our gut flora: the internist’s guide...movement of the talk describe the microbiome microbiology...
TRANSCRIPT
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Our Gut Flora: The Internist’s Guide
Walter J. Coyle, MD, FACP,FACG
Scripps Clinic Grand Rounds 2012
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Disclosures
Speaker’s Bureau: Takeda Pharmaceuticals
Consultant: CSA Medical
Specifically, I have no financial relationships with any probiotic or prebiotic company
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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
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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
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Anatomic Regions of the Gut
Upper GI tract: 102 – 104 cells/ml Lactobacilli, streptococci, H pylori
Ileum: 106-1012 cells /ml, upper bacteria plus Faculative anaerobes: Enterobacteriaceae Obligate anaerobes: Bacteroides, Veillonella,
Fusobacterium 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
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Microbes and Humans
Dethlefsen Nature 2007; 449:812-818
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Micro 101
Pace in Science 1997 276:735-740
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The Human Gut Flora
DiBiase, et al. Mayo Clin Proc 2008;83:460-469
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Mouse intestine, mucus gel layerHome to billions of organisms
Sonnenburg J, et al. in NatureImmunology 2004:5
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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 ;124:837Ley, et al. PNAS. 2005, 102: 11070 Edwards, et al. Br J Nutr. 2002
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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
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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
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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 ;124:837Ley, et al. Science 2005; 307: 1915
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Ley R, et al in Cell 2006:124:837-848
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Gut Flora and Metabolism
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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
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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
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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
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Gut Flora Metabolic Reactions
Metabolic reactions
Goldin BR in Gastrointestinal Microbiology 138-154, 2006
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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
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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
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The Human Microbiome
Immunologically active environment Protection: 2 levelsColonization resistance:
Competition, pH, gases, bacteriocins, etc… Stimulate immune function:
Indigenous bacteria stimulate mucosal defense and immune development
GALT: Gut-associated lymphoid tissue
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J Allergy Clin Immunol 2011;127:1087-94
J Allergy Clin Immunol 2011;128:646-52.)
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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;127:1087-94J Allergy Clin Immunol 2011;128:646-52
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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
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Obesity and the Gut Flora
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Ley, et al. PNAS. 2005, 102: 11070-75
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The Microbiome: Changes in flora
Mice and humans have different gut flora but the two largest divisions are shared in commonBacteroidetes (Gram -) Firmicutes (Gram +)
These flora change in response to diet and obesity of host
Ley, et al. PNAS. 2005, 102: 11070-75
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Ley, et al. PNAS. 2005, 102: 11070-75
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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
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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
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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
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Gordon J, et al, PNAS 2007;104:983
Gut Flora and Obesity
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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
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Hippocrates
“Let food be thy medicine and medicine be thy food.”
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The Microbiome: transplant experiments
Turnbaugh, et al. Nature 2006; 444:1027
Ob Ob mouseWild type mouse
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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
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Ley R, et al in Nature 2006:444
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%)
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Ley R, et al in Nature 2006:444
Human Flora and Obesity
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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)
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Blaser, et al. Nature 2012
Murine model Given various antibiotics early in life Found changes in fat composition
Metabolic changes also occurred The mix of flora changed
Firmacutes >> Bacteroidetes
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Antibiotic exposure in mice
Blaser, et al. Nature 2012
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International J of Obesity; 2012: 1-8
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…
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Blaser, et al. Nature 2012
Antibiotics in children and obesity
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Proposed Mechanisms in Obesity
DiBiase, et al. Mayo Clin Proc 2008;83:460-469
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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”
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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?
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CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2011;9:1044–1049
Fecal Transplant Workgroup Good review and provides “cookbook”
recipe for performing fecal transplant
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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
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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 patientsStool 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
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Colonoscopy Stool Transplants
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Coyle’s Corollary
It is better to be a stool donor
than a recipient.
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Stool donor cards will be made available after this
lecture.
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Stool Donor Card
Share your stool; stop obesity
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CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2010;8:471–473
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Coming to your neighborhood soon….
Stool transplants done here.
Donations accepted.
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Probiotics
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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) Bifidobacterial species and Lactobacillus species Survive transit through stomach and duodenum
Others include: non-pathogenic streptococci, enterococci, E coli Nissle 1917, Saccharomycesboulardii (yeast)
Fooks, et al. Int Dairy J, 1999 Sheil, et al. In Gastrointestinal Microbiology, 2006
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Common Probiotics
Khedkar and Ouwehand in Gastrointest Microbiology 315-334, 2006
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Probiotics
VSL #3 4 lactobacilli
L. plantarum, casei, acidopholus, delbrueckii spp
3 bidifobacteria B. infantis, breve, longum
1 streptococcus Streptococcus salivarius ssp.
thermophilusSmall, Rand, PC studies have shown efficacy in pouchitis and IBSNewer studies shown some efficacy in mild/mod UC
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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
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Probiotics
12 different species
Mostly Lactobacillus and Bidifobacter species
Clinical data with these combinations lacking
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Probiotics
Bifidobacterium infantis35624 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; 101O’Mahoney L, et al. Gastro 2005;128
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Probiotics: E. coli Nissle 1917
Discovered in 1917 by Professor Alfred Nissle
Well studied Some data for use in
IBD, IBS, and Abassociated diarrhea
Excellent safety profile
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Probiotics
Saccharomycesboulardii
Other minor ingredients Shown in Rand / PC
trials to help prevent recurrent C. difficileinfection
Decreases antibiotic associated diarrhea
Am J Gastroenterol. 2006 Apr;101(4):812-22
McFarland, et al. JAMA 1994;271:1913-8
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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.”
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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
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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
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IBD and the Gut: A target for Probiotics
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Hypothesis for flora and IBD
Gastro 2011;140:1720–1728
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Probiotics and prebiotics in maintenance of remission in Crohn’s disease
Study Groups Relapse Rate (%)
n Intervention Comparator Dur Intervention Comparator p
Guslandi(2000)
32 S. Bouliardii+
Mesalamine
Mesalamine 6 6 38 0.04
Campieri(2000)
40 VSL #3 Mesalamine 12 20 40 NR
Prantera(2002)
45 LGG Placebo 12 17 11 0.3
Schultz (2004)
11 LGG Placebo 6 60 67 NS
Bousvaros (2005)
75 LGG Placebo 24 31 17 0.18
Marleau(2006)
98 L. johnsonii Placebo 6 49 64 0.15
Van Gossum(2007)
70 L. johnsonii Placebo 3 15 14 0.91
Chermesh (2007)
30 Synbiotic2000
Placebo 24 25 20 NS
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Probiotics and IBD
Studies in France have shown that Crohn’s pts may be lacking in bacteria from Clostridium leptumgroup Their studies also support F. prausnitzii as key
In mice and men have shown that F. prausnitziidecreases recurrence of Crohn’s and decreases gut inflammation.
? Use as probioticSokol, et al. Proceedings of the National Acad Science, Oct 2008
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1. F. Prausnitzii is a major member of the Clostridium leptum group (firmacutes)
2. MAM bacteria (mucosa-associated microbiota): probably more important than luminal bacteria
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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
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Clostridium difficile and altered microbiota
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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.
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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
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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
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Cochrane Review
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
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Irritable Bowel Syndrome(IBS), the
Microbiome, and Probiotics
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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
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Gut flora and IBS: an hypothesis
Talley and Fodor inGastro 2011.09.019Editorials
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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
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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
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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
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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
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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
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Prebiotics
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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.
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Prebiotics
Inulin: plant polymers mainly comprising fructose units, have a terminal glucose
Indigestable fiber Gut flora produce H2,
CO2, methane gas from inulin
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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”
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Prebiotics
Feed your flora!!!
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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
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Synbiotics
Combination of a probiotic and prebiotic Little data supports efficacy Prebiotic has been inulin, fatty acid, other carrier
molecules More to come…
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Designing Probiotics: The Future?
GASTROENTEROLOGY 2009;136:2015–2031
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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.
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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!
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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.
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Questions
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Name this famous scientist
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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