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
www.neonatalresearch.netImproving outcomes
Newcastle Neonatal Research Team
Research focus: www.neonatalresearch.net
Gut function, nutrition & microbiome
Long-term metabolic outcome
Work with parents: 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
www.neonatalresearch.netImproving outcomes
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
www.neonatalresearch.netImproving outcomes
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
www.neonatalresearch.netImproving outcomes
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
www.neonatalresearch.netImproving outcomes
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
www.neonatalresearch.netImproving outcomes
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
www.neonatalresearch.netImproving outcomes
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 …..
www.neonatalresearch.netImproving outcomes
How do we study the microbiome?
www.neonatalresearch.netImproving outcomes
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)
www.neonatalresearch.netImproving outcomes
Challenges: massive datasets
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Adj. P = 2.9e−61
Observed OTUs Shannon
0
10
20
30
40
50
0
1
2
A B C D E F A B C D E F
Alph
a Di
vers
ity PGSTletterABCDEF
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
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)• Not enough detail
• Does not identify most
bacteria
• ‘Expensive’
• Too much information
• Identifies bacteria we
do not ‘know’
• Complex to analyse
www.neonatalresearch.netImproving outcomes
Challenge: defining ‘normal’ e.g. urine culture
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Adj. P = 2.9e−61
Observed OTUs Shannon
0
10
20
30
40
50
0
1
2
A B C D E F A B C D E F
Alph
a Di
vers
ity PGSTletterABCDEF
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
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
• Urinary tract infection
What is ‘normal’
• Sterile
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●
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● ●
●●●●
●
Adj. P = 2.9e−61
Observed OTUs Shannon
0
10
20
30
40
50
0
1
2
A B C D E F A B C D E F
Alph
a Di
vers
ity PGSTletterABCDEF
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
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
●
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Adj. P = 2.9e−61
Observed OTUs Shannon
0
10
20
30
40
50
0
1
2
A B C D E F A B C D E F
Alph
a Di
vers
ity PGSTletterABCDEF
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
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
●
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Adj. P = 1.7e−13
● ●
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●
Adj. P = 2.9e−61
Observed OTUs Shannon
0
10
20
30
40
50
0
1
2
A B C D E F A B C D E F
Alph
a Di
vers
ity PGSTletterABCDEF
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
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
www.neonatalresearch.netImproving outcomes
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
www.neonatalresearch.netImproving outcomes
Bacteria live in communities, just like
animals
Based on idea from Rob Knight’s TED talk
www.neonatalresearch.netImproving outcomes
Community looks similar: a little more or less;
pattern characteristic of that niche
www.neonatalresearch.netImproving outcomes
These communities are very different: they
come from different ecological niche
www.neonatalresearch.netImproving outcomes
Same data – use a circle to represent a
community
www.neonatalresearch.netImproving outcomes
Replace the animals with colour circles:
each circle represents a ‘community’
www.neonatalresearch.netImproving outcomes
Circles that are close are more similar
Each circle represents a community
?
www.neonatalresearch.netImproving outcomes
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
www.neonatalresearch.netImproving outcomes
Early colonisation is key life event
www.neonatalresearch.netImproving outcomes
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)
Credit: Rob Knight (TED talk)
Baby stool
At birth – baby stool microbiome reflects
delivery method (vaginal)
Credit: Rob Knight (TED talk)
Baby stool
By 2 years age –
resembles adult stool
Effect of antibiotics
Credit: Rob Knight (TED talk)
Baby stool
How might breast milk modify the
microbiota?
www.neonatalresearch.netImproving outcomes
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
www.neonatalresearch.netImproving outcomes
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
www.neonatalresearch.netImproving outcomes
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
www.neonatalresearch.netImproving outcomes
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
www.neonatalresearch.netImproving outcomes
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?
www.neonatalresearch.netImproving outcomes
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
www.neonatalresearch.netImproving outcomes
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
www.neonatalresearch.netImproving outcomes
What is going on in the gut lumen?
Gut microbial
communitiesNutrients
www.neonatalresearch.netImproving outcomes
Key nutrients promote microbes
Gut microbial
ecology
Amino acids
Urea Lactoferrin
Fatty acids
Carbohydrates
HMOsHuman milk
oligosacharides
www.neonatalresearch.netImproving outcomes
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
www.neonatalresearch.netImproving outcomes
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
www.neonatalresearch.netImproving outcomes
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?
www.neonatalresearch.netImproving outcomes
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
www.neonatalresearch.netImproving outcomes
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
www.neonatalresearch.netImproving outcomes
Choline
Clostridia ↑Choline
compounds
Complex role• Cell membrane• Neurotransmitter• Methyl donor
Trimethylamine OxideBacteroidetes ↓
TMAO increased risk
adverse cardiovascular
events
www.neonatalresearch.netImproving outcomes
Summary schema
Different types of
bacteria which…
Dietary
nutrients
affect…. Differences in pattern
of metabolites……
Patterns of health
and disease
www.neonatalresearch.netImproving outcomes
The potential range & complexity is enormous
>500 bacterial
species in preterm
Breast milk
proteins,
HMOs, FAs >50,000 metabolites
Every baby is
unique
www.neonatalresearch.netImproving outcomes
Breast milk nutrients promote health: more ‘healthy’ gut microbial patterns
Gut microbial
ecology
Amino acidsUrea
Lactoferrin
Fatty acids & MFGM
Carbohydrates
HMOs
Breast milk
microbes
www.neonatalresearch.netImproving outcomes
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
www.neonatalresearch.netImproving outcomes
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
www.neonatalresearch.netImproving outcomes
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
www.neonatalresearch.netImproving outcomes
Lactoferrin functions
• Lactoferrin → lactoferricin
• Direct antimicrobial effects
– bacterial, viral, fungal
• Modification of host immune response
– Gut lymphoid tissue
• Direct epithelial effects
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
Underwood et al. Peds Res 2015
www.neonatalresearch.netImproving outcomes
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
www.neonatalresearch.netImproving outcomes
Bifidobacterium longum subspecies infantis:
champion coloniser of the infant gut.
Underwood et al. Peds Res 2015
Human Milk Oligosaccharide structure is
key to Bifidobacterium growth & function
www.neonatalresearch.netImproving outcomes
Human milk has high degree of
oligosaccharide polymerization
Underwood et al. Peds Res 2015
MammalsPrimatesHumans
70% fucosylated
<20% sialylated >70% sialylated
<5%% fucosylated
GalactoseN-acetylglucosamine Glucose
N-acetylneuraminic
acid
Fucose
www.neonatalresearch.netImproving outcomes
Human milk has higher degree of
oligosaccharide polymerization
MammalsPrimatesHumans
70% fucosylated
<20% sialylated >70% sialylated
<5%% fucosylated
GalactoseN-acetylglucosamine Glucose
N-acetylneuraminic
acid
Fucose
www.neonatalresearch.netImproving outcomes
Human milk has higher degree of
oligosaccharide polymerization
Underwood et al. Peds Res 2015
MammalsPrimatesHumans
70% fucosylated
<20% sialylated >70% sialylated
<5%% fucosylated
GalactoseN-acetylglucosamine Glucose
N-acetylneuraminic
acid
Fucose
www.neonatalresearch.netImproving outcomes
Complex HMOs
• Unique structures in humans
• Individual differences
• Appear key to promoting growth of Bifidobacteria
• Does it matter which Bifidobacteria?
www.neonatalresearch.netImproving outcomes
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
Underwood et al. Peds Res 2015
Fucosylated HMOs Sialylated HMOs
5 different HMOs
www.neonatalresearch.netImproving outcomes
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
Underwood et al. Peds Res 2015
Fucosylated HMOs
<10%
10-40%
>40%
Utilisation
Really good at utilising HMOs
Less good at utilising HMOs
www.neonatalresearch.netImproving outcomes
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
Underwood et al. Peds Res 2015
<10%
10-40%
>40%
Utilisation
www.neonatalresearch.netImproving outcomes
Formula fed also colonised by Bifidobacteria,
more diversity & species ‘typical’ of adults
Underwood et al. Peds Res 2015
B infantis
B longum
B breve
+ others
B infantis
B longum
B breve
B adolescentis
++++ others
www.neonatalresearch.netImproving outcomes
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?
www.neonatalresearch.netImproving outcomes
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
www.neonatalresearch.netImproving outcomes
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?
www.neonatalresearch.netImproving outcomes
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
www.neonatalresearch.netImproving outcomes
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?
www.neonatalresearch.netImproving outcomes
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
Adapted from Tamburini et al. 2016
• Diverse microbes
• Presence of ‘healthy’ microbes
• Absence of ‘un-healthy’ microbes• Healthy mucus layer
• Active/receptive epithelium
• Healthy blood flow/oxygenation
Dysbiosis
Adapted from Tamburini et al. 2016
Dysbiosis
Adapted from Tamburini et al. 2016
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’
www.neonatalresearch.netImproving outcomes
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
www.neonatalresearch.netImproving outcomes
Dysbiosis pre-NEC
Warner et al. 2016 Lancet
NEC Control
Age (days)
Gammaproteobacteria
www.neonatalresearch.netImproving outcomes
Early colonisation: abnormal in preterm infants
1 2 3
Dysbiosis precedes NEC – association or
causation?
?
www.neonatalresearch.netImproving outcomes
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?
www.neonatalresearch.netImproving outcomes
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
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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
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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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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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?
www.neonatalresearch.netImproving outcomes
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)
www.neonatalresearch.netImproving outcomes
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.
www.neonatalresearch.netImproving outcomes
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
www.neonatalresearch.netImproving outcomes
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
www.neonatalresearch.netImproving outcomes
• Increased urinary & salivary IgA
• Decreased salivary TGF
Oropharyngeal colostrum administration
in extremely preterm infants
IgA
Saliva
LF TGF
Colostrum Colostrum ColostrumWater Water Water
www.neonatalresearch.netImproving outcomes
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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