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Gut Microbiome in the Early Years: What is it doing and should we mess with it?

Gail A.M. Cresci, PhD, RD, LD, CNSC

Faculty Staff, Pediatric Gastroenterology & Inflammation & Immunity

Director of Nutrition Research Center for Human Nutrition

Cleveland Clinic

crescig@ccf.org

Financial support provided by an educational grant from Nestlé Health Science

Objectives

• Describe the pediatric gut microbiome

• Discuss factors that affect gut microbiome during the early years of life

• Define prebiotic, probiotic, synbiotic and evidence for their use in pediatrics

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Gut Microbiome versus Gut Microbiota

• Microbiota: composite organism• Composed of 10-100 trillion microbial cells

• bacteria, viruses, fungi, yeast, parasites

• Microbiome: Genomic catalog of microbiota• Composed of ~3.3 million nonredundant genes

• Terms often used interchangeably

• ~800 species, >7,000 strains

• 30-40 species comprise 90% total

population

• Bacteroides and Firmicutes comprise

90%

Human Gut Microbiota

• Mapped 39 samples from 6 nationalities

• 3 clusters: Bacteroides, Prevotella,

Ruminococcus

• No one “ideal” microbiota

• Limited number of well-balanced host-

microbial symbiotic states

Nature, 2011

Healthy Adult vs Pediatric

A) Phylum level relative abundance via 16SrRNA gene sequencing

B) 16S sequencing

C) Shotgun metagenomics sequencing

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Metabolome

Metabolome: total number of metabolites present within an organism, cell or tissue

Importance of Metabolome

• Every organism strives for metabolic homeostasis

• Produces variety of metabolites to achieve biological balance

• Disease, genetic mutations, environmental factors can disturb biological systems

• Result: Metabolites change

• Can be used as biomarker

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Developmental Origins of Health and Disease

• Early life microbiome is crucial factor for • Proper immune development

• Long-term health

• Transient microbial dysbiosis during this time is associated with• Development of immune-mediated, metabolic and neurodevelopmental disorders

• Colonization begins in utero – Maternal-to-Fetal Microbe Exchange

• amniotic fluid, fetal membranes, umbilical cord, placenta, meconium

• Diversity and richness increase significantly first 2 yrs

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Delivery Mode

• Vaginal – resemble mom’s vaginal microbiota

• Lactobacillus, Prevotella, or Sneathia spp

• C-Section – resemble skin microbes

• Staphylococcus, Corynebacterium, and Propionibacterium spp.

• C-section do not receive first vaginal maternal inoculum –susceptible to certain pathogens

• Atopic diseases, allergies, asthma, skin infection (MRSA)

Diet - Maternal• Maternal diet during pregnancy influences infant microbiome

• Vaginal delivery-• 3 distinct clusters (genera Bifidobacterium, Streptococcus, Clostridium)

• 2.73x w/fruit

• C-Section –• high Bifidobacterium and Clostridium genera; low Streptococcus and Ruminococcus; family

Enterobacteriacea, genus Ruminococcus, and family Lachnospiraceae

• Maternal dairy and odds of clustering Enterobacteriaceae (species E. Coli);

• negative association with general Bifidobacterium, Pseudomonas, and Bacteroides;

• decreased Lactobacillus (associated with dairy allergies)

• Mother-infant dyads (n=26) – high fat maternal gestational diet

• Associated with distinct variations neonatal gut microbes

• Persisted until 4-6 weeks of age

• Animal model – maternal high fiber diet – increased SCFA production offspring

• Increased thymic T-reg cells

• Animal model – maternal high fat, energy, fructose diet – decreased SCFA in offspring

Diet - Infant

• Formula-Fed Microbiome• Enriched in Roseburia, Clostridium, Proteobacteria

• Greater quantities associated with inflammation, more rapid maturation toward adult-type composition

• Breast-fed Microbiome• Enriched in Lactobacillus, Staphylococcus, Bifidobacterium

• Reduced microbial diversity than formula-fed infants

• Decreased diversity associated with increase in genes that degrade oligosaccharides

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Mother’s Milk

Influenced by gestational age at parturition and postnatal age

• Nutrients

• Hormones

• Growth factors

• Immunoglobulins

• Cytokines

• Enzymes

• Endocannabinoids

Nutr Res Rev, 2010

Mother’s Milk –Prebiotics

Oligosaccharides – 3rd largest solid component (> 200 types; ~5-20 g/L)

• Neutral and fucosylated oligosaccharides• Lactose core; elongated by N-acetyllactosamine units;

structural diversity via fucosylation/sialylation

• Formula supplemented with: • FOS, GOS, inulin (structurally different)

• 2’-Fucosyllactose (FL) HMO

• Stimulate growth of bifidobacteria and lactobacilli, reduce pathogen growth

• SCFA pattern similar to breast-fed infants

• Effects on intestinal physiology largely unknown

Bode, Nutr Rev, 2016Bode, Front Pediatrics, 2018

HMO Vs

GOS and FOS

Mother’s Milk - is NOT STERILE!~600 different bacterial species

Country Bacteria Methodology

United States Staphylococcus, Streptococcus, Serratia, Pseudomonas, Corynebacteria

pyrosequencing DNA encoding V1-V2 16SrRNA

Finland Mature milk: Leuconostoc, Weisella, Lactococcus, Staphylococcus predominant mature milkColostrum: Streptococcus

V1-V3 regions

Canada Actinobacter, Stenotrophomonas, Pseudomonas, Streptococcus, Staphylococcus, Lactobacillus

V6 region

Spain* Staphylococcus, Pseudomonas, Streptococcus, Acinetobacter

McGuire, Curr Opin Biotech, 2017

*Median bacterial load ~106 CFU/mLSuggests breastfed infants consume 7-8 x109 CFU/day

Potential Influencing Factors

Methodology

Delivery Mode

Gestational Age

Long-term BMI

Maternal antibiotics

Medical treatments (chemo)

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Antibiotics

• Prenatal maternal antibiotics alter diversity neonatal and maternal microbiotas• Intrapartum antibiotics alter infant oral microbiome composition

• Decreased streptococcaceae and gemellaceae, lactobacillus• Increased proteobacteria phylum• Varied by antibiotic delivery (cocktail vs 1 antibiotic)

• Postnatal antibiotics to infant first 3-9 months• Alter Ruminococcus and Clostridiales• 6-12 months of life associated with decreased maturation infant microbiota

• Epidemiologic studies• Antibiotic-induced dysbiosis promotes noncommunicable disease development later in

childhood and adulthood (obesity, IBD, asthma)• Causality uncertain –

• if diseases related to early life antibiotic use or • reflect early life immune deficiencies or propensity for infections

• Cohort study US DOD TRICARE beneficiaries• 2006-2013• Prescription antibiotics, H2RA, PPI medications in first

2 yr of life• Single event analysis obesity

COX proportional hazards regression

RESULTS• Total 333,353 children

-241,502 antibiotic -39,488 H2RA-11,089 PPI

• Antibiotic associated with obesity(HR 1.26;95% CI 1.23-1.28)

• Persisted regardless antibiotic class• Strengthened with each antibiotic• H2RA and PPI also associated with obesity (30+ d

supply)20

Lifestyle-Induced Variations in Microbiome Composition and Function

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Diet and Gut Microbiota

• Changes in diet influence gut microbial patterns• Decreased fiber (fruits, vegetables, other plants)

• More varied the diet – more diversified gut microbiota

• Globally different among populations and cultures

• Children of Rual Africa vs Western Europe (Italy)

• African children• Breast-fed until 2 yrs, then mainly vegetarian diet

• Low fat and animal protein, high in starch, plant polysaccharides, fiber

• Resources produced locally

• European children• Breast-fed up to 1 yr, then Western Diet

• High in animal protein, sugar, starch, fat; low in fiber

• Gut microbiota analysis – 16S rRNA gene pyrosequencing• 4 phyla represented in both (Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria)

• Proportions varied:

• African: Higher Actinobacteria, Bacteroidetes

• European: Higher: Firmicutes, Proteobacteria

• Africans - Higher total SCFA (butyrate, propionate 4x)• Energy harvest from polysaccharides

DeFilippo, PNAS, 2010

versus

Therapeutic Manipulations of Gut Microbiota

• Eradication of Helicobacter pylori • Manage peptic ulcer disease

• H. pylori-associated gastric lymphoma

• Fecal Microbiota Transplant• Treat recurrent Clostridium difficile infection

• Probiotics

• Prebiotics

• Synbiotics

• Fermented Foods

Fecal Microbiota Transplant• ~90% effective at curing recurrent C. difficile infection

• Restoration of fecal community structure and function

• Source of fecal sample• Individual source, stool banks, frozen encapsulated oral formulas, synthetic microbial and metabolic cocktails

• Donor screening policies vary

• FDA in 2013 issued guidance to practice enforcement discretion of standard IND regulations for refractory CDI – patient consent required

• FMT safety – long-term not yet established• AE – abdominal discomfort, bloating, diarrhea, constipation, transient fever

• SAE – 2% (upper) 6.1% (lower) GI routes

• Data limited in pediatrics

• June 2019 – FDA release potential risk of serious or life-threatening infections with FMT• Bacterial infections caused by multi-drug resistant organisms (MDRO)

• Immunocompromised adults (n=2, 1 died) with FMT developed invasive infections• extended-spectrum beta-lactamase (ESBL)-producing E. Coli

• Donor stool not tested prior, and it contained the organisms

• FMT proposed for IBD, MDRO, allergic colitis, autism spectrum disorder – sparse data, efficacy unclear

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Probiotic

“Live microorganisms which when administered in adequate amounts confer a health benefit on the host”.

Food and Agricultural Org., WHO, 2001

“Good Little Bugs”

Desirable Properties of a Probiotic Strain

Human Origin Species-dependent health effects, maintenance of

viability, applicability to fermented foods

Resistant to acid and bile Survival into the intestine, maintenance of

adhesiveness

Attachment to human epithelial cells Immune modulation, competitive exclusion of

pathogens, good acidity profile

Colonization of the human intestine Survives fecal bacteria, multiplication in the GI tract,

immune modulation

Production of antimicrobial substances Pathogen inactivation in intestine, normalization of

gut flora, good storage stability

Antagonism against pathogenic bacteria Prevention of pathogen adhesion, normalization of

gut flora, sustains processing methods

Safety in food and clinical use Accurate strain identification (genus, species),

documented safety

Clinically validated and documented beneficial effects

Dose-response data for minimum effective dosage

Prebiotics

• Three Necessary Criteria of Ingredient• Non-digestible by host enzymes• Fermented in GI tract • Selective stimulation of gut microbiota and metabolic activity• Demonstration of criterion is difficult

• A prebiotic is not available to all bacterial species that inhabit GI ecosystem• Lactobacillus and Bifidobacterium considered indicator organisms

• Naturally occurring or synthetic sugars, starches• Used as a carbon source by certain colonic bacteria for growth and

metabolism• Examples: Inulin, fructooligosaccharides (FOS), galactooligosaccharides

(GOS), lactulose

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Synbiotic

• Combination of probiotic and prebiotic• Meets criteria of probiotic and prebiotic

• The prebiotic selectively supports the growth of the probiotic component

• General Aim: • Support the probiotic and other indigenous beneficial organisms by

providing a preferred carbon and energy source to promote its growth

• Provide substrate for optimal or desired fermentation byproducts of probiotic

Fermented Foods

• Foods or beverages made through controlled microbial growth and enzymatic conversions of major and minor food components

• Examples:Yogurt, kefir, kimchi, sauerkraut, wine, beer, olives, tempeh,

soy sauce, miso, cheese, sour cream, sourdough bread

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Dietary Carbohydrate and Bacterial FermentationHealthy Gut Microbiota

SLC5A8

MCT

GPR109A

Butyrate

Copyright ©2001 The American Society for Nutrition

Cummings, J. H et al. Am J Clin Nutr 2001;73:415S-420S

Molar ratios of acetate, propionate, and butyrate produced by carbohydrate fermentation of slurries ofmixed human fecal bacteria

Biological Role of SCFAs

• Ion transport (Na+, HCO3-)

• Metabolic fuel source for colonic epithelium

• Modulate intracellular pH and cell volume

• Regulators of • Proliferation• Differentiation• Gene expression

• Butyrate• Contributes to differentiation of epithelial cells• Enhancement of electrolyte and water absorption• Promotes angiogenesis• Modulates immune function• Associated with decreased incidence of colorectal CA and IBD

• Inhibitor of HDACs

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Probiotics, Prebiotics, Synbiotics Biologic Effects and Mechanisms of Action

Strain Specific Mechanisms

ProbioticsGastrointestinal Disorders

• Inflammatory Bowel Disease (IBD)• Crohn’s Disease, Ulcerative colitis

• Pouchitis*• Irritable Bowel Syndrome* (IBS)• Diarrhea*

• Antibiotic Associated• Infectious (e.g., Clostridium Difficile)• Traveler’s

• Helicobacter pylori*• Small intestinal bacterial overgrowth*• Necrotizing enterocolitis*• Pancreatitis*• Liver transplant*

Non-Gastrointestinal Disorders

• Critical Illness*

• Surgery*

• Dermatology*• Eczema

• Urology

• Dentistry*• Dental caries, periodontal diseases

• Gynecology*• Vaginal candidiasis

• Oncology*

• Obesity*

*Positive outcomes – strain specific

Probiotics

• Limitations:• Study Variability

• Small sample sizes• Dosing (method, timing) • Strains provided

• Viability of product• Fermentation substrate lacking• Transient colonization

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Probiotics in Pediatrics

• Treatment acute gastroenteritis

• Prevention antibiotic associated diarrhea

• Prevention of infections

Treatment Acute Gastroenteritis

• Decrease stool consistency (liquid/loose) and/or increase in frequency (>3 stools/d)

• ± vomiting or fever• Duration 7-14 d

• Major causes: rotovirus, norovirus• Treatment: prevent dehydration,

shorten diarrhea duration• Probiotic strains: Lactobacillus GG

and Saccharomyces boulardii + Rehydration Therapy (ESPGHAN)

• LGG reduced mean duration (27 h) 1, 2

• S. boulardii reduced risk of diarrhea lasting >4d 1,3

• L. reuteri DSM 17938 (3 RCTs) –reduced diarrhea duration (32 h) 4,5

• No effect on stool volume

1) Allen, Cochrane Database Syst Rev 2010(11) CD0030482) Szajewska Aliment Pharm Ther 2013;38:467-763) Dinleyici, Expert Opin Biol Ther 2012;12:395-4104) Szajewska Benef Microbes 2014;5:285-93.5) Dinleyici, J Pediatr 2015;91:392-6.

Prevention Antibiotic Associated Diarrhea

• Diarrhea occurring in relation to antibiotic treatment

• Antibiotics targeting anaerobic bacteria & disrupt microbiota

• Clindamycin, penicillin, amoxicillin, clavulanic acid, etc

• Mild diarrhea to pseudomembranous colitis (C. difficile)

• Limit inciting antibiotic, probiotics

• Probiotics: LGG, S. boulardii (ESPGHAN)

• LGG – 5 RCTs (n=445)• Reduced risk AAD from 23% to

9.6% 1

• No effect on CD (1 trial tested) 2

• S. boulardii – 6 RCTs (n=1653) 1

• Reduced risk AAD from 20.9% to 8.8%

• Reduced risk CDI – 2 RCTs (n=579)

1) Szajewska, J Ped Gastro Nutr 2016;62:495-5062) Arvola, Pediatrics 1999;104:e64

Ideal Candidates for Probiotics with AntibioticsChildren who:• Are Younger aged • Hospitalized• Experienced AAD or CD-associated diarrhea in past

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Infection PreventionDay Care Centers

• Respiratory and GI infections

• Good hand hygiene, absenteeism of ill child, vaccination for influenza and rotavirus

• Probiotic: LGG (ESPGHAN) – no cost-effective analysis

Respiratory infections

• LGG – 3 RCTs (n=1375)- positive 1-3

• B. animalis subsp. lactis – 4 RCTs -negative 2,

• Inconclusive data for GI Infections

Ideal Candidates for Probiotics Children who:• Are Younger aged • Experience recurrent respiratory infections in winter

months

1) Hatakka, BMJ 2001; 322:3272) Hojsak, Clin Nutr 2010; 29:312-63)Kumpu, Eur J Clin Nutr 2012;66:1024) Merenstein Eur J Clin Nutr 2011;65:447-535) Merenstein, Eur J Clin Nutr 2010;64:669-776) Merenstein, Eur J Clin Nutr 2010, 64:685-91-3

Infection PreventionNosocomial Infections

• Develop during hospital stay within 48 hr

• 5-10% GI and respiratory incidence

• Hand hygiene, isolation

• Probiotics: LGG (ESPGHAN)

• Nosocomial Diarrhea• LGG – 3 RCTs

• Reduced risk 13.9% to 5.2%

• L. reuteri DSM 17938 – negative results

• Nosocomial Respiratory Tract Infections (RTI)

• LGG – reduced risk upper RTI

• B. animalis subsp. lactis, no benefitIdeal Candidates for ProbioticsChildren who:Have extended hospital length of stay

Prebiotics

GI Disorders

• Colic

• Functional Constipation

• Functional abdominal pain & Irritable bowel syndrome

Overall Study Conclusions

• Colic – limited studies, some benefit with prebiotics

• FC-Studies highly heterogeneous• Trend towards softer stools

• FAP/IBS – no RCTs • FODMAP – small studies, short-

term

• but show benefit

Fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAP)

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Summary

• Evidence for gut microbiota affecting health

• Demonstration of causality of microbiota and disease state• Animal models show for some disease states (obesity)

• A better understanding of gut microbiome development in healthy versus diseases needed

• Modification of gut microbiota via diet or nutritional supplements to improve microbiome of high interest

• Targeted approach needed

• Personalized approach – algorithms for predictive responses to given food/supplement based on their microbiome