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Probiotics – A New Frontier

Doron D. Kahana, MDCenter for Digestive Health & Nutritional Excellence

dkahana@gmail.com www.KahanaMD.com

October 11, 2014

Scientific Basis, Clinical Application

Disclosures

• Sigma Tau – Speaker (Probiotics)

• Nutricia – Speaker (Eosinophilic Esophagitis)

• NPS – Speaker, Board Advisory Committee forGattex (teduglutide, GLP-2 agonist for shortbowel syndrome)

• Abbott – Speaker (EE and food allergy)

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Probiotics - Outline

I. Microbes

a. Historical Perspective

b. A New Germ Theory

II. The Microbiome

a. You’re Not Alone

b. Microbial Recognition

c. Toll-like Receptors

III. Probiotics

a. Mechanism of Action

b. Clinical Application

c. Safety Concerns

IV. Summary

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Listener Participation

Question: Which scientific field is considered to bemost modern:

a) Zoology

b) Botany

c) Entomology

d) Microbiology

e) I still believe in spontaneous generation

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The Microbial World

• The existence of micro-organisms wasalways suspected

• Microscope was invented in the 17th century

– Robert Hooke (1635-1703)

– Antonie van Leeuwenhoek (1632-1723)

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Microbiology

• Louis Pasteur (1822-1895) disproved the widely-held theory ofspontaneous generation.

– Food preservation (i.e. pasteurization)

– Vaccination (e.g. anthrax, cholera, rabies).

• Robert Koch (1843-1910) developed the germ theory of disease.

– Specific pathogens cause specific diseases (e.g. anthrax, tuberculosis).

– Koch postulates (e.g. presence, isolation, reproduction, recovery).

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A New Germ Theory

• New microbes discovered through genetic sequencing

• Helped explain previous “idiopathic” diseases

– Lyme disease, Whipple disease, Ehrlichiosis

• As well as conditions previously thought to be non-infectious

– PUD (H. pylori), GBS (C. jejuni), HUS (E. coli O157:H7),cervical cancer (HPV), HCC (HCV), atherosclerosis (?)

– Microbes not only cause disease directly, but may also doso indirectly (e.g. via activation of host immune defenses)

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-Ewald P, Evolution of infectious disease. Oxford Univ Press 1996-Lorber B, Are all diseases infectious? Ann Intern Med 1996-Koeth RA, Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat,promotes atherosclerosis. Nature Medicine 2013

DNA Microarray Analysis

• DNA microarray analyzer

– Rapid sequencing/profiling ofmicrobial populations

Berkeley Lab(http://www.lbl.gov/Tech-transfer/techs/lbnl2229.html)

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Phylogenetic Trees

• White branches – core

• Colored portions - variable

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Phyla, Species, Strains

• Firmicutes:

– Bacillus

– Listeria

– Staphylococcus

– Streptococcus

– Enterococcus

– Lactobacillus

– Clostridium

– Heliobacterium

– Mycoplasma

– Faecalibacteriumprausnitzii

• Bacteroidetes:

– Bacteroides

– Flavobacterium

– Sphingobacterium

– Flexibacterium

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• Actinobacteria:

– Bifidobacterium

– Mycobacterium

– Corynebacterium

– Streptomyces

• Fusobacteria

• Cyanobacteria

• Verrucomicrobia

• Chlamydiae

• Proteobacteria:

– Escherichia coli

– Enterobacterium

– Haemophilus

– Pseudomonas

– Neisseria

– Aeromonas

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• Sum collection of allmicrobes living within ourbodies

• “In a sense, you aren‘treally you”

• Superorganism

The Microbiome

-Colin Nickerson, Boston Globe 2/25/2008

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10x90% of the cellscontained inthe humanbody belong tonon-humanorganisms

• We are born (relatively) sterile

• Colonized as we exit the womb

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Human Microbiome Project

- Sequence genomes ofbacteria that makeupmicrobiome.

>100x

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The human bodysupports a uniqueecology.

100 trillion (1014)microbial cells

10 trillion (1013)human cells

10-folddifference

GI lumen

103/g stomach

108-9/g colon

Densest biologicalecosystem on earth

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The Gut Microbiome

Digestion

Fermentation of food residue

Assimilation of minerals and traceelements

Detoxification (nitrogen compounds)

Immunity

Antigen processing and presentation

Training of self vs. foreign

Enhancing innate immunity

Memory training (adaptive immunity)

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The Gut Microbiome

Metabolic Production

Vitamins (biotin, folate, B12, K)

Digestive enzymes (lactase)

Short-chain fatty acids (buryrate)

Metabolites (phenols, toxins)

Regulate Inflammation

Physiologic repair and regeneration –Epithelial homeostasis

Maintenance care – Promotes cellularsurvival, enhances barrier function

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-O’Keefe, Curr Op Gastro 2008-Medzhitov, Cell 2004

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Toll-like Receptors (TLRs)

Pattern-recognition receptors (PRR) - sense conserved, microbialstructures (motifs) in nature.

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• TLR engagement initiates a signalingcascade that results in expression ofnuclear factors that mediate tissuerepair and inflammation.

– MyD88IL-1R-assoc-protein-kinases (IRAK)

TGF-beta-activate kinase (TAK)

TNF receptor-assoc factor (TRAF)

TAK1-binding protein (TAB)

Mitogen-activated protein (MAP)

– NF-κB

TLRs Initiate Signaling Cascades

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Immunological Balance

InjuryRepair

Microbial Signaling

ImmuneSystem

Inflammation

Dysbiosis and Inflammation

Good bacteria

Stimulate immune system

Maintain regulation

Promote cellular repair andtissue remodeling

Bad bacteria and toxins

Compromise epithelium

Activate immune pathways

Spark inflammation

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TLRs

TLRs

-Round, Nat Immunol 2009 -Rachmilewitz D, Gastro 2004 -Walker WA, CID 2008

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-Round JL, Nature Immunology 2009

Probiotics? Eubiosis

Obesity

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• 400 million obese adults globally in 2005, projected to increaseto more than 700 million in 2015

• Important inheritable component estimated at 40-70%, withmore than 50 validated genome-wide significant loci associatedwith adiposity and body composition

• An increase in the phylum Firmicutes and a decrease inBacteroides is associated with obesity

• Low bacterial richness is characterized by more marked overalladiposity, insulin resistance, dyslipidemia, and a morepronounced inflammatory phenotype when compared with highbacterial richness

-Le Chatelier E, Nature 2013

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Obesity

23-Le Chatelier E, Nature 2013

Recap

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• Microbiology is a relatively modern scientific field

• New molecular technique has allowed us to detect newmicrobes in places we never thought were hospitable to life

• Microbes are responsible for disease both directly and indirectly

• We are a colony of creatures—a multitude of entities

– Microbial cells outnumber our own 10:1

• The gut microbiome is essential for digestion, immunity, repair,tissue homeostasis, and metabolic activity

– Microbial pattern-recognition is evolutionarily conserved

– Richness of microbiome correlates with overall health

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Listener Participation

Question: The first successful clinical trial of probioticswas conducted for the treatment of:

a) Lactose intolerance

b) Rotavirus gastroenteritis

c) Inflammatory bowel disease

d) Irritable bowel syndrome

e) Probiotics are a fad unworthy of clinical trials

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The Father of Probiotics

• Bulgarian peasants who ate large quantities of sour milk lived longer.

• “The accumulation of waste matter, retained in the large intestine forconsiderable periods, becomes a nidus for microbes which producefermentations and putrefaction harmful to the organism.” (p. 67)

• Metchnikoff believed that lactic acid prevents “the multiplication of themicrobes which cause putrefaction.” (p. 170)

26--Metchnikoff E. The Prolongation of Life: Optimistic Studies. Putnam Sons 1908

• 1908 – Élie Metchnikoff, Russian scientist andsub-Director of the Pasteur Institute in Paris,published a classic text entitled:

Prolongation of Life: Optimistic Studies

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Probiotics Defined

• Live microorganisms which confer a health benefit to the host.

• Probiotic: Greek word meaning “supporting or favoring life”

• Alfred Nissle isolated an E. coli strain from the stool of a WWIsoldier who was unaffected by an outbreak of Shigellosis in1917, then used it to treat infectious diarrhea.

• Broad clinical use of probiotics gained momentum in the ‘70s.

• Lactose intolerance, recurrent UTIs, infectious diarrhea

• First successful trial of probiotics was to treat rotavirusgastroenteritis in children in the 1990s.

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-Nissle A, Z Klin Med 1951 -Lilly & Stillwell, Science 1965-Isolauri 1991 and 1994 -Kaila 1995 -Majamaa 1995

--U.N. Food and AgricultureOrganization and WHO, 2001

Mechanism of Action

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-Adopted from Ramakrishna BS, Trop Gastro 2009

Influence Outcome Implication

Lactic acid Decreases stool pH Habitat formation

Short-chain fatty acids(butyrate)

Epithelial proliferation, full differentiation Mucosal energy(Cancer prevention)

Bacteriocins Microcidal Direct Protection

Mucins Prevent pathogen binding Colonization resistance

Enzymes Improve digestion Enhance absorption

Barrier Function Increase production of tight junction proteins Protection from leaky gut(bacterial translocation)

Detoxification Nitrogen and sulfur bindingConsumption of reactive oxygen species (ROS)

Cancer prevention

Immune conditioning Enhance oral tolerance (T regulatory cells, IL-10)Defensin production (Paneth cells)Secretory IgA production

Attenuate inflammationPrevent bacterial overgrowthSystemic clearance of bacteria (UTI)

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29-Vanderpool C, IBD 2008

1. Block pathogens

– Competitive inhibition

– Bacteriocins

2. Regulate immuneresponses

– Enhance innate immunity

– Modulate inflammation(TLR signaling pathways)

3. Regulate epithelialhomeostasis

– Promote cell survival

– Enhance barrier function

– Stimulate protectiveresponses

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Mechanism of Action

• Colonization Resistance

– Competitive inhibition

– Lactic acid production

– Bacteriocin production

• Immune Conditioning

– Enhances oral tolerance

– Stimulates T-regulatory cells

– Increases immunoglobulinproduction

– Stimulates TLR expression

30-Walker WA, CID 2008

• Mucosal Stimulation

– Mucin proteins

– Defensins

– Tight junction proteins

– Regulate apoptosis(programmed cell death)

– Promote repair

• Genetic Coordination

– Up-regulation of host genes

– Expression of bacterial genes

– Sharing of genetic info?

-McNulty NP, Sci Trans Med 2011

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The Interactome

31-Bisanz JE, Sci Trans Med 2011

• Effect of probiotics in vivo on mucosal gene expression

– Prospective, double-blind, placebo-controlled study

– L. acidophilus, L. casei, L. rhamnosus GG in 1010

concentration

– Healthy volunteers; acute response (6 hrs)

– EGD with mucosal biopsies of duodenum

– Different probiotic lactobacilli led to markedly differentexpression profiles of human mucosal genes

32-van Baarlen, PNAS 2011

Genetic Influence

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• Consumption of probiotics lead to altered mucosalgene expression

– Immune response: IL-1β, IL17B, IRAK2, multiple chemokinesand cytokines, intracellular adhesion molecules (ICAM-1)

• Increased expression of surface receptors in antigen-presenting cells

– Hormonal regulation of tissue growth and development,water and ion homeostasis

• Angiogenin, oxytocin, apelin, POMC, bone morphogeneticprotein (BMP2), IGF1, IL-23

33-van Baarlen, PNAS 2011

Genetic Influence

• Probiotics work in methods that go beyond hostcolonization

– Direct antimicrobial effect

– Immune conditioning

– Mucosal stimulation

– Transcriptional modification

– Epigenetic influence

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Recap

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Clinical Application

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Case Presentations

• 29-year-old male with UC on maintenance therapy with 5-ASA(mesalamine), experiencing mild-moderate disease activity

• 56-year-old male with long-standing UC, s/p total colectomywith ileo-anal pull-through, now with episodes of pouchitis

• 13-year-old female with Crohn’s disease involving TI and rightcolon, on maintenance with 6-MP, showing short stature

• 5-year-old male with recurrent acute otitis media (middle earinfection), prescribed amoxicillin for 10 days

• 23-month-old female in preschool with low-grade fever,diarrhea, mild dehydration; sent home with parents

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Case Presentations

• 38-year-old male with s/p liver transplant, onimmunosuppression, with recurrent C. diff colitis

• 26-year-old female with bloating, irregular bowel habits, andintermittent pain; colonoscopy normal

• 27-week-old premature infant with feeding intolerance,abdominal distention, concern for NEC

• 32-year-old pregnant woman with a strong family history ofatopy and eczema; concerned about unborn child

• 76-year-old male with colon cancer s/p resection; concernedabout recurrence

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38-Fedorak, J Clin Gastro 2008

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Pouchitis

• Probiotics are effective in maintaining antibiotic-inducedremission in patients with recurrent or refractory pouchitis

– 40 adults with chronic relapsing pouchitis, in remission with antibiotics

– Randomized to receive probiotics vs. placebo

– After 9 months, relapse of 15% in probiotics group vs. 100% in placebo

– All patients relapsed within 3 months of stopping probiotics

• Probiotics were also effective in preventing onset of pouchitiswithin the first year after surgery (ileal-pouch creation)

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-Gionchetti et al Gastro 2000 -Mimura, Gut 2004-Kuehbacher et al Gut 2006 -Kuisma et al Aliment Pharmacol Ther 2003

Functional Bowel Disorders

• Complex interaction—biological, psychological, social factors

• Abdominal pain

• Bloating/gassiness

• Constipation

• Indigestion

• Nausea

• Diarrhea

• Urgency

• Irritable bowel syndrome

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Irritable Bowel Syndrome

• Common in developed world

– 10 to 20% of general population

– Higher incidence and increased severity in females

– Altered motility, visceral hypersensitivity, abnormal brain-gutinteraction, food intolerance, maldigestion/malabsorption, alteredmicrobiota, post-infectious, reactive and inflammatory changes

• Abdominal pain, irregular bowel movements, bloating

– Defined by symptom criteria (no pathognomonic finding)

• Associated with bacterial overgrowth in the small intestine

– Often responds to antibiotic therapy

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-DiLorenzo C, ROME III -Baber, JPGN 2008-Ringel, Clin Gastro Hepatol 2009 -Pimentel M, Ann Intern Med 2006

Etiology• Alterations in proportions and

diversity of commensal bacteria

• Related changes in metabolicoutput and ecological milieu

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2001 2012

Ford AC, BMJ 2012

Horwitz BJ, NEJM2001

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Enterotypes and IBS

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• L. acidophilus, B. lactis, B. bifidum at 25 B CFU ⁄ capsule• Symptom severity: probiotic intervention vs. control group (*P < 0.05)

-Aliment Pharm Ther 2009

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45-Moayyedi, Gut 2010

Antibiotic-Associated Diarrhea

• Frequency of AAD varies greatly, up to 30% commonlyreported (DD 1998, BMJ 2002)

• CDAD accounts for 15-39% of cases of AAD

– Pseudomembranous colitis

– Toxic megacolon

– High case-fatality

– Cumulative antibiotic exposure increases risk

• Multiple randomized controlled trials conclude that co-administration of Abx with probiotics is superior to placebo

• Risk reduction of diarrhea is usually 50-60%

46-Vanderhoof 1999 -D’Souza 2002 -Jirapinyo 2002 -Kotowska 2005-Correa 2005 -Szajewska 2007 -Johnston 2007

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-D’Souza AL, BMJ 2002

48-Szajewska, J Ped 2007

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• Meta-analysis of63 RCTs with11,811 subjectsshowed astatisticallysignificant assocof probioticsadministrationwith reduction inAAD– RR 0.58; 95% CI,

0.50-0.68;(P<0.001)

– NNT 13; 95% CI,10.3 to 19.1

49-Hempel S, JAMA 2012

Clostridium Difficile

• Probiotics reduce risk ofrecurrent C. diff >50%compared to placebo

• Probiotics improvetreatment results whenused in combination withantibiotic therapycompared to antibioticsalone

50-McFarland, Anaerobe 2009

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AAD and CDAD• PLACIDE: Probiotic

lactobacilli andbifidobacteria inAAD and C. difficilediarrhea in theelderly (PLACIDE)

• Patients >65 yearswith 1 or more oralor parenteral Abx inthe preceding 7days or about tobegin Abx therapy

• L. acidophilus, B.bifidum, B. lactis

51-Allen SJ, Lancet 2013

Infectious Diarrhea - Treatment

• Meta-analysis of 8 randomized, placebo-controlled trialsconcluded that probiotics significantly reduced duration ofacute infectious diarrhea in children

– Average reduction of 1.1 days (95% CI -1.9 to -0.3)

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-Szajewska 2006-Michail 2006-Sazawal 2006

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Infectious Diarrhea - Prophylaxis

• Population of 986preschoolers receivingprobiotics vs. placebo in adaycare setting

– Short = 3 months

– Long = 7 months

• Multiple probiotic group(black) with significantreduction in GI infection

– 42% rate reduction

– Short and long-term therapywith similar results

53-Lin, Vaccine 2009

Probiotic Therapy in Diarrhea

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-Guandalini S, J Clin Gastro 2008

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Atopic Disease

• Asthma, allergic rhinitis, atopic dermatitis, and food allergyhave increased in prevalence in recent decades.

– Epidemic proportions in developed countries with up to 20-30%prevalence.

– #1 cause of chronic disease in children.

– Hygiene hypothesis

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-Grammatikos AP Ann Med 2008-Bach JF NEJM 2002

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-Grammatikos AP Ann Med 2008

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57-Lee, J All Clin Immunol 2008

Atopic Disease• Dysbiosis is assoc with

increased prevalence offood allergy.

• Clostridia strainsisolated from healthyhumans induce T-regsupon transfer to GFmice.

• Tolerance-inducingprotocols.

– Clostridiaenrichmentpotentiates antigen-specific tolerance

58-Stefka AT, PNAS 2014

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Atopic Disease

59-Stefka AT, PNAS 2014

Other Conditions

• Colon cancer

• Hepatic encephalopathy

• Fatty liver disease

• Urinary tract infections

• Vulvo-vaginal candidiasis and bacterial vaginosis

• Otitis media/respiratory disease

• Dental disease

• Lactose intolerance

• Critical illness

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Listener Participation

Question: The reported adverse effects of probioticsinclude the following, EXCEPT:

a) Necrotizing pancreatitis

b) Diarrhea

c) Nausea

d) Bacteremia

e) D-Lactic acidosis

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Safety

• Common side effects include nausea and diarrhea

• D-lactic acidosis has been reported

– Only in patients with short bowel syndrome; strain specific

• Reports of bacteremia and fungemia are sparse

– Immunocompromised patients

– Central venous access or other hardware (artificial heart valves)

– Hand contamination is strongly implicated

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-Boyle J, Am J Clin Nutr 2006 -Rayes, Am J Transplant 2005 -Munakata S, Brain & Dev 2010-Anukam KC, J Clin Gastro 2008 -Madsen K, J Clin Gastro 2008

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63-Boyle J, Am J Clin Nutr 2006

Safety

• Many prospective clinicaltrials of probioticsshowed no significantadverse events

• Some trials looked atvulnerable populations

– Critical illness

– IBD

– Transplant recipients

– Liver failure

– HIV

64-Snydman DR, Clin Infect Dis 2008

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Safety

• Recent Dutch RCT of probiotics in severe acute pancreatitisreported increased mortality vs. placebo

– 296 patients: 152 in probiotic group, 144 in placebo group

– Increased mortality (16% vs. 6%; RR 2.53, 95% CI 1.22-5.25)

• Bowel ischemia in probiotic group only (8 deaths)

– Multi-stain synbiotic product, 10 B CFU

– Nasojejunal route BID, mixed in formula, continuous feeds

– All cases occurred within first 14 days

– No mesenteric occlusion or more severe pancreatic disease

– No difference in infectious complications

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Conclusions

• We are a complex ecosystem inhabited by a multitude oforganisms which exhibit a symbiotic biological existence

• Probiotics are beneficial organisms that engage both host andcommensal components of our being, exerting health benefitsthat go beyond colonization

• Microbes have tremendous therapeutic potential andrepresent a new frontier in medicine

• Probiotics promote digestion, enhance intestinal integrity,stimulate immunity, and exert numerous benefits to the host

• Probiotics are largely safe and have an enormous upside topharmacological therapy

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Thank You!

Questions?

dkahana@gmail.com

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