et al. In Gastrointestinal Microbiology, 2006 Common Probiotics
Khedkar and Ouwehand in Gastrointest Microbiology 315-334, 2006 Probiotics
VSL #3 4 lactobacilli L. plantarum, casei, acidopholus, delbrueckii spp 3 bidifobacteria B. infantis, breve, longum 1 streptococcus Streptococcus salivarius ssp. thermophilus Small, Rand, PC studies have shown efficacy in pouchitis and IBS Newer studies shown some efficacy in mild/mod UC Probiotics
Digestive Advantage Ganeden BC30 Bacillus coagulans Erythritol Cellulose Other minor ingredients Some data for IBS Mostly bloating
Postgrad Med, Vol. 121, Issue 2, March 2009 Probiotics
12 different species
Mostly Lactobacillus and Bidifobacter species
Clinical data with these combinations lacking Probiotics
Bifidobacterium infantis 35624 aka Bifantis “Patented” strain of probiotic in Align Decreased symptoms in two moderate sized trials in subjects with IBS* Bloating decreased Less loose stools
*Whorwell P, et al. Am J Gastro 2006; 101 O’Mahoney L, et al. Gastro 2005;128 Probiotics: E. coli Nissle 1917
Discovered in 1917 by Professor Alfred Nissle Well studied Some data for use in IBD, IBS, and Ab associated diarrhea Excellent safety profile Probiotics
Saccharomyces boulardii Other minor ingredients Shown in Rand / PC trials to help prevent recurrent C. difficile infection Decreases antibiotic associated diarrhea
Am J Gastroenterol. 2006 Apr;101(4):812-22 McFarland, et al. JAMA 1994;271:1913-8 Probiotics in Food (Actimel)
L. casei Immunitas™ Claim it is scientifically proven to be effective “Each bottle contains 10 billion live” bacteria “that survive and remain active in the digestive tract.” Probiotics in Food (Activia)
Contains Bifidus regularis Bifidobacterium animus Scientific trials show increased transit time in adults and women “Helps with slow transit in women and the elderly”
Bioscience and Microflora, 2001;20:43-48, Aliment Pharm Ther 2002;16:587-93 Probiotics for Immune System???
Lactobacillus rhamnosus GG (ATCC 53103) Patented by Gorbach and Goldin Various studies have shown it to be better than placebo for diarrheal illnesses Proven to survive the stomach, produces lactic acid and binds to human colonocytes
BMJ 2007; 335 : 340-345 IBD and the Gut: A target for Probiotics Hypothesis for flora and IBD
Gastro 2011;140:1720–1728 Probiotics and prebiotics in maintenance of remission in Crohn’s disease
Study Groups Relapse Rate (%) n Intervention Comparator Dur Intervention Comparator p
Guslandi 32 S. Bouliardii Mesalamine 6 6 38 0.04 (2000) + Mesalamine Campieri 40 VSL #3 Mesalamine 12 20 40 NR (2000) Prantera 45 LGG Placebo 12 17 11 0.3 (2002) Schultz 11 LGG Placebo 6 60 67 NS (2004) Bousvaro 75 LGG Placebo 24 31 17 0.18 s (2005) Marleau 98 L. johnsonii Placebo 6 49 64 0.15 (2006) Van 70 L. johnsonii Placebo 3 15 14 0.91 Gossum (2007) Chermes 30 Synbiotic Placebo 24 25 20 NS h(2007) 2000 Probiotics and IBD
Studies in France have shown that Crohn’s pts may be lacking in bacteria from Clostridium leptum group Their studies also support F. prausnitzii as key In mice and men have shown that F. prausnitzii decreases recurrence of Crohn’s and decreases gut inflammation. ? Use as probiotic Sokol, et al. Proceedings of the National Acad Science, Oct 2008 group (firmacutes) is a major member of the
Clostridium leptum
1. Prausnitzii F. (mucosa-associated 2. MAM bacteria microbiota): probably more important than luminal bacteria Probiotics and Ulcerative Colitis
Prosp, Rand/PC trial of 144 pts with mild to mod UC: VSL #3 (3,600 Billion) vs PBO Standard Rx same, 8 wk duration Outcome: 50% decline in UCDAI, remission Results: 50% decline 63% vs 41% (P=.01 PP) Remission: 47% vs 32% (P=.069 PP) Safety: no major AEs, bloating most common CAUTION: Large dose, PP ok, ITT less so Tursi A, et al. AJG 2010; 218:1-10 and altered microbiota
Clostridium difficile Recurrence: Probiotic Treatment
Probiotics Saccharomyces boulardii: 500 mg bid for 4-6 wks Modest evidence One DB / PC trials show fair efficacy Lactobacilli: 1 g qid for 4-6 weeks Evidence not as convincing PO nontoxicogenic C Diff: experimental Effective but only case reports to date Gastro 2006;130 Ann Intern Med 2006; 145 Am J Gastroenterol 2006; 101:812–822. Probiotics and C. Difficile
124 Adults with C difficile (Rand, PC) 64 1st episode, 60 recurrent CDAD Standard Ab with S. boulardii or PBO Outcome: Recurrence of CDAD 1st Episode: 19.3% vs 24.2% (P=.86) Rec CDAD: 34.6% vs 64.7% (P=.04) S. boulardii reduced risk for recurrence in subjects with recurrent C difficile (small numbers)
McFarland, et al. JAMA 1994;271:1913-8 Probiotics and Diarrhea
135 hospitalized pts given antibiotics DB, PC, Rand trial Probiotic Yogurt (Actimel) or PC BID Diarrhea: 34% PC vs 12% active (NNT:5) C Diff: Less often in Rx arm (NNT: 6) First rand trial to show prevention of C diff with probiotics
Hickson M, et al. BMJ, 2007:335-80 16 studies, 3432 children (ages 2wks-17yrs) Great heterogeneity, short studies, low quality Overall protective effect seen (NNT:7) High dose (>5 billion CFU) L rhamnosus and S boulardii had best data
Better studies needed Cochrane Review Irritable Bowel Syndrome(IBS), the Microbiome, and Probiotics The Microbiome and IBS
Hydrogen gas production is a by-product of bacterial fermentation Elimination is dependent on methanogenic and sulfate-reducing bacteria (making either methane or hydrogen sulfide). Competitive species: usually 1 predominates in your gut flora
Strocchi, et al. Gut 1994; 35:1098 Gut flora and IBS: an hypothesis
Talley and Fodor in Gastro 2011.09.019 Editorials The Microbiome and IBS
Methane gas produced in higher quantities in constipation predominant IBS. Methane shown to decrease contractility of smooth muscle. Suggests that your IBS type is based largely on your type of gut flora Predominance of Methane producers = IBS-C Predominance of Sulfide producers = IBS-D
Pimental, et al. DDS 2003; 48: 86-92 Probiotics in IBS
103 IBS pts (Rome 2) 6 month, DB, PC, Rand trial Probiotic Capsule: 5 bacteria (Lacto, Bifido, Priopionibacter, etc) 42% reduction of Total Symptom score in treatment vs 6 % for placebo Largest decline was in Borborygmi
Alimen Pharm and Therapeutics, 2005:22(5):387 Probiotics in IBS
Similar study in 59 children using VSL #3 Placebo controlled, crossover IBS like scale as primary outcome Safe and effective for relief of Sx Guandalini S. et al. Abstract 73rd ACG 2008 Probiotics in IBS
Large , systemic review of best trials for IBS Only 16 trials met the rigorous criteria 11 still had one or more major flaws Only two trials showed significant improvement in a primary endpoint vs placebo Both used Bifidobacterium infantis 35624 Better trials required, need to use Rome criteria
Brenner D, Am J Gastro 2009;104:1033-1049 Probiotics in IBS
Recent Meta-analysis on probiotics and IBS 18 RCTs found (N=1650) 11 trials high quality, 4 very high Different organisms and outcomes Most improvement was in bloating No sign. Adverse events reported “Probiotics are effective in IBS” Caution: Best 4 studies> No difference; Rome criteria not used, all studies have small #s
Moayyedi P, Talley N, et al. Gut, 2010;59:325-32 Prebiotics Prebiotics
Ingested substances that selectively stimulate the proliferation and/or activity of desirable bacterial populations present in the host intestinal tract. Usually target bifidobacteria and lactobacilli Bifidogenic or bifidus factors explored in the 50s Usually are non-digestible oligosaccharides (NDOs) Lactulose, galacto-oligosaccharides, lactosucrose…
Crittenden and Playne. In Gastrointestinal Microbiology, 2006, pg 285-314. Prebiotics
Inulin: plant polymers mainly comprising fructose units, have a terminal glucose Indigestable fiber Gut flora produce H2, CO2, methane gas from inulin Prebiotics
Inulin: plant polymers mainly comprising fructose units, use have a terminal glucose Indigestable fiber Gut flora produce H2, CO2, methane gas from inulin “Breakfast of Flatulance” Prebiotics
Feed your flora!!! Prebiotics
Is is possible to design a food, sugar, protein, or fat that would alter your gut flora to promote weight loss? More likely possibility is to give a prebiotic that decreases your “Energy Harvest” of colonic bacteria ie. lose weight by making your gut flora less efficient at digesting your left over food Synbiotics
Combination of a probiotic and prebiotic Little data supports efficacy Prebiotic has been inulin, fatty acid, other carrier molecules More to come… Designing Probiotics: The Future?
GASTROENTEROLOGY 2009;136:2015–2031 Conclusions
The human microbiome and the Microbiome Project: research just beginning… Gut flora by their genes, by-products, and metabolic activity influence our metabolism, weight, activity, immunity, health and disease. Manipulation of gut flora may be an integral part of weight loss programs and different disease treatments in the future. Conclusions
Future studies must focus on the mechanisms of influence of our gut flora. Studies must be placebo controlled and high quality. Truly need translational science to work at the levels of the petri dish, genomics, and clinical outcomes. Much more to come! The Future
Define who is there: in healthy subjects and those with disease Study at Scripps in subjects with diet change, NASH, and IBD Elucidate the gut flora host interaction Design trials that truly assess the potential for probiotics, prebiotics, and synbiotics to make a difference in health and disease. Questions Name this famous scientist Antonie van Leeuwenhoek
Father of microbiology Born in Delft, Netherlands on October 24, 1632 Studied the bacteria in his own stools Invented the first microscope