preterm gut microbiome disease outcomes and nutrient ... · disease outcomes and nutrient...

Preterm gut microbiome

disease outcomes and nutrient

interactions

Dr Nicholas Embleton, Consultant Neonatal Paediatrician,

Newcastle upon Tyne, UK

Neonatal Conference, Palais des Papes, Avignon

September 2017

www.neonatalresearch.netImproving outcomes

Conflict of Interest

• Research funding

– UK National Institutes for Health Research

– Nestle Nutrition, Danone/Nutricia, Prolacta Bioscience

– Charities (Tiny Lives, Special Trustees)

• No shares, financial holdings


Summary

• Gut microbiome in early life

• Gut function & dysbiosis

• Interactions between nutrients and microbes

• Milk nutrients – lactoferrin, HMOs etc

www.neonatalresearch.net

Concepts & ideas

“Data light” !

Our bodies are full of microbes

• 80% antibody producing cells located

in gastrointestinal tract

• Gut = most important part of the

immune system

• Microbial > human cells

• Microbial >>> human genes

Are we human?

We have more than 1000 different types of

micro-organisms living inside our bodies

We have more than 300x as many

microbial genes as human genes

We are full of microbes

• Humans are a “superorganism”

• Many metabolic & immune processes

• Human development impossible

without microbes

• ~1.5kg microbes

…... si usted está preocupado sobre su peso no importa: 1.5kg no es usted!

World evolution (4.5 billion years) into

one month …...

• Most of month: only microbes

• Animals & plants appear - 27th

• Humans appear – last day, last hourAt least 65% of

human genome

evolved in microbes

Rook et al. 2017

Human evolution & development:

“tug of war”

Identical to challenge for neonatal gut:

Tolerance v Activate immunity

Rook et al. 2017

Manage beneficial microbes

Exclude harmful microbes


Strong evidence that early life

microbiome exposures are important

Exposure Outcomes

C-section

Asthma, Arthritis, IBD, immune

deficiencies, leukaemia, obesity,

BMI, food allergies

Antibiotics

Asthma, allergy, eczema,

overweight, obesity, IBD, weight gain

Pets Reduced risk preclinical type 1

diabetes

Tamburini et al. 2016

Body site: different microbiomes

Firmicutes• Staphylococcus

• Lactobacilli

• Clostridia

Actinobacteria• Bifidobacteria

Proteobacteria• E coli

Bacteroidetes• Bacteroides

• Prevotella

Colon

Stomach

Vagina

Skin

Hair

Oesophagus

Oral

Nose

Microbiomes – vary with

geography, history, diet, age …..

Embleton 2009Credit: Science 2005

Gut – just for nutrition?

Drink milk → absorb nutrients → make stool?

Fluid Nutrients

Food Waste

Gut – The Inner Tube of Life

Fluid Nutrients

Immune Hormones Microbes

Metabolites

FoodBreast milk: developmentally regulated maternal-infant biochemical

signalling pathway


Focus on preterm baby in NICU

• What affects the microbiome = ‘exposures’

• How to increase/decrease risks of disease

• How do we

– Promote ‘healthy’ growth

– Decrease risk of NEC, sepsis etc.

What are the interactions between nutrients,

metabolites & microbes?

Nutrients = food component used as building block for tissue e.g. AA

Immunonutrients = food component with function e.g. lactoferrin

Metabolite = may have functional effect e.g. signalling molecule


Gut interactions: nutrition, microbes

and immunity = gut ‘health’

Nutrients

Immune system

Microbes


Early colonisation is key life event



“Pioneer” species

sustain low oxygen

environment

Anaerobes – most abundant early on

• E coli

• Bifidobacteria

• Bacteroides



“Pioneer” species

sustain low oxygen

environment

Anaerobes – most abundant early on

• Bifidobacteria

• Bacteroides

“2nd wave” of colonisation

Human life-course: baby to adult

Rook et al. 2017

Bacteroides

Bifidobacteria

E coli

Clostridia

Faecalibacterium &

Eubacterium

Week 1 Solid food Weaning Adult


Early colonisation: abnormal in preterm infants

1 2 3


Preterm microbiota are different

from term infants

• Increased

– Firmicutes e.g. Staph, Enterococcus

– Enterobacteriaceae e.g. E. coli & Klebsiella

– Propionibacterium

• Decreased

– Bacteroidetes

– Bifidobacterium

– Lactobacilli

More ‘extreme’

differences in

preterm infants who

develop NEC

Dysbiosis precedes NEC – association

or causation?

?


NEC: breakdown of interactions between

microbes, metabolites & immune system

NEC

Nutrients

Immune system

Microbes

NEC

Gastrointestinal dysfunction

Injury Repair

Exogenous

Endogenous

Gastrointestinal dysfunction

Injury Repair

Exogenous

Endogenous

•Pathogens

•Food antigens

•GI secretions

•Microbes

•Immuno-nutrients

•Growth factors (EBM)

•Protective factors - sIgA

•Hypoxia

•Vascular

•Poor nutrition

•Immune dysfunction

•Good nutrition

•Motility

•Immune cell cross talk

•Systemic hormones

Unique properties of mothers’

own milk

Injury Repair

Exogenous

Endogenous

•Pathogens

•Food antigens

•GI secretions

•Microbes

•Immuno-nutrients

•Growth factors (EBM)

•Protective factors - sIgA

•Hypoxia

•Vascular

•Poor nutrition

•Immune dysfunction

•Good nutrition

•Motility

•Immune cell cross talk

•Systemic hormones

Milk ‘nutrients’

Healthy gut

Adapted from Tamburini et al. 2016

• Diverse microbes

• Presence of ‘healthy’ microbes

• Absence of ‘un-healthy’ microbes

Healthy gut


• Diverse microbes

• Presence of ‘healthy’ microbes

• Absence of ‘un-healthy’ microbes

• Healthy mucus layer

• Active/receptive epithelium

• Healthy blood flow/oxygenation

Dysbiosis


Dysbiosis


Loss of microbial diversity & key taxa

Increasing ‘pathogens’

Dysbiotic

Thinning &

disruption of

mucus layer


Epithelial damage

Microbial metabolites

Changing metabolites (function)

Anti- Pro-

Loss of microbial diversity & key taxa

Increasing ‘pathogens’

Dysmetabolic

Thinning &

disruption of

mucus layer


Epithelial damage

Changing metabolites (function)

Anti- Pro-


Epithelial development in gut – need to learn to …..

Commensal Pathogens

Develop tolerance Activate immune system

• Microbiota are key determinant of gut health

• Modulate effects via metabolites & nutrients


What is going on in the gut lumen?

Gut microbial

communities

Nutrients &

metabolites


Milk nutrients promote microbes

Supplements?

Gut microbial

ecology

Amino acids

Polyamines

Urea Lactoferrin

Fatty acids

Lactose

HMOsHuman milk

oligosacharides

RCT of iron supplementation: term

infants in Kenya

E coli / Shigella Clostridia

Firmicutes Bifidobacteria

Jaeggi et al. 2015)

Term infants – not preterm

Example


Summary

• Microbiota patterns associated with disease (NEC,

sepsis) etc.

• Nutrient intake affects

– Microbiota patterns

– Gut health in other ways e.g. EGF

• Microbiota exert effect via different mechanisms

– Nutrient & metabolite production


Microbes produce key nutrients

Gut microbial

metabolismBile acids

Amino acids

Phenols

Choline

Short chain fatty acids

e.g. butyrate

Vitamin B, K


Microbes produce much more than nutrients

Gut microbial

metabolism

Signalling

molecules

Anti- or Pro-inflammatory

molecules

Nutrients, peptides etc.

~50,000 metabolites:

we don’t know what

they do or we cannot

yet identify


Neonatal microbiome - function

• Interested in microbial patterns

• But more important is what microbes do

– What are the microbial products

– How do they interact with our cells

• Epithelial health & stimulate immune system

• Produce signalling molecules e.g. for brain

What is relationship between microbes & metabolism?


Host-microbe metabolic axes

Host-microbe metabolic axes “multi-directional interactive chemical communication highway”

J K Nicholson et al. Science 2012;336:1262-1267

Gut microbesMetabolic &

immune function

Examples of microbially derived metabolites

J Nicholson 2012


Short chain fatty acids (SCFAs)

Clostridia,

Eubacteria

RoseburiaHMO, lactate,

linoleic acid

etc.

Gene expression: multi-organ targets

• Promotion of Treg cells

• Cytokines

• Antimicrobial peptides

• Mucous production

• Gut brain axis

• Etc.

SCFAs


Choline

Clostridia ↑Choline

compounds

Complex role• Cell membrane• Neurotransmitter• Methyl donor

Trimethylamine OxideBacteroidetes ↓

TMAO increased risk

adverse cardiovascular

events


Summary schema

Different types of

bacteria which…Dietary

nutrients

affect…. Differences in pattern

of metabolites……

Patterns of health

and disease


The potential range & complexity is enormous

>500 bacterial

species in pretermBreast milk

proteins,

HMOs, FAs>50,000 metabolites

Every baby is

unique


How do breastmilk nutrients affect

microbiome & gut health in early life

• Major challenge – almost every study is observational

– Control for other factors

– Causation v association

– ..does it matter?

• Lactoferrin

• Human Milk Oligosaccharides (HMOs)


Human milk proteins: mucin, casein

& whey

• Mucins = milk fat globule membrane (MFGM) proteins

– small % but important functional activity

• Early lactation – mainly whey protein (esp. lactoferrin)

– Casein virtually undetectable during first day

– Milk concentration increases as synthesis develops

• No “fixed” ratio of whey to casein in human milk

– 80:20 early to 50:50 late

– Therefore, amino acid content also changes


Lactoferrin

• Antimicrobial glycoprotein

• Colostrum, breast milk, tears, saliva

• Acid proteolysis lactoferricin

Enteral lactoferrin in Neonates

Lonnerdal et al. 2015

Lactoferrin - high in colostrum

g/100mL

0.5

0Cow

Mature

human

Colostrum

Lactoferrin

concentration

Structure is highly

conserved


Lactoferrin functions

• Lactoferrin → lactoferricin

• Direct antimicrobial effects

– bacterial, viral, fungal

• Modification of host immune response

• Direct epithelial effects

• Decreased sepsis & possibly NEC

– (Manzoni et al 2009)

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Lactoferrin- antimicrobial actions

Cell membrane disruption

Iron sequestration

Inhibition of microbial adhesion

Prevention of biofilm formation

Lactoferrin in human milk is key part of our immune

system – but preterm babies do not get much milk

If lactoferrin is similar - can we get benefit from

bovine lactoferrin?


Enteral lactoferrin in Neonates

(ELFIN)

• Large NIHR collaborative RCT

• 25 NICUs across UK

• Recruited 2150 preterm infants so far (total = 2200)

• Outcome = sepsis & NEC

LactoferrinDisease

NEC

Sepsis

Does lactoferrin reduce sepsis?


Mechanisms Affecting the Gut of Preterm

Infants in Enteral feeding studies

• Embedded mechanistic study = MAGPIE

• Explore microbiome-metabolome (n = 480/2200)

Lactoferrin

Immune

Microbiota

Disease

NEC

Sepsis

How does

lactoferrin reduce

sepsis?

RCT of human recombinant lactoferrin

Sherman et al. (2016) Tal-Lf decreased

• Staph in stool

• Infections with Staph

• Enterobacter hormaechei

• (?ID issue…)

• ..but more Citrobacter spp

HMOs: evolutionary advantages of

breast milk composition

HMOs3rd largest component

1. Lactose 70g/L

2. Fat 40g/L

3. HMOs 10g/L

We lack glycosidases to

cleave HMO linkages

Underwood et al. Peds Res 2015

Evolutionary advantages of breast

milk composition

• Bifidobacterium

• Bacteroidetes

HMOs

Lack glycosidases to

cleave HMO linkages

• Distal small intestine

• Colon



Oligosaccharides and Bifidobacteria

• >100 HMOs – unique to breast milk

• >30 species of Bifidobacteria

– Utilise HMOs differently

– Different effects

• HMOs – direct epithelial/signalling i.e. not just microbiome

Relationship between HMOs & microbiome is very

complex


Bifidobacterium longum subspecies infantis:

champion coloniser of the infant gut.


Human Milk Oligosaccharide structure is

key to Bifidobacterium growth & function


Human milk has high degree of

oligosaccharide polymerization


MammalsPrimatesHumans

70% fucosylated

<20% sialylated >70% sialylated

<5%% fucosylated

GalactoseN-acetylglucosamine Glucose

N-acetylneuraminic

acid

Fucose


Human milk has higher degree of

oligosaccharide polymerization


MammalsPrimatesHumans

70% fucosylated

<20% sialylated >70% sialylated

<5%% fucosylated

GalactoseN-acetylglucosamine Glucose

N-acetylneuraminic

acid

Fucose


HMOs

• Unique structures in humans

• Individual differences >100 different structures

• Important for growth of Bifidobacteria

• HMOs: key role in immunity & related to risk of

necrotising enterocolitis (NEC)


Human milk: high degree of oligosaccharide

polymerization

Humans

GalactoseN-acetylglucosamine Glucose N-acetylneuraminic

acid

Fucose

• >100 different HMOs identified

• Ethnic/genetic factors

• ‘Secretor’ status (Lewis blood group)

• Each mother’s pattern unique

o 5-10 HMOs dominate

o Many more in lower concentrations

DSLNT

LNT

LSTb

3’FL

3’SL

Etc………….


HMO composition predicts risk of NEC

in preterm infants.

• 200 VLBW infant/mother pairs

• Expressed breastmilk (EBM) collected

• HMO concentration in EBM fed to infants 0-28 days

• Matched 8 NEC cases to 40 controls (5 controls/case)

• DSLNT abundance identifies NEC cases prior to onset

Autran et al. 2017

DSLNT level (ug/mL)

NEC Bells 3

NEC Bells 2

Bells 2/3Bells 1Control

DSLNT in babies with NEC is

much lower

Bells 2/3Bells 1Control

On each day [DSLNT] lower in those

babies who subsequently developed NEC

NEC Bells 3 NEC Bells 2

• Breastmilk sample collected

each day

• Grey circle shows DSLNT

HMO concentration in baby

who stayed well

• Red and yellow – show

concentration of DSLNT

HMO in the EBM from

babies who got NEC

Babies who got NEC had

always received breastmilk

with the lowest amount of

DSLNT

abundant HMO, which represent > 95% of the HMO in human

milk. In this panel, additional HMO (ie, LNFP1 and DFLNT)

were also found to provide a lesser contribution. It is important to

note that we did not measure total milk volume fed to each infant

per feeding or per day. The analysis purely focuses on HMO

concentrationsand not on absolute HMO amountsreceived.

The role of DSLNT and other HM O were robustly quantified

using GEE. DSLNT deficiency is significantly associated

(p= 0.001) with NEC onset with an OR of 0.84. LNFP1 and

DFLNT were also found to have a significant protective

(OR 0.91) and harmful (OR 1.14) contribution, respectively

(figure 3C). These contributions were stable across all

Figure 2 Disialyllacto-N-tetraose (DSLNT) concentrations are uniquely and consistently low in necrotising enterocolitis (NEC) cases (left) whencompared with controls (right). Samples in each row are case-control matched by study site, gestational age, birth weight and other NEC-relevantfactors. For each milk sample collected over the first 28 days post partum, the fold difference of human milk oligosaccharides (HMO) concentrationrelative to the associated matched control sample average is illustrated for DSLNT (A), sialyllacto-N-tetraose (LSTb) (B), lacto-N-tetraose (LNT) (C)and the sum of all integrated HMO(D). DSLNTwas lowest in cases with a Bell stage of 3 and 2 at concentrations an order of magnitude lower thanmatched control averages. Bell stage 1 cases showed slightly lower concentrations than their matched controls. Structurally similar HMOs withreduced sialylation, such as LSTb and LNT, failed to exhibit consistent variations in concentration in NECcases compared with matched controls.Number in parentheses after case codes denotes NECBell stage. (*) denotes the day of NEConset, (+) denotes the day of death due to NEC.Oligosaccharide structure nomenclature: blue circles: glucose; yellow circles: galactose; blue squares: N-acetylgucosamine; purple diamonds: sialicacid.

Autran CA, et al. Gut 2017;0:1–7. doi:10.1136/gutjnl-2016-312819 5

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group.bmj.com on April 5, 2017 - Published by http://gut.bmj.com/Downloaded from

Autran et al. 2017















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Similar structure HMOs -

difference is one sialic acid residue

Autran et al. 2017

Associated with lower NEC

Not associated with lower NEC















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multivariate models (see online supplementary table S1).

Furthermore, these HM O were consistently dysregulated in

NEC cases. When considering dysregulation over multiple con-

secutive days, the separation between cases and controls

increased (see online supplementary figures S2–S4), suggesting

that prolonged dysregulation of HM O is more indicative of

NEC onset. Interestingly, NEC Bell stage 1 did not correlate

with DSLNT deficiency, supporting the lack of specificity of Bell

stage 1 in diagnosing NEC or suggesting that DSLNT deficiency

only impacts infants’ risk for more advanced NEC.

The underlying mechanisms of how HMO such as DSLNT

attenuate NEC risk remain to be elucidated. Although HMO have

profound effects on infant microbiota composition,18–20 the

importance of microbiota composition on NEC onset and devel-

opment is poorly understood.21–26 Whether microbial dysbiosis is

a causative event or merely a marker of intestinal disease remains

unknown.27 Instead, HMO may have direct effectson infant intes-

tinal epithelial or immune cells, which might directly attenuate

NEC risk, and also indirectly alter microbiota composition. The

observation that the effects of DSLNT are highly structure-specific

(removal of just onesialic acid renderstheoligosaccharide ineffect-

ive in neonatal rats11 and these truncated oligosaccharides are no

longer associated with NEC risk in the cohort study) indicates a

potentially receptor-mediated mechanism.

The study recruited 200 mothers and their VLBW infants, of

which 8 (4%) developed NEC Bell stage 2 or 3. NEC incidence

in VLBW infants in North America typically varies between < 5

and up to 10%, but that includes both human milk-fed as well

as formula-fed infants. Since NEC incidence is 6-fold to 10-fold

lower in predominately human milk-fed infants compared with

formula-fed infants,3–5 the 4% NEC incidence reported in this

study is well within the anticipated range.

While the results from this study indicate that higher DSLNT

concentrations in mother ’s milk lower the infant’s risk to

develop NEC, larger cohort studies with more detailed maternal

data will be needed to identify maternal factors (genetics, nutri-

tion, stress, etc) that influence DSLNT synthesis.

Although selection bias is a common limitation of case-

control studies, this has been minimised in two ways: first by

Figure 3 Univariate logistic regression screening of (A) birthcharacteristics (ie, birth weight and gestational age) were effectivelycontrolled through case-control matching, and therefore, no clinicalcovariate showed a significant association with necrotising enterocolitis(NEC). (B) Univariate temporal logistic generalised estimating equation(GEE) screening of human milk oligosaccharides (HMOs) revealedseveral candidate associations, with disialyllacto-N-tetraose (DSLNT)being the predominant HMO. (C) The final multivariate temporal GEEmodel demonstrated that DSLNT, lacto-N-fucopentaose (LNFP1) anddifucosyl-LNT (DFLNT) each contribute significantly to a finalmultivariate model. The ORis the exponentiated coefficient and the95% CI describes the range of possible OR. For panel A, the p valuerepresents the significance based on the χ2 distribution, while p valuesin panels B and Cwere calculated from the Wald statistic of eachcoefficient, evaluated along a normal distribution.

Figure 4 Aggregation of disialyllacto-N-tetraose (DSLNT)concentration for multiple days enhances the identification of high-riskinfants. Infants who will develop necrotising enterocolitis (NEC) aremore readily identifiable when DSLNT concentration from multipleconsecutive milk samples for each subject is aggregated using thegeometric mean. When we combine the computed odds for 2, 4 and 6consecutive milk samples from the same individual, separation of casesand controls increased and variance in the average odds decreased.

6 Autran CA, et al. Gut 2017;0:1–7. doi:10.1136/gutjnl-2016-312819

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OR for developing NEC: univariate

OR for developing NEC: multivariate

Autran et al. 2017


HMO composition predicts risk of NEC

in preterm infants: interpretations

1. DSLNT content in breastmilk associated with NEC

2. HMO functions are highly specific

3. HMOs involved in non-microbiomic mechanisms

4. Relationship: HMO & Bifidobacteria is complex

5. >30 different Bifido species: functional effects differ


Is NEC the only important outcome of microbiome?

NEC


We need to consider ‘gut health’ -not only NEC

NECFeed toleranceFull feedsDuration of PNAntibioticsSepsisGrowthMetabolismCognition

That’s enough theory…anything

practical for 2017?

Gut microbial

metabolism

Signalling

molecules

Anti- or Pro-inflammatory

molecules

Nutrients, peptides etc.

~50,000 metabolites: we

don’t know what they do

or we cannot yet identify

Gut microbial

ecology

Amino acidsUrea

Lactoferrin

Fatty acids

Carbohydrates

HMOs

Human milk

oligosacharides

Nutrients

Immune system

Microbes

NEC

lactoferrin HMOs

NEC

BrainMetabolismSepsisFeeding etc.

Final slide - 8 practical ways to improve

gut health in preterm infants

?

Educate

Minimise

Promote

Breastmilk day 0

Encourage

Reflect

Think about NICU

Audit, guidelines etc.

Practice

Avoid

preterm gut microbiome disease outcomes and nutrient ... · disease outcomes and nutrient...

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