gut microbiota in infants: health, disease & nutrition · gut microbiota in infants: health,...

Gut Microbiota in Infants: Health,

Disease & Nutrition

Dr Nicholas Embleton

Consultant Neonatal Paediatrician, Newcastle, UK

UNICEF BFI annual meeting

Telford, UK

November 2017

www.neonatalresearch.netImproving outcomes

Conflicts of interest

• Research funding

– NIHR – HTA, EME

– MRC, Charities (Bliss, Tiny Lives)

– Industry - Nutricia Research, Prolacta Bioscience US

• I hold no shares, financial benefits etc.

• Views do not represent those of employers / affiliated organisations

– UK Neonatal Nutrition Network (N3)

– Committee of Nutrition, ESPGHAN

– BAPM working group on Donor Breast Milk


Newcastle Neonatal Research Team

Research focus: www.neonatalresearch.net

Gut function, nutrition & microbiome

Long-term metabolic outcome

Work with parents: www.neonatalbutterflyproject.org

http://www.neonatalresearch.net

http://www.neonatalbutterflyproject.org

www.neonatalbutterflyproject.org

Films with parents who suffered loss of a co-twin baby

Free to download guidelines, resources, teaching packs

Embleton 2009


Necrotising enterocolitis

• 10% of infants born <30 weeks

• Fear-of-NEC dominates early feeding

• More deaths from NEC than all childhood leukaemia

• Overwhelming evidence: mother’s own breastmilk

reduces the risk of NEC

• Single, most important reason to make provision of

mother’s own milk focus of neonatal careBut it’s not just NEC


Longer term benefits – major importance

for term and preterm infants

Lower rates of

• Obesity, leukemia & diabetes

• Stroke, heart attack & hypertension

• Osteoporosis and fractures in later life

• …and less cancer & osteoporosis in the mother!

And better

• Brains – higher IQ, employment, income …

Is breast milk ‘food’?

Drink milk → absorb nutrients → make stool?

Fluid Nutrients

Food Waste

Breast milk: allows the mother to

signal to the baby

Fluid Nutrients

Immune Hormones Microbes

Metabolites

Food Breast milk is more than food


Microbes acquired from mother and breast

milk are important

Nuestros cuerpos están llenos de microbios

• 80% antibody producing cells located

in gastrointestinal tract

• Gut = most important part of the

immune system

• More microbial > human cells

• Microbial >>> human genes

Are we human?

>1000 different types of micro-

organisms living inside our bodies

300x as many microbial as human genes

We are full of microbes

• Humans are a “superorganism”

• Many metabolic & immune processes

• Human development impossible

without microbes

• ~1.5kg microbes

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 microbesRook 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


What is the microbiome?

• Microbiome = all microbial life and genes

• Microbiota = microbes in specific site e.g. gut

• Microbiome affects vast range of diseases

• Early life microbiome has long-lasting effects

Human life-course: baby to adult

Rook et al. 2017

Bacteroides

BifidobacteriaE coli

Clostridia

Faecalibacterium &

Eubacterium

Week 1 Solid food Weaning Adult


Gut microbiota – health & disease

• New area of scientific study

• Understanding limited – especially in preterm infants

• Most gut microbes cannot be cultured

• Scientific advancement since the 1980’s

– Molecular techniques

– Revealed complexity of the microbiome

– Provided new insights into health & disease


What is evidence that early life

microbiome exposures are important?Exposure Numbers & ages Outcomes

C-section 1.9 million (0-15 years)

2,803 (0-2 years)

Asthma, arthritis, IBD, leukemia,

obesity, food allergies

Antibiotics 163,820 (2-18 years)

9 million (0-18 years)Asthma, allergy, eczema, obesity, IBD

Iron 139 (6-14 years) Intestinal inflammation

Pets3,143 (0-1 years) Reduced risk preclinical type 1 diabetes

Tamburini et al. 2016

What about ‘hygiene’ hypothesis for later

allergy: Adults who clean baby’s pacifier

• n=184; reviewed at 6 months

– Cleaned by sucking

– Did not suck/clean

• Oral microbiota at 4 months

– Significantly different

• Lower risk of asthma/eczema

– 18 months & 36 months

Hesselmar et al. 2013

Body site: different microbiota

Firmicutes• Staphylococcus

• Lactobacilli

• Clostridia

Actinobacteria• Bifidobacteria

Proteobacteria• E coli

Bacteroidetes• Bacteroides

• Prevotella

Colon

Stomach

Vagina

Skin

Hair

Oesophagus

Oral

Nose

Not only bacteria

Bacteria

Fungi

Viruses

Bacterio-phages

Body sites – not only bacteria

Microbiota – vary with geography,

history, diet, age …..


How do we study the microbiome?


1980’s – 2010’s: new molecular techniques

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*** *** ****** ****** *** ****** ***

4%

16%

36%

64%

100%

K.oxytocea

E.coliE.faecalis

S.aureus

Bifidobacter ium

Clostr idium_sensu_stricto

Streptococcus

Lactobacillus

Morganella

Veillonella

Relat

ive A

bund

ance PGSTletter

A

B

C

D

E

F

123456

A B

C

StatusPGCT

PGCT

Culture Genetic sequencing

(16s microbial genes)Typical result

• 3-4 species

• Light v heavy growth

Typical result

• >100 species

• Relative % (not absolute)


Challenges: massive datasets

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Adj. P = 2.9e−61


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40

50

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1

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Alph

a Di

vers


1 2 3 4 5 6 1 2 3 4 5 6PGCT PGCT

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*** *** ****** ****** *** ****** ***

4%

16%

36%

64%

100%

K.oxytocea

E.coliE.faecalis

S.aureus

Bifidobacter ium


Streptococcus

Lactobacillus

Morganella

Veillonella

Relat

ive A

bund

ance PGSTletter

A

B

C

D

E

F

123456

A B

C

StatusPGCT

PGCT

Culture Genetic sequencing

(16s microbial genes)• Not enough detail

• Does not identify most

bacteria

• ‘Expensive’

• Too much information

• Identifies bacteria we

do not ‘know’

• Complex to analyse


Challenge: defining ‘normal’ e.g. urine culture

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Adj. P = 2.9e−61


0

10

20

30

40

50

0

1

2


Alph

a Di

vers


1 2 3 4 5 6 1 2 3 4 5 6PGCT PGCT

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*** *** ****** ****** *** ****** ***

4%

16%

36%

64%

100%

K.oxytocea

E.coliE.faecalis

S.aureus

Bifidobacter ium


Streptococcus

Lactobacillus

Morganella

Veillonella

Relat

ive A

bund

ance PGSTletter

A

B

C

D

E

F

123456

A B

C

StatusPGCT

PGCT

• Urinary tract infection

What is ‘normal’

• Sterile

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● ●

●●●●

●

Adj. P = 2.9e−61


0

10

20

30

40

50

0

1

2


Alph

a Di

vers


1 2 3 4 5 6 1 2 3 4 5 6PGCT PGCT

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*** *** ****** ****** *** ****** ***

4%

16%

36%

64%

100%

K.oxytocea

E.coliE.faecalis

S.aureus

Bifidobacter ium


Streptococcus

Lactobacillus

Morganella

Veillonella

Relat

ive A

bund

ance PGSTletter

A

B

C

D

E

F

123456

A B

C

StatusPGCT

PGCT

●

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Adj. P = 1.7e−13

● ●

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Adj. P = 2.9e−61


0

10

20

30

40

50

0

1

2


Alph

a Di

vers


1 2 3 4 5 6 1 2 3 4 5 6PGCT PGCT

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*** *** ****** ****** *** ****** ***

4%

16%

36%

64%

100%

K.oxytocea

E.coliE.faecalis

S.aureus

Bifidobacter ium


Streptococcus

Lactobacillus

Morganella

Veillonella

Relat

ive A

bund

ance PGSTletter

A

B

C

D

E

F

123456

A B

C

StatusPGCT

PGCT

●

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● ●

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●

Adj. P = 2.9e−61


0

10

20

30

40

50

0

1

2


Alph

a Di

vers


1 2 3 4 5 6 1 2 3 4 5 6PGCT PGCT

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*** *** ****** ****** *** ****** ***

4%

16%

36%

64%

100%

K.oxytocea

E.coliE.faecalis

S.aureus

Bifidobacter ium


Streptococcus

Lactobacillus

Morganella

Veillonella

Relat

ive A

bund

ance PGSTletter

A

B

C

D

E

F

123456

A B

C

StatusPGCT

PGCT


Microbiota patterns: how different are

two samples?

• Trillions of bacteria from hundreds of different species

• Use a Principal Coordinates Analysis (PCA) plot

• Statistically very complex but

– “Spreads out” the data

– Each dot = a bacterial community (millions of bacteria)

– Dots close together = community ‘pattern’ is similar

• i.e. Similar types & proportions of bacteria


Bacteria live in communities, just like

animals

Based on idea from Rob Knight’s TED talk


Community looks similar: a little more or less;

pattern characteristic of that niche


These communities are very different: they

come from different ecological niche


Same data – use a circle to represent a

community


Replace the animals with colour circles:

each circle represents a ‘community’


Circles that are close are more similar

Each circle represents a community

?


Communities different: this is what a PCA

plot looks like

?

Mouth

Stool

Skin on arm

Genital

Example of a PCA plot: oral microbiota in

infant

Interpretation: PCA plot show ‘community’ is different

Does not tell us ‘how’ they are different

Points that are further apart are ‘more different’

Suck to cleanDo not suck

Delivery mode important in term infants

Dominguez-Bello et al.

Vagina

Oral

Skin

Mother’s microbiome

Mother

microbiome

Delivery mode important in term infants

Dominguez-Bello et al.

Vagina

Oral

Skin

Mother’s microbiome

Baby microbiome

Vaginal delivery

C-sectionBaby stool

microbiome

Mother

microbiome


Early colonisation is key life event


Early colonisation is key life event

“Pioneer” species

sustain low oxygen

environment

Anaerobes – most abundant early on

• Bifidobacteria

• Bacteroides

“2nd wave” of colonisation

Another PCA plot – adult microbiomes

Credit: Rob Knight (TED talk)

At birth – baby stool microbiome reflects delivery

method (vaginal)


Baby stool

At birth – baby stool microbiome reflects

delivery method (vaginal)


Baby stool

By 2 years age –

resembles adult stool

Effect of antibiotics


Baby stool

How might breast milk modify the

microbiota?


PROBIT trial

• PROmotion of Breastfeeding Intervention Trial

• Cluster randomization: 29 hospitals

• 17,046 infants - hospitals were BFI or control

Kramer et al. JAMA 2001; 285: 413-20


PROBIT trial

• Key results:

– Reduced GI infections (OR 0.60)

– Higher verbal IQ at 6.5 y (+7.5)

Kramer et al. JAMA 2001; 285: 413-20

Kramer et al. AJCN 2007; 86: 1717-21

Kramer et al. BMJ 2007; 335: 815

Kramer et al. Arch Gen Psychiatr 2008; 65: 578

Martin et al. Circulation 2014


Breastfeeding and infections

in high-income countries

• Breast feeding reduces risk of

– Acute otitis media

– Respiratory tract infections

– Diarrhea

• i.e. Not just due to poor sanitation…

Bowatte G. Acta Paediatrica 2015; 104: 85-95

Uhari M. Clin Infect Dis. 1996;22:1079


Breast milk is not just food

• It contains everything a baby would need in a food

– Protein, Carbohydrates, Fat, Water, Minerals,

Vitamins, Micronutrients etc.

– You can get similar nutrients in formula milk

But breast milk FUNCTIONAL

COMPONENTS are a key reason

it is better for babies


What are the key “anti-infective”,

functional or microbiota components?

• Multiple potential components

• Difficult to ‘prove’ which individual nutrients most

important – breast-milk comes as a package

• …and it may not matter

• But, how did breast-milk evolve anti-infective properties

• Did early humans die from infections all the time?


There are literally 1000’s of functional

components to explore

• Millions of years ago – before mammals & humans

• Breast-milk started to evolve when ‘we’ laid eggs ?!

• Early “milk” provided functional components & no nutrition

Evolution of breast milk in one slide!

Dehydration Anti-fungal Broad antimicrobial

Placenta Growth constraints Bipedalism

Complex society - slow growth Husbandry / civilization


What components might decrease risk of

NEC or infections?

• Microbes (‘healthy bacteria’)

• Proteins & peptides

• Immunoglobulin, lysozyme & hormones

• Lipids & Carbohydrates

• Human milk oligosaccharides (HMOs)

….probably 1000’s of components

Lots of reasons why NEC/sepsis is less common if

breast fed

Anti-infective & immune aspects: what

happens in the gut?

Intestinal lumen

Gut immune system

Systemic immune system

Adapted fromCorthésy B et al. J. Nutr. 2007;137:781S-790S

Gut mucosa & epithelium


What is going on in the gut lumen?

Gut microbial

communitiesNutrients


Key nutrients promote microbes

Gut microbial

ecology

Amino acids

Urea Lactoferrin

Fatty acids

Carbohydrates

HMOsHuman milk

oligosacharides


Microbes important for ‘health’: 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.

1000’s of other

metabolites: we don’t

know what they do


Neonatal microbiome - function

• Interested in microbial patterns

• … more important is what microbes do

– What are microbes producing

• Vitamins, amino acids, small molecules etc.

– How do they interact with our cells

• Epithelium, immune system, 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

Roseburia

HMO, lactate,

linoleic acid

etc.

Gene expression: multi-organ targets

• Promotion of Treg cells

• Cytokines

• Antimicrobial peptides

• Mucous production

• Gut brain axis

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 preterm

Breast milk

proteins,

HMOs, FAs >50,000 metabolites

Every baby is

unique


Breast milk nutrients promote health: more ‘healthy’ gut microbial patterns

Gut microbial

ecology

Amino acidsUrea

Lactoferrin

Fatty acids & MFGM

Carbohydrates

HMOs

Breast milk

microbes


Human milk proteins: mucin, casein & whey

• Mucins = milk fat globule membrane (MFGM) proteins

– small % but important functional activity

• Whey protein (e.g. lactoferrin) - early

• Casein virtually undetectable on day 1

– concentration increases as synthesis develops

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

– 80:20 early to 50:50 late


Milk fat globule membrane (MFGM)

• Fat droplet in human milk very large

• Formula - small

Formula Breast-milk

MFGM – trilayer structure

Layer 1 from

Endoplasmic

Reticulum

Layers 2 & 3 from apical

plasma membrane of

mammary cells

• Mucins

• Other proteins

• Glycolipids

• Sphingomyelin

• Cholesterol

• Phospholipids

• Gangliosides


Lactoferrin

• Antimicrobial glycoprotein

• Colostrum, breast milk, tears, saliva

• Acid proteolysis lactoferricin

Lactoferrin

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

– Gut lymphoid tissue

• Direct epithelial effects

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Lactoferrin- antimicrobial actions

Cell membrane disruption

Iron sequestration

Disruption of virulence proteins

Inhibition of microbial adhesion

Prevention of biofilm formation

..might be especially important in preterm infants

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

More HMOs than protein?!?

HMOs

• Bifidobacterium

• Bacteroidetes

HMOs

Lack glycosidases to

cleave HMO linkages

• Distal small intestine

• Colon



Oligosaccharides and Bifidobacteria

Lots of HMOs and lots of Bifidobacteria

They are all different

• >200 HMOs – unique to breast milk

• >30 species of Bifidobacteria

– Utilise HMOs differently

– Different effects


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



70% fucosylated


<5%% fucosylated


N-acetylneuraminic

acid

Fucose


Human milk has higher degree of




70% fucosylated


<5%% fucosylated


N-acetylneuraminic

acid

Fucose


Complex HMOs

• Unique structures in humans

• Individual differences

• Appear key to promoting growth of Bifidobacteria

• Does it matter which Bifidobacteria?


Which bacteria are able to utilise HMOs as a

food source?

Species

tested2’FL 3-FL LDFT 3’SL 6’SL

E coli 1

Clostridium 2

Lactobacillus 2 +/- +/- ++/-

Enterobacter 2

Enterococcus 2 +/- +/-

Staphylococcus 2

Streptococcus 1 + +

Bacteroides 3

Bifidobacterium 10


Fucosylated HMOs Sialylated HMOs

5 different HMOs


Which bacteria are able to utilise 2’ FL HMOs

as a food source?

Species

tested2’FL

E coli 1

Clostridium 2

Lactobacillus 2 +/- +/- ++/-

Enterobacter 2

Enterococcus 2 +/- +/-

Staphylococcus 2

Streptococcus 1 + +

Bacteroides 3

Bifidobacterium 10


Fucosylated HMOs

<10%

10-40%

>40%

Utilisation

Really good at utilising HMOs

Less good at utilising HMOs


Only Bifidobacteria & Bacteroides able to

comprehensively use HMOs as food source

Species

tested2’FL

E coli 1

Clostridium 2

Lactobacillus 2 +/-

Enterobacter 2

Enterococcus 2 +/-

Staphylococcus 2

Streptococcus 1 +

Bacteroides 3

Bifidobacterium 10


<10%

10-40%

>40%

Utilisation


Formula fed also colonised by Bifidobacteria,

more diversity & species ‘typical’ of adults


B infantis

B longum

B breve

+ others

B infantis

B longum

B breve

B adolescentis

++++ others


HMOs

• Unique structures in humans

– Individual differences >100 different structures

– Important for growth of Bifidobacteria

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

• Other breastmilk components also vital

– lactoferrin, MFGM, IgA, lysozyme, fatty acids etc.

What about breast milk microbes?


Maternal milk microbiome

• Breast milk - 104/mL microbes

• 100mL = >106 bacteria per day

• Microbes from

• Maternal gut travel through blood/lymphoid tissue

• From skin on breast

• From suckling baby

Breast Milk Bacterial Communities and Development of

Infant Gut Microbiome. Pannaraj et al. JAMA Peds. 2017

• Breast-milk microbiome

reflected in infant’s stool

• Every mother-baby dyad

unique

• ‘Normal’ to have microbes on

areolar skin & in breast-milk


Early feeding & microbes

• Important – preterm & term infants

• Multiple risks for ‘abnormal’ microbiome

• Breast-milk

– ‘Normal’ for term infants

– ‘Best’ for preterm infants

• Health & disease: not a simple nutrient or bacterial effect

What is effect of breast-milk on gut microbiota?


A meta-genomic study of diet-dependent interaction

between gut microbiota and host in infants

Schwartz et al. Genome Biology 2012

Firmicutes Actino- Proteo- Bacteroidetes

Formula fed – ↑ firmicutes (Staph, Strep etc.)

Breast fed – ↑ bacteroidetes

Schwartz et al. Genome Biology 2012

• Immunity & mucosal defense-related genes

• Related to microbiome patterns

• Genes that modulate gut motility & growth

• up-regulated in breast fed

• Genes that prime mucosal inflammatory

responses

• down-regulated in breast fed

• up-regulated formula infants

Identified human genes (RNA) from

baby stool


Preterm infants

• Easy to see relevance of Microbiome to preterm infants

– NEC, sepsis, mortality

• Preterm gut microbiota

– “Dysbiotic” i.e. pattern is abnormal

– What do we mean by this?

• HMOs and NEC – is there a link?


Preterm infants are Dysbiotic

• Lack of diversity

• Absence of key species e.g. Bifidobacteria

• Presence of pathogens e.g. Enterobacter cloacae

• Dysbiosis associated with inflammation

• Remember – microbes have functional effects

– Dysbiosis is partly due to changes in metabolic

products from the bacteria

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’

Dysbiosis

Thinning &

disruption of

mucus layerAdapted from Tamburini et al. 2016

Epithelial damage

Microbial metabolites

Changing metabolites (function)

Anti- Pro-

Loss of microbial diversity & key taxa

Increasing ‘pathogens’


Dysbiosis in preterm infants

• Define dysbiosis in different ways

– Less ‘healthy’

– More ‘pathogenic’

– Less diversity

• Preterm infants – different from term infants

– Patterns change preceding NEC & Sepsis


Dysbiosis pre-NEC

Warner et al. 2016 Lancet

NEC Control

Age (days)

Gammaproteobacteria


Early colonisation: abnormal in preterm infants

1 2 3

Dysbiosis precedes NEC – association or

causation?

?


Necrotising enterocolitis (NEC) and milk

feeding

• Strong evidence for benefits of mother’s breast-milk

• Nutrition = nutrient and functional

– Nutrients: unique fatty acid & protein structures

– Functional: proteins, peptides, enzymes, HMOs

• HMOs – do they play a role in NEC?


HMO composition predicts risk of NEC in

preterm infants

• N = 200 VLBW infant/mother pairs

• Expressed breast-milk (EBM) collected

• HMO concentration in EBM fed to infants

– Range of HMOs: DSLNT, LNFP1, 2’FL etc.

• Compared NEC to controls

• DSLNT ↓↓ in milk samples from NEC cases

Autran et al. 2017

DSLNT level (ug/mL)

NEC Bells 3NEC 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

• Breast-milk sample collected

each day

• Grey circle: DSLNT concentration

in EBM from healthy baby

• Red/yellow: DSLNT in EBM from

babies who got NEC

• Babies who got NEC had

always received breast-milk

with lowest 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

Nut rit ion

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

Similar structure HMOs -

difference is one sialic acid residue

Autran et al. 2017

Associated with lower NEC

Not associated with lower NEC

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

Nut rit ion


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

Nut rit ion


Risk of developing NEC

NEC less like NEC more likely

Autran et al. 2017

• Risk of developing NEC related to one or two

specific HMOs

• Does this explain why breast-milk appears

more/less effective in different studies?


HMO composition predicts risk of NEC in

preterm infants. Autran et al. 2017

Conclusions:

1. DSLNT in EBM may identify infants at risk of NEC

2. HMO functions are highly specific

3. HMOs may be involved in other mechanisms (not

purely microbiomic)


Summary

• Early life exposures & colonisations

• Microbiome changes over human life-course

• Multiple diseases linked to the early microbiome

• May be one explanation why breastmilk beneficial over

the life-course

• Gut health in infants

– Microbes, metabolites & nutrient interactions

– Breastmilk – MFGM, lactoferrin, HMOs, microbes etc.


Improving breast milk provision for

preterm infants: early colostrum

• Fresh to EVERY baby as soon as available

– Colostrum by syringe/spoon immediately (<12hours)

– Oro-pharyngeal immune system

– <0.5mL – place in mouth


Oropharyngeal colostrum administration in

extremely preterm infants

• n=48 <28w gestation; starting day 2-4

• 0.2ml colostrum oro-pharynx every 3 hours versus water

• Looked at immune & inflammatory markers

– Salivary IgA, TGF-B1, IL-8

– Urinary IgA, Lactoferrin, IL-1B

Lee et al. Pediatrics 2015


• Increased urinary & salivary IgA

• Decreased salivary TGF

Oropharyngeal colostrum administration

in extremely preterm infants

IgA

Saliva

LF TGF

Colostrum Colostrum ColostrumWater Water Water


Summary

• Breast milk reduces

– NEC & sepsis in preterm infants

– Infections in early life in term infants

• Nutrient and ‘non-nutrient’ (functional) mechanisms

• Multiple components

– Lactoferrin, HMOs, MFGM etc.

– Unique structures & composition will never be replicated

– May not matter why or how breast milk works

8 ways to promote a normal microbiome

in early life?

?

Breastfeeding Skin-skin Avoid

Home environment Family diet Reduce

Reduce preterm births Live in a cave

Water

Carbohydrates

Lactose

Corn maltodextrin

Protein

Partially hydrolyzed reduced

minerals whey protein

concentrate (from cow’s milk)

Fats

Palm olein

Soybean oil

Coconut oil

High oleic safflower oil

M. alpina oil (Fungal DHA)

C.cohnii oil (Algal ARA)

Minerals

Potassium citrate

Potassium phosphate

Calcium chloride

Tricalcium phosphate

Sodium citrate

Magnesium chloride

Ferrous sulphate

Zinc sulphate

Sodium chloride

Copper sulphate

Potassium iodide

Manganese sulphate

Sodium selenate

Vitamins

Sodium ascorbate

Inositol

Choline bitartrate

Alpha-Tocopheryl acetate

Niacinamide

Calcium pantothenate

Riboflavin

Vitamin A acetate

Pyridoxine hydrochloride

Thiamine mononitrate

Folic acid

Vitamin D3

Vitamin B12

Enzyme

Trypsin

Amino acid

Taurine

L-Carnitine

Nucleotides

Cytidine 5-MP

Disodium uridine 5-MP

Adenosine 5-MP

Disodium guanosine 5-MP

Soy LecithinFormula

Breast milk

Water

Carbohydrates

Lactose

Corn maltodextrin

Protein

Partially hydrolyzed reduced

minerals whey protein

concentrate (from cow’s milk)

Fats

Palm olein

Soybean oil

Coconut oil

High oleic safflower oil

M. alpina oil (Fungal DHA)

C.cohnii oil (Algal ARA)

Minerals

Potassium citrate

Potassium phosphate

Calcium chloride

Tricalcium phosphate

Sodium citrate

Magnesium chloride

Ferrous sulphate

Zinc sulphate

Sodium chloride

Copper sulphate

Potassium iodide

Manganese sulphate

Sodium selenate

Vitamins

Sodium ascorbate

Inositol

Choline bitartrate

Alpha-Tocopheryl acetate

Niacinamide

Calcium pantothenate

Riboflavin

Vitamin A acetate

Pyridoxine hydrochloride

Thiamine mononitrate

Folic acid

Vitamin D3

Vitamin B12

Enzyme

Trypsin

Amino acid

Taurine

L-Carnitine

Nucleotides

Cytidine 5-MP

Disodium uridine 5-MP

Adenosine 5-MP

Disodium guanosine 5-MP

Soy Lecithin

Formula

Water

Carbohydrates

Lactose

Oligosaccharides

Carboxylic acid

Alpha hydroxy acid

Lactic acid

Proteins

Whey protein

Alpha-lactalbumin

HAMLET (Human Alpha-

lactalbumin Made Lethal to

Tumour cells)

Lactoferrin

antimicrobial factors

Casein

Serum albumin

Non-protein nitrogens

Creatine

Creatinine

Urea

Uric acid

Peptides

Breast milk

Amino Acids: Alanine, Arginine,

Aspartate, Glycine, Cystine,

Glutamate, Histidine, Isoleucine,

Leucine, Methionine, Phenylalanine,

Proline, Serine, Taurine, Theronine,

Tryptophan, Tyrosine, Valine

Carnitine

Nucleotides (chemical compounds that are

the structural units of RNA and DNA)

5’-Adenosine monophosphate (5”-AMP)

3’:5’-Cyclic adenosine monophosphate

(3’:5’-cyclic AMP)

5’-Cytidine monophosphate (5’-CMP)

Cytidine diphosphate choline (CDP choline)

Guanosine diphosphate (UDP)

Guanosine diphosphate - mannose

3’- Uridine monophosphate (3’-UMP)

5’-Uridine monophosphate (5’-UMP)

Uridine diphosphate (UDP)

Uridine diphosphate hexose (UDPH)

Uridine diphosphate-N-acetyl-hexosamine

(UDPAH)

Uridine diphosphoglucuronic acid (UDPGA)

Fats

Triglycerides

Long-chain PUFA

Docosahexaenoic acid (DHA)

Arachidonic acid (AHA)

Linoleic acid

Alpha-linolenic acid (ALA)

Eicosapentaenoic acid (EPA)

Conjugated linoleic acid (Rumenic acid)

Free Fatty Acids

Monounsaturated fatty acids

Oleic acid

Palmitoleic acid

Heptadecenoic acid

Saturated fatty acids

Stearic

Palmitic acid

Phospholipids

Phosphatidylcholine

Phosphatidylethanolamine

Phosphatidylinositol

Lysophosphatidylcholine

Lysophosphatidylethanolamine

Plasmalogens

Sphingolipids

Sphingomyelin

Gangliosides

GM1

GM2

GM3

Glucosylceramide

Glycosphingolipids

Galactosylceramide

Lactosylceramide

Globotriaosylceramide (GB3)

Globoside (GB4)

Sterols

Squalene

Lanosterol

Dimethylsterol

Methosterol

Lathosterol

Desmosterol

Triacylglycerol

Cholesterol

7-dehydrocholesterol

Stigma-and campesterol

7-ketocholesterol

Sitosterol

β-lathosterol

Vitamin D metabolites

Steroid hormones

Vitamins

Vitamin A

Beta carotene

Vitamin B6

Vitamin B8 (Inositol)

Vitamin B12

a-Tocopherol

Vitamin K

Thiamine

Riboflavin

Niacin

Folic acid

Pantothenic acid

Biotin

Minerals

Calcium

Sodium

Potassium

Iron

Zinc

Chloride

Phosphorus

Magnesium

Copper

Manganese

Iodine

Selenium

Choline

Sulpher

Chromium

Cobalt

Fluorine

Nickel

Metal

Molybdenum

Growth Factors

Cytokines

interleukin-1β (IL-1β)

IL-2

IL-4

IL-6

IL-8

IL-10

Granulocyte-colony stimulating factor (G-CSF)

Macrophage-colony stimulating factor (M-CSF)

Platelet derived growth factors (PDGF)

Vascular endothelial growth factor (VEGF)

Hepatocyte growth factor -α (HGF-α)

HGF-β

Tumor necrosis factor-α

Interferon-γ

Epithelial growth factor (EGF)

Transforming growth factor-α (TGF-α)

TGF β1

TGF-β2

Insulin-like growth factor- II

Nerve growth factor (NGF)

Erythropoietin

Peptides (combinations of amino

acids)

HMGF I (Human growth factor)

HMGF II

HMGF III

Cholecystokinin (CCK)

β-endorphins

Parathyroid hormone (PTH)

Parathyroid hormone-related peptide

(PTHrP)

β-defensin-1

Calcitonin

Gastrin

Motilin

Bombesin

Neurotensin

Somatostatin

Cortisol

Triiodothyronine (T3)

Thyroxine (T4)

TSH

TRH)

Prolactin

Oxytocin

Insulin

Corticosterone

Thrombopoietin

GnRH

GRH

Leptin

Ghrelin

Adiponectin

Feedback inhibitor of

lactation (FIL)

Eicosanoids

Prostaglandins

PG-E1 , PG-E2 , PG-F2

Leukotrienes

Thromboxanes

Prostacyclins

Amylase

Arysulfatase

Catalase

Histaminase

Lipase

Lysozyme

PAF-acetylhydrolase

Phosphatase

Xanthine oxidase

Antiproteases

a-1-antitrypsin

a-1-antichymotrypsinLeukocytes

Phagocytes

Basophils

Neutrophils

Eoisinophils

Macrophages

Lymphocytes

B lymphocytes

T lymphocytes

sIgA

IgA2

IgG

IgD

IgM

IgE

Complement

C1 , C2 ,

C3 , C4,

C5, C6,

C7, C8,

C9

Glycoproteins

Mucins

Lactadherin

Alpha-lactoglobulin

Alpha-2 macroglobulin

Lewis antigens

Haemagglutinin inhibitors

Bifidus Factor

Lactoferrin

Lactoperoxidase

B12 binding protein

Fibronectin

Oligosaccharides (>200

different kinds!)


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