Our Gut Flora: The Internist’s Guide - scripps.org Gut Flora: The Internist’s Guide Walter J. Coyle, MD, FACP,FACG Scripps Clinic Grand Rounds 2012

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 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

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 layerHome to billions of organisms

Sonnenburg J, et al. in NatureImmunology 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 ;124:837Ley, et al. PNAS. 2005, 102: 11070 Edwards, et al. Br J Nutr. 2002


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


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

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


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


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 levelsColonization 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;127:1087-94J 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 commonBacteroidetes (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


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





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!


Hippocrates

“Let food be thy medicine and medicine be thy food.”

The Microbiome: transplant experiments

Turnbaugh, et al. Nature 2006; 444:1027

Ob Ob mouseWild type mouse

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

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%)





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)

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

Antibiotic exposure in mice


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…


Antibiotics in children and obesity

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?

CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2011;9:1044–1049

Fecal Transplant Workgroup Good review and provides “cookbook”

recipe for performing fecal transplant

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 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

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) 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

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.

thermophilusSmall, Rand, PC studies have shown efficacy in pouchitis and IBSNewer 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 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

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

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

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(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

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

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

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

Clostridium difficile and altered microbiota

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

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

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 inGastro 2011.09.019Editorials

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

Documents

Our Gut Flora: The Internist’s Guide - scripps.org Gut Flora: The Internist’s Guide Walter J. Coyle, MD, FACP,FACG Scripps Clinic Grand Rounds 2012