Vol. 78, Suppl.

2, 20-21

Gut Microbiota: No Longer

the Forgotten Organ

Editor
Hania Szajewska, Warsaw

Editorial Board
Andrew Prentice, Banjul/London
Raanan Shamir, Tel Aviv
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 Vol. 78, Suppl. 2, 20-21

Contents

11 Editorial
Szajewska, H. (Warsaw)

Gut Microbiota: No Longer the Forgotten Organ

13 Focus on: Microbiome and Infection: A Case for “Selective

Depletion”

14 Microbiome and Infection: A Case for “Selective Depletion”

Hill, C. (Cork)

10 Focus on: Impact of Delivery Mode on Infant Gut Microbiota

11 Impact of Delivery Mode on Infant Gut Microbiota

Korpela, K. (Helsinki)

20 Focus on: Gut Microbiota Development: Influence of Diet

from Infancy to Toddlerhood

21 Gut Microbiota Development: Influence of Diet from Infancy

to Toddlerhood
Laursen, M.F. (Kongens Lyngby)

35 Focus on: Microbiomes and Childhood Malnutrition: What Is

the Evidence?

36 Microbiomes and Childhood Malnutrition: What Is the

Evidence?
McGuire, M.K.; McGuire, M.A. (Moscow, ID)

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S. Karger AG, Basel
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 Editorial

Reprint with permission from:

Ann Nutr Metab 2021;77(suppl 3):1–2
DOI: 10.1159/000519223

Gut Microbiota: No Longer the

Forgotten Organ
Hania Szajewska
The Department of Paediatrics, The Medical University of Warsaw, Warsaw, Poland

While the gut microbiota was once called “the forgotten
organ” [1], it is no longer forgotten. On the contrary, it is a hot
topic for research, as documented by the rapidly increasing
number of scientific papers on this subject as well as coverage
in the lay press, TV/radio programs, and social media.
The aim of this new series is to highlight recent develop-
ments and hot topics in gut microbiome/microbiota research.
These two terms are often used interchangeably. However,
there is a subtle difference between them [2]. The term mi-
crobiome refers to all the microorganisms (and their genes)
living in a specific environment such as the gut. These micro-
organisms include bacteria, archaea, eukaryotes, and viruses.
The term microbiota, in principle, refers to the microbes
themselves. If only the genes are of interest, the term metage-
nome is used.
Regardless of the terminology, a balanced gut microbiota
is crucial for health. Conversely, dysbiosis, which refers to al-
tered gut microbiota diversity and composition (i.e., changes
at the level of phylum, genus, or species) [3], contributes to
the development of gastrointestinal and extraintestinal dis-
eases. Among others, the microbiota mediates colonization
resistance and influences susceptibility to infectious diseases.
The first paper, by Colin Hill [4] from the APC Microbiome Ire-
land and School of Microbiology, University College Cork, Ire-
land, reviews advances in the research on infections and the
microbiome, which provides a barrier to infection but can also
serve as a source of novel antimicrobials. He also introduces
the concept of “selective depletion,” where pathogens can be
reduced by narrow-spectrum inhibitors, such as bacteriocins
or bacteriophages (phages). The microbiome is protected and
can play a role in reducing the spread of infection by sup-
pressing the growth of pathogens.
The mode of delivery affects the gut microbiome. The
World Health Organization estimates the cesarean section
rate as 10–15% of all births if only used when medically nec-
essary. However, cesarean delivery is increasing in prevalence
worldwide. Based on data from 169 countries that included
over 98% of births worldwide, cesarean section almost dou-
bled in 2015 compared with 2000 (21.1% vs. 12.1%, respec-
tively) [5]. The second paper by Katri Korpela [6] from the Uni-
versity of Helsinki, Finland, discusses the impact of delivery
mode on the infant gut microbiota, its consequences, and the
interventions to restore the microbial dysbiosis created by ce-
sarean section.
Diet is a key factor that has a substantial impact on the
composition and function of the gut microbiota. The third
paper, written by Martin Laursen [7] from the National Food
Institute, Technical University of Denmark, covers the most
important factors controlling the succession and establish-
ment of the gut microbiota in infants after birth. Considering
how the early-life gut microbiota impacts our immunity, a
better understanding of the process could potentially lead to
strategies modifying the risk of diseases later in life.

karger@karger.com © 2021 Nestlé Nutrition Institute, Switzerland/ Correspondence to:

S. Karger AG, Basel Hania Szajewska, hszajewska @ wum.edu.pl
 Some children have too little to eat, while others consume
too many calories via junk food and beverages. Consequent-
ly, malnutrition consisting of undernutrition, overweight, and
obesity is a major public health concern affecting all countries
– low-, middle-, and high-income – although in different
proportions [8, 9]. In the fourth paper written by Michelle Mc-
Guire and Mark McGuire [10] from the University of Idaho,
Moscow, ID, USA, the authors discuss data linking the com-
References
1 Marchesi JR, Adams DH, Fava F, Hermes GD, Hirschfield GM, Hold
G, et al. The gut microbiota and host health: a new clinical frontier.
Gut. 2016 Feb;65(2):330–9.
2 Quigley EM, Ghoshal UC, editors. WGO Handbook on the Gut Mi-
crobiome. World Gastroenterology Organisation; 2020 [cited Aug
22, 2021]. Available from: https://www.worldgastroenterology.
org/wgo-foundation/wdhd/wdhd-2020/tools-and-
resources#Handbook.
3 Hooks KB, O’Malley MA. Dysbiosis and Its Discontents. MBio. 2017
Oct;8(5):e01492–17.
4 Hill C. Microbiome and infection: a case for “selective depletion”.
Ann Nutr Metab. doi: 10.1159/000516399.
5 Boerma T, Ronsmans C, Melesse DY, Barros AJD, Barros FC, Juan
L, et al. Global epidemiology of use of and disparities in caesarean
sections. Lancet. 2018 Oct;392(10155):1341–48.
2 Reprint with permission from:
Ann Nutr Metab 2021;77(suppl 3):1–2
DOI: 10.1159/000519223
position of the gut microbiota and the regulation of body
weight.
In summary, gut microbiome/microbiota research is a hot
topic and is booming. This series also documents that the gut
microbiota is indeed no longer forgotten and is increasingly
recognized as a potential key player in the pathogenesis of
several diseases.
6 Korpela K. Impact of delivery mode on infant gut microbiota. Ann
Nutr Metab. doi: 10.1159/000518498.
7 Laursen MF. Gut Microbiota development: influence of diet from
infancy to toddlerhood. Ann Nutr Metab. doi: 10.1159/000517912.
8 Hawkes C, Ruel MT, Salm L, Sinclair B, Branca F. Double-duty ac-
tions: seizing programme and policy opportunities to address
malnutrition in all its forms. Lancet. 2020 Jan;395(10218):142–55.
Erratum in: Lancet. 2020 Feb 1;395.
9 The double burden of malnutrition. Lancet. Dec 16, 2019 [cited
Aug 22, 2021]. Available from: https://www.thelancet.com/series/
double-burden-malnutrition.
10 McGuire MK, McGuire MA. Microbiomes and childhood mal-
nutrition: what is the evidence? Ann Nutr Metab. doi: 10.1159/
000519001.
Szajewska
 Focus
The future of smart antimicrobial therapies may not involve pathogen
elimination with collateral damage to the microbiome (broad-spectrum
approaches), but may well lie in restricting the growth of pathogens to a
point where they cannot cause disease while harnessing the power of the
microbiome to ensure a return to a healthy state

Reprinted with permission from: Ann Nutr Metab 2021;77(suppl 3):4–9

Microbiome and Infection: A Case for “Selective Depletion”

Colin Hill

Key insights Fecal microbiota

transplantation Selective microbial
Microbial infections can be recognized as a disruption of the consortiums

normal homeostatic relationship between a particular

microbe and the host. There are many instances where the
pathogen is not acquired but is already a member of the
microbiome, e.g., Clostridioides difficile, Staphylococcus
aureus, or certain strains of Escherichia coli. Often, these
organisms only cause disease when the surrounding
microbiome is compromised, enabling the pathogen to grow
and result in disease symptoms in the host. Thus, the
microbiome itself should be treated as a virtual organ and
protected during therapeutic interventions. A targeted
approach towards eliminating unwanted microbial pathogens
is termed “selective depletion,” whereby pathogen numbers
are curtailed by a narrow-spectrum inhibitor, but the
microbiome is protected and can thus play a role in restoring
health and suppressing pathogen outgrowth in the infected
patient.
Current knowledge
From birth to adulthood, all our exposed bodily surfaces be-
come colonized by thousands of different microbial strains.
The human body consists of hundreds of micro-environ-
ments that facilitate the growth of various microbes (the mi-
crobiome). In turn, the microbiome fulfills many functions
that are critical for human health. Under a normal state of
homeostasis, the microbiome acts as a “virtual organ,” con-
stantly signaling to the digestive, immune, nervous, and en-
docrine systems in a process that maintains health and pre-
vents disease. Infection can be viewed as any interaction be-
tween a microbe and the host that results in damage severe
enough to be manifested in the form of disease symptoms.
Healthy
Probiotics microbiome
Purified
bacteriocins
Bacteriophages
Bacteriophage lysins
Mining the microbiome has yielded a variety of therapeutic strategies
for the treatment of infections and chronic diseases.
Practical implications
What is it about the microbiome that keeps pathogens in check,
and can we harness the microbiome itself for selective therapies
against specific pathogens? Microbiome-derived anti-infection
strategies have ranged from using an entire intact microbiome,
a selected microbial consortium, or single biological entities.
Recent advances in mining the microbiome have taken this one
step further. A targeted approach involves the isolation and pu-
rification of bacteriocins, toxins produced by bacteria that in-
hibit the growth of related bacterial strains. For example, thuri-
cin CD is produced by a strain of Bacillus thuringiensis and is a
very narrow-spectrum bacteriocin targeting C. difficile. This ap-
proach paves the way for selective depletion of pathogens with-
out collateral damage to the microbiome.
Recommended reading
De Maesschalck V, Gutiérrez D, Paeshuyse J, Lavigne R, Briers
Y. Advanced engineering of third-generation lysins and for-
mulation strategies for clinical applications. Crit Rev Micro-
biol. 2020;46:548–64.

karger@karger.com © 2021 Nestlé Nutrition Institute, Switzerland/

S. Karger AG, Basel
 Review Article

Reprint with permission from:

Ann Nutr Metab 2021;77(suppl 3):4–9
DOI: 10.1159/000516399

Microbiome and Infection:

A Case for “Selective Depletion”
Colin Hill  

APC Microbiome Ireland and School of Microbiology, University College Cork, Cork, Ireland

Key Messages virtual organ within the human body, and we would surely
hesitate to advance any therapeutic approach that would
• The microbiome is a virtual organ that should be protected in cause substantial damage to one of our organs. This is one
therapeutic interventions.
consequence of many broad-spectrum antimicrobial thera-
• Narrow-spectrum inhibitors can “selectively deplete” the
pathogen while protecting the microbiome. pies. There may be instances where a more precise approach
• The microbiome itself can be a valuable source of antimicro- would be useful. I have termed this “selective depletion”; a
bials. concept where pathogen numbers are curtailed by a narrow-
spectrum inhibitor but the microbiome is protected and can
play a role in restoring health and suppressing the outgrowth
Keywords of the pathogen in the infected patient. It may well be that the
Bacteriocin · Bacteriophage · Probiotics · Infection · best reservoir of microbiome-friendly antimicrobial agents is
Microbiome the microbiome itself, and I provide examples of where the
microbiome has been mined for novel precision antimicrobi-
als. © 2021 Nestlé Nutrition Institute, Switzerland/
Abstract S. Karger AG, Basel

In most instances where a pathogen has initiated an infection,

the primary goal of the treating physician or pharmacist is to
eliminate the pathogen. In the absence of knowledge of the
precise identity of the problem-causing microbe, a broad-
spectrum antimicrobial gives the best chance of success. This
approach has saved many lives and is an invaluable tool in
fighting infections. However, perhaps our current apprecia-
tion of the importance of the microbiome in human health
should give us pause. We can regard the microbiome as a
Microbiome, Pathogens, and Host
As a rule, microbes are good for us. We interact with trillions
of microbes every day, and almost all these interactions are
mutually beneficial, or at the very least are benign. Our most
important microbial interaction is that between us and our
microbiome, our fellow travellers on our journey through life.

karger@karger.com © 2021 Nestlé Nutrition Institute, Switzerland/ Correspondence to:

S. Karger AG, Basel Colin Hill, c.hill @ ucc.ie
 Colonisation resistance
Pathogen
Microbiome
Host
epithelium
Immune
system
Access to
other organs
Fig. 1. A diverse abundant microbiome (left) can act as a barrier to infection through a variety of mechanisms, includ-
ing direct inhibition, colonisation resistance, or educating the immune system. A damaged microbiome (right) may
be more susceptible to infectious bacteria causing damage.
We are born into a microbial world, and as adults, all our ex-
posed surfaces are colonised with thousands of microbial
strains selected from the millions we have encountered since
birth [1]. We provide our microbes with a somewhat protect-
ed, albeit highly competitive, environment. It is more correct
to state that we provide hundreds of micro-environments or
niches. We as hosts provide sustenance and permissive envi-
ronmental conditions, while our microbial partners fulfil many
functions important to human health. We share the same
chemical language, and so there is constant communication
between our microbes and our nervous system, our immune
systems, and our mucosal surfaces. In a healthy human, these
interactions with our microbial “virtual organ” [2] is important
for maintaining health and preventing disease. The microbi-
ome is a valuable partner and, as with any other organ, should
be protected insofar as possible.
In this context, we can see microbial infections as an aber-
ration where a specific microbe has broken with the normal-
ly benign or beneficial relationship between microbes and
hosts. We call these microbes pathogens or opportunistic
pathogens. Infection describes any interaction between a mi-
crobe and a host that results in damage that is severe enough
to be manifested in the form of symptoms. Infection usually
follows a very prescribed path, initiated by acquisition or ex-
posure to the infectious microbe (pathogen), engagement
A Case for Selective Depletion in Treating
Infectious Disease
Infection
between the pathogen and the host in terms of survival, colo-
nisation, growth, and ultimately damage mediated by viru-
lence factors such as invasive mechanisms and/or the pro-
duction of toxins. The damage caused can result in mild
symptoms or death depending on a myriad of factors. These
factors include the identity of the pathogen, including the ge-
nus, species, and even the genetic complement of a given
strain; the possession and expression of virulence factors; the
dose; the circumstances surrounding the exposure in terms
of food carriers or breach of barriers (skin or mucosal surface);
the genetics of the host; the hosts immune status; and of
course, the commensal microbes present at the site of infec-
tion (the microbiome).
There are many instances where the pathogen is not ac-
quired but is already a member of our microbiome, for exam-
ple, Clostridioides difficile, Staphylococcus aureus, or certain
strains of Escherichia coli. These pathogens often only cause
disease when the surrounding microbiome is compromised in
some way, and the pathogen manages to grow and use its
virulence factors to cause damage to the host. We can regard
the microbiome as a barrier to infection (Fig.  1). Antibiotic
treatment can be an effective solution to reduce or even elim-
inate a pathogen but can also lead to further damage to the
microbiome. For C. difficile, this can result in recurring bouts
of diarrhoea and even fatal outcomes. Most microbiomes will
Reprint with permission from: 5
Ann Nutr Metab 2021;77(suppl 3):4–9
DOI: 10.1159/000516399
 Faecal Microbiota Transplantation
Faecal microbiota transplantation (FMT) is perhaps the most
FMT
(Microbial consortia) dramatic example of mining the microbiome for solutions to
infections. A recent systematic review and meta-analysis con-
Probiotics cluded that there is high-quality evidence that FMT is effective
(Postbiotics) in breaking the vicious cycle of recurring C. difficile infections
[4], although a similar success rate has not been reported for
Phage non-infectious diseases such as irritable bowel syndrome [5].
(Phage lysins) Essentially, FMT involves collecting the entire microbiome
from one individual in the form of a faecal donation and trans-
Microbiome Bacteriocins planting it with minimal processing to the colon of a patient
RWKHU£DQWLPLFURELDOV
Fig. 2. Mining the microbiome for selective depletion strategies.
FMT, faecal microbiota transplantation.
recover from antibiotic exposure and will revert to a level of
complexity and community structure that approximates the
pre-treatment microbiome, but of course, there will also be an
undesirable selection pressure for the emergence of antibi-
otic resistance in both target and nontarget species. A link be-
tween microbiome composition and sensitivity to viral infec-
tion in humans has also been reported recently [3].
What is it about the microbiome that keeps these patho-
gens in check, and can we develop effective therapies aimed
at selectively depleting the pathogen during an infection? I
use the term “selective depletion” to describe any treatment
that causes minimal damage to the microbiome but reduces
the pathogen to levels that do not lead to symptoms or cause
long-term damage to the host. What kind of strategies would
meet this objective of selective depletion? Perhaps the best
place to start is the microbiome itself. Can we explore the mi-
crobiome for strategies that can prevent and even treat infec-
tions? I will draw upon some of our own work in this area to
illustrate some of the possibilities of “mining the microbiome”
for novel anti-infectives.
Mining the Microbiome
Microbiome-derived anti-infection strategies have ranged
from using an entire intact microbiome or a microbial con-
sortium to selecting single biological entities (bacteria, fungi,
bacteriophage, or bacterial metabolites) (Fig. 2). In most in-
stances, the objective is one of selective depletion, rather than
the broad-spectrum approach typical of many antibiotic or
antiviral strategies. Much of the data generated to date have
been acquired from animal models since it can be difficult to
design and perform experiments on humans with respect to
infection.
6 Reprint with permission from:
Ann Nutr Metab 2021;77(suppl 3):4–9
DOI: 10.1159/000516399
with recurring C. difficile infections. The generally accepted
opinion is that FMT “repairs” a microbiome that has been dam-
aged by the antibiotic therapies used to/try to control the ini-
tial infection(s) and allows the recipient to use this more di-
verse microbiome to suppress subsequent outgrowth of the
pathogen. Another strategy linked to FMT is using an unde-
fined microbial consortium composed of spores purified from
a faecal sample and transferred to a patient with a similar ob-
jective of restoring diversity and suppressing pathogen-in-
duced damage. This intervention had less successful results
in published phase 2 clinical trials [6] but has the potential to
be the platform for a range of microbiome-based intervention
strategies.
Probiotics
Probiotics are live microorganisms that, when administered in
adequate amounts, confer a health benefit on the host [7].
There is evidence that probiotics can be used to prevent and
treat infection, and some of these data have been generated
in humans. The strength of the data is not entirely consistent,
since most meta-analyses and systematic reviews are per-
formed across a range of different probiotic interventions,
dose ranges, and disease outcomes. Nonetheless, there have
been positive outcomes. A recent meta-analysis on the abil-
ity of probiotics to prevent necrotizing enterocolitis in pre-
term neonates concluded that there are “significant benefits
of probiotic supplements in reducing death and disease in
preterm neonates” [8]. A similar analysis for the use of probi-
otics in upper respiratory tract infections concluded that
“low-quality evidence provides support that probiotics have
potential efficacy for preventing upper respiratory tract infec-
tion episodes in adults” [9]. Yet another meta-analysis con-
ducted on the use of probiotics in children with acute diar-
rhoea “supports the potential beneficial roles of probiotics
and synbiotics for acute diarrhoea in children” [10]. There are
also single clinical trials with impressive evidence, for exam-
ple, a randomised clinical trial in India involving over 45,000
children using a symbiotic composed of Lactiplantibacillus
plantarum together with fructooligosaccharides reported a
Hill
significant reduction in sepsis and death in the treatment co-
hort [11].
Mechanistic insights have also been generated in animal
models. We were able to demonstrate that bacteriocin pro-
duction is responsible for the protective effect of Ligilactoba-
cillus salivarius UCC118 against deliberate infection of mice
with Listeria monocytogenes [12]. We showed that the bacte-
riocin was highly active against L. monocytogenes and that a
non-bacteriocin-producing genetic knockout had no protec-
tive effect against the pathogen in the same model. Other
anti-infective mechanisms could include interacting with the
immune system or improving barrier function [13]. Bacterio-
cins represent another example of the “selective depletion”
concept in that they are usually narrow spectrum and should
be rapidly broken down by proteases and peptidases in the
gut and therefore unlikely to select for resistance.
Bacteriocins
Concentrated or purified bacteriocins have also been ex-
plored for their potential in preventing or treating infection
(reviewed by [14–16]). Bacteriocins have been largely tested
in laboratory animal models, although a broad-spectrum lan-
tibiotic, nisin, has been used commercially to prevent mastitis
in dairy cattle [17, 18]. Some bacteriocins such as nisin are
relatively broad spectrum, but an attempt has also been made
to use narrow-spectrum bacteriocins for “selective depletion”
of specific pathogens. One example is the discovery of thuri-
cin CD, a very narrow-spectrum bacteriocin targeting C. dif-
ficile [19, 20]. Thuricin CD is produced by a strain of Bacillus
thuringiensis that was mined from the human gut microbiome
in an extensive screening program. It was subsequently dem-
onstrated in improvised models of the human colon (faecal
fermentations) that thuricin CD could dramatically reduce C.
difficile levels in a highly complex microbiome without caus-
ing substantial collateral damage to the other members of the
community. Antibiotics or broad-spectrum bacteriocins de-
ployed in the same model also reduced levels of C. difficile
but also caused significant shifts in microbiome composition
[20]. While this approach has not been validated in humans or
animals, it provides a glimpse of future developments in this
exciting area.
Bacteriocins also have a potential as antimicrobials for
treating skin infections, some as broad-spectrum inhibitors
such as garvicin KS and micrococcin P1 [21, 22], but others as
potentially narrow-spectrum inhibitors that could be used in
selective depletion strategies. Because bacteriocins are gene
encoded, they can be engineered to improve their physico-
chemical characteristics and/or inhibition spectrum [23]. Bac-
teriocins can also be used in combination with other antimi-
crobials for greater effect [24].
A Case for Selective Depletion in Treating
Infectious Disease
Bacteriophage and Bacteriophage Lysins
Bacteriophages (phages) are bacterial viruses that often have
a very narrow-spectrum of inhibition. There are several ad-
vantages and disadvantages to using bacteriocins to treat in-
fections, a concept often referred to as phage therapy [25].
Disadvantages include the narrow host ranges, although, of
course, this could be regarded as an advantage in selective
depletion strategies. This disadvantage can be addressed by
using cocktails of phages to cover a wider spectrum of target
bacteria, or by carefully selecting the correct phage from an
existing phage bank to use against a specific pathogenic strain
infecting a patient. Another disadvantage is the ease with
which bacteria can develop resistance to the phage attack,
again an issue that may be overcome by using multiple phag-
es in cocktails. Advantages include their widespread distribu-
tion in nature and subsequent ease of isolation in many in-
stances, and, of course, their ability to multiply at the site of
infection. However, the clinical evidence is scarce. Phages
have been used for decades in eastern Europe, and it is logical
to assume they must have benefits in that they have survived
as frontline treatments for so many years [26]. In the West,
phages have been used in some high-profile instances in sin-
gle patients with infections that were recalcitrant to antibi-
otic therapies [27, 28]. Phages can potentially be applied on
the skin, by inhalation, or by ingestion [29].
Lysins are enzymes produced by bacteriophages that are
responsible for lysing the target bacterium following multipli-
cation of phage particles. Phage lysins target the cell wall and
can also act from outside the cell. Lysins can be broader spec-
trum than their carrying phages, but are still limited to the spe-
cies or genus level, making them good candidates for selec-
tive depletion strategies. There have been many attempts to
use phage lysins to target specific pathogens (reviewed by [30,
31], particularly on the skin [32] and in the respiratory tract [33].
As is the case for bacteriocins, these gene-encoded antimi-
crobials can readily be engineered for greater efficacy [34].
Conclusions
The microbiome provides both a barrier to infection and a
source that can be mined for novel antimicrobials. While
these antimicrobials, including FMT, microbial consortia, pro-
biotics, bacteriocins, and bacteriophages (and their lysins),
have often been analysed for their solo impact on infection,
there is no reason why these could not be used as combina-
torial treatments. Perhaps the most attractive feature of these
microbiome-based inhibitors is their potential narrow spec-
trum of inhibition that would act to selectively deplete the
pathogen, while preserving the complexity of the surrounding
Reprint with permission from: 7
Ann Nutr Metab 2021;77(suppl 3):4–9
DOI: 10.1159/000516399
microbiome. This might require precise identification of the
pathogen before initiating treatment, but this is not a signifi-
cant problem with chronic infections and is becoming less of
a problem with speedy high-throughput bacterial identifica-
tion becoming more accessible for acute infections [35]. The
future of smart antimicrobial therapies may not involve patho-
gen elimination with collateral damage to the microbiome
(broad-spectrum approaches) but may well lie in restricting
the growth of pathogens to a point where they cannot cause
disease while harnessing the power of the microbiome to en-
sure a return to a healthy state – a strategy I designate as se-
lective depletion.
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Statements of Ethics
No ethical approval is required as there is no original data in this ar-
ticle.
Conflict of Interest Statement
The writing of this article was supported by Nestlé Nutrition Institute
and the author declares no other conflicts of interest.
Funding Sources
The author is an investigator at APC Microbiome Ireland that receives
funding from the Science Foundation Ireland (SFI) (Grant No. SFI/12/
RC/2273).
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A Case for Selective Depletion in Treating Reprint with permission from: 9

Infectious Disease Ann Nutr Metab 2021;77(suppl 3):4–9
DOI: 10.1159/000516399
 Focus
Emerging evidence suggests that antibiotic exposure during
birth can be likened to a chemical C-section from the
microbial perspective, eliminating the natural transmission of
gut microbiota, especially bifidobacteria, from mother to infant

Reprinted with permission from: Ann Nutr Metab 2021;77(suppl 3):11–19

Impact of Delivery Mode on Infant Gut Microbiota

Katri Korpela

Key Insight
Exposure to maternal gut microbes
Infant gut is colonized
C-section birth or exposure to intrapartum antibiotics by specific maternal
Infant Vaginal birth microbes
eliminates the normal mother-to-infant transmission of (bifidobacterial and
beneficial microbes. This alters the development of the infant Bacteroides species)
gut microbiota, with negative consequences on immune and Exposure to skin microbes, microbes
metabolic development. C-section deliveries are increasing from the hospital environment
Lower abundance of
worldwide, affecting over 50% of infants in certain regions. In bifidobacterial and
Infant C-section birth
developed countries, over 30% of infants are also exposed to almost total lack of
Bacteroides species
antibiotics during vaginal birth. This suggests that natural
microbial colonization at birth is currently disrupted in up to
half of infants, even in regions with low C-section rates and
C-section birth eliminates the normal mother-to-infant transmission
good antimicrobial stewardship.
of beneficial microbes.

Current knowledge
During vaginal birth, the infant is exposed to diverse maternal
microbes, of which specific fecal microbes colonize the in-
fant’s gut. However, not all maternal gut bacteria have the
ability to colonize infants. It is important to note that the ma-
ternal gut microbiota is not transmitted as an entire commu-
nity: only specific members are able to establish persistent
populations in the infant. The dominant maternally-derived
colonizers of the infant gut are bifidobacteria and Bacteroi-
des. Infants born by C-section are colonized by bacteria from
the environment, including potential pathogens from the
hospital environment. Therefore, there is a pressing need to
restore normal gut microbiota composition in C-section-
born and antibiotic-exposed infants.
addressing the microbiota imbalance in infants born via C-
section and in infants exposed to antibiotics before or during
birth. The impact of antibiotic exposure can be modified by
feeding practices: evidence shows that formula-fed infants
who are on antibiotics are more severely affected by antibi-
otic exposure. Breastfeeding and probiotics are particularly
important for infants with disrupted gut colonization, as these
measures enhance the growth of beneficial bifidobacteria. Fe-
cal microbiota transplant from mother to neonate has also
been shown to restore normal gut microbiota in C-section-
born infants but requires careful screening of the mother.
Recommended reading

Practical implications Korpela K, Helve O, Kolho K, Saisto T, Skogberg K, Dikareva E,

et al. Maternal fecal microbiota transplantation in cesarean-
Gut microbiota development is an important part of the phys- born infants rapidly restores normal gut microbial develop-
iological development of infants and should be considered in ment: a Proof-of-Concept Study. Cell. 2020;183:324–34.e5.
clinical practice. Attention should therefore be paid towards

karger@karger.com © 2021 Nestlé Nutrition Institute, Switzerland/

S. Karger AG, Basel
 Review Article
Reprint with permission from:
Ann Nutr Metab 2021;77(suppl 3):11–19
DOI: 10.1159/000518498
Impact of Delivery Mode on Infant
Gut Microbiota
Katri Korpela  
Human Microbiome Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
Key Messages
• Gut microbiota development is part of the physiological
development of infants and should be considered in clinical
practice.
• A large proportion of infants are exposed to C-section birth
or intrapartum antibiotics that eliminate normal mother-to-
infant transmission of beneficial microbes, altering the
development of their gut microbiota and consequently
immune and metabolic development.
• Breastfeeding and probiotics are especially important for
infants with disrupted gut microbiota colonization.
• Faecal microbiota transplant from mother to neonate has
been shown to restore normal gut microbiota in C-section-
born infants but requires careful screening of the mother.
Keywords
Antibiotics · Bifidobacteria · Breastfeeding · C-section ·
Infancy and Childhood · Microbiota · Pediatrics · Probiotics
Abstract
Microbial colonization of the neonate is an important feature
of normal birth. The gut microbiota has a central role in the
programming of the host’s metabolism and immune func-
tion, with both immediate and long-term health conse-
quences. During vaginal birth, the infant is exposed to diverse
maternal microbes, of which specific faecal microbes colo-
nize the infant’s gut. C-section eliminates the infant’s contact
karger@karger.com © 2021 Nestlé Nutrition Institute, Switzerland/
S. Karger AG, Basel
with maternal microbes, preventing vertical transmission of
gut microbes. Consequently, infants are colonized by bacte-
ria from the environment, including potential pathogens from
the hospital environment. Recent studies have shown that
intrapartum antibiotic exposure has a C-section-like effect
on the infant gut microbiota. While the composition of the
gut microbiota largely normalizes during the first year of life,
epidemiological studies suggest that the aberrant early mi-
crobial exposures have long-term immunological and meta-
bolic consequences. Because of the high prevalence of pro-
cedures that prevent normal gut microbiota development,
effective methods to normalize the gut microbiota of neo-
nates are urgently needed. Even more importantly, attention
should be paid to the microbiota imbalance in C-section-
born and antibiotic-exposed infants in clinical practice.
Breastfeeding and probiotics are particularly important for in-
fants with disrupted gut colonization.
© 2021 Nestlé Nutrition Institute, Switzerland/
S. Karger AG, Basel
Colonization and Early Development of Infant
Gut Microbiota
Although the notion of infants being born sterile has been
contested by the detection of both bacteria and bacterial DNA
in the placenta, amniotic fluid, and meconium passed within
hours of birth, the expert consensus is currently that micro-
Correspondence to:
Katri Korpela, katri.korpela @ helsinki.fi
 Microbial signals to the host Microbial signals to the host

Clostridia Enterobacteria

Relative abundances

Relative abundances
Bifidobacteria Clostridia
Enterobacteria

Bifidobacteria
Bacteroides
Bacteroides

Age Age
a Weaning b Weaning

Fig. 1. Temporal development of the relative abundances of the dominant classes of bacteria in the infant gut in
vaginally born, breastfed infants (a) and CS-born infants (b). CS, C-section.

bial colonization of the infant, that is, the establishment of
replicating microbial cells, does not commonly occur in
healthy pregnancies [1, 2]. In any case, all infants are colonized
by microbes during birth. The microbes that have been ob-
served in utero do not include the normal infant gut microbes,
which must be acquired during birth.
During birth, infants are exposed to maternal vaginal, fae-
cal, and skin bacteria, which colonize the infant body sites that
provide a suitable habitat and nutrients. Each bacterial species
has specific habitat requirements and cannot permanently
colonize inhospitable body sites. The infant gut is colonized
at birth by maternal gut microbes [3–8], while maternal vagi-
nal microbes do not colonize the infant gut [7, 9, 10]. Not all
maternal gut bacteria have the ability to colonize infants, and
it is important to note that the gut microbiota is not transmit-
ted as a whole community, but only specific members are
able to establish persistent populations. The dominant mater-
nally derived colonizers of the infant gut are bifidobacteria
and Bacteroides [6, 7]. Several species of Bifidobacterium and
Bacteroides have the capacity to ferment human milk oligo-
saccharides (HMOs) [11], which very likely explains their suc-
cess in colonizing the infant gut [5]. While certain infant gut-
adapted Bifidobacterium strains, such as B. longum subsp. in-
fantis, B. bifidum, and B. breve, are specialized in HMO
utilization [12], Bacteroides spp. are substrate generalists.
Bacteroides fragilis, thetaiotaomicron, and vulgatus are effi-
cient HMO utilizers [11]. They exploit the same pathways for
host mucus and HMO degradation and are able to degrade a
broad range of HMOs [13]. In contrast, Firmicutes and Proteo-
bacteria generally do not degrade HMOs, with the exception
of certain Lactobacillus, Streptococcus, and Enterococcus
strains with narrow HMO utilization spectra [11].
The importance of early maternal colonization is exempli-
fied by the finding that maternally derived microbes colonize
the infant essentially permanently, while non-maternal mi-
crobes are more transient [6]. This is suggestive of special
compatibility between infant and maternal microbes, poten-
tially related to partly genetically determined factors such as
breast milk oligosaccharide composition, gut mucus compo-
sition, and immune system.
While the gut microbiota is generally characterized by tre-
mendous interindividual variation, the microbiota develop-
ment during infancy follows a fairly clear and uniform pattern,
dependent on birth mode (Fig. 1). During the first year of life,
ecological succession takes place in the infant gut with a con-
sistent temporal pattern across cultures [14]. The first stools
harbour a highly diverse microbiota of mostly transient pass-
ers-by [7]. After the first few days, the diversity decreases as
specific microbes bloom while others disappear [7]. Initially
dominant species include aerotolerant taxa such as staphylo-
cocci, enterococci, and streptococci as well as enterobacte-
ria [12, 14]. Within weeks, the composition changes as breast
milk oligosaccharide-degrading bifidobacteria outgrow. Bifi-
dobacteria are usually the dominant group until weaning after
which taxa that utilize plant-based carbohydrates, mainly
members of the clostridial families Lachnospiraceae and Ru-

12 Reprint with permission from: Korpela

Ann Nutr Metab 2021;77(suppl 3):11–19
DOI: 10.1159/000518498
 Table 1. Consistent gut microbiota patterns observed in CS-born infants compared to vaginally born
Bacteroides
Bifidobacteria
Clostridia
Gram-negative pathogens
Gram-positive pathogens
minococcaceae, begin to gradually increase in relative abun-
dance [14]. The abundance of Bacteroides can be high already
very early and remains fairly stable through the early develop-
ment [14]. This likely reflects the generalist nature of Bacte-
roides species in terms of substrate utilization potential, in-
cluding utilization of both diverse dietary polysaccharides and
host-derived glycans [13]. There are some geographical dif-
ferences in the average gut microbiota composition of in-
fants. African infants show an increased relative abundance of
Bacilli, while North American infants have more Bacteroidetes
and less Actinobacteria than infants in other continents [14].
Whether these differences are caused by technical or bio-
logical reasons is unknown.
Impact of C-Section Birth on Gut Microbiota
The natural microbiota transmission from mother to infant is
disrupted by C-section (CS) birth [6], resulting in a deviation
of microbiota development, most notably in the first 6–12
months of life [14]. In fact, CS birth is perhaps the factor with
the most uniform effects on the gut microbiota across indi-
viduals and studies. A large study on nearly 600 infants found
delivery mode to be the most important factor influencing
infant gut microbiota composition, with a stronger effect than
postnatal antibiotic treatments [15]. A particularly prominent
and ubiquitous feature of the CS-born infant’s gut microbiota
is the low relative abundance of bifidobacteria and almost to-
tal lack of Bacteroides [14], that is, the taxa that would nor-
mally be obtained from the mother at birth and that are large-
ly responsible for breast milk oligosaccharide fermentation in
the infant gut. These patterns are observed in practically all
studies investigating the impact of CS on infant gut microbi-
ota (Table 1), including several large cohort studies of 600–
1,000 infants each [15–18]. In addition, the relative abundance
of pathogenic bacteria is often increased in CS-born infants.
These taxa are common in the hospital and are transmitted
from hospital surfaces to new-born infants [19].
Impact of Delivery Mode
Increased in CS Decreased in CS Reference (large birth cohort
studies or meta-analyses)
x [12, 14, 16, 17]
x [12, 14, 16]
x [12]
x [14, 18]
x [14, 16, 18]
It has been suggested that in terms of microbial contact,
CS delivery after rupture of membranes would more closely
resemble vaginal delivery than elective CS in which the infant
has no contact with maternal birth canal microbiota. While
the exposure of mother and infant to the labour process un-
doubtedly has physiological effects, there is no reason to ex-
pect much benefit for the microbial colonization from labour,
even with rupture of membranes, as maternal faecal microbes
are unlikely to be present in the birth canal of healthy moth-
ers. Indeed, the hypothesis has not received much support in
the scientific literature. Only a few studies have compared the
effect of different CS types on infant gut microbiota. Emer-
gency CS seems to result in more drastic and long-lasting
changes in infant gut microbiota [18, 20], possibly due to
health differences. To conclude, there is currently no evi-
dence suggesting a beneficial effect of rupture of membranes
or CS during labour versus pre-labour CS on infant gut micro-
biota.
Thus far, only a few studies have looked into the viral com-
ponent of the gut microbiota, the so-called gut virome, in
early infancy. Recent results show that most of the viruses in
the neonate gut are phages, that is, virus-infecting bacteria
[21]. There is evidence that phages are transmitted from the
mother at birth together with their host bacteria [5, 22]. Con-
sequently, total virus and phage diversity has been shown to
be reduced in CS-born infants at the age of 1 year, with par-
ticularly low abundances of Anelloviridae and Caudovirales in
CS-born infants [23]. In fact, the effect of birth mode on the
gut virome was much more prominent at 1 year than the ef-
fect on the bacteriome [23]. This indicates that birth is crucial
for the acquisition of the gut phageome, and the potential for
postnatal colonization, for example, via breast milk or the en-
vironment, is limited.
A meta-analysis of the dominant bacterial classes revealed
that the differences between birth modes gradually even out
and mostly disappear by the age of 12 months [14]. However,
only a few studies have investigated the associations between
birth mode and gut microbiota development after infancy.
Reduced diversity of Bacteroides spp. has been reported up
Reprint with permission from: 13
Ann Nutr Metab 2021;77(suppl 3):11–19
DOI: 10.1159/000518498
to the age of 2 years [24]. Another study found reduced rela-
tive abundances of several members of Lachnospiraceae and
Ruminococcaceae in CS-born children at the age of 4 years
[25]. Comparably, a study using fluorescent in situ hybridiza-
tion found a reduced abundance of clostridia in CS-born
compared to vaginally born 7 year olds [26]. A study using
quantitative PCR found significant differences in gut micro-
biota composition between CS-born and vaginally born
young adults [27]. In particular, relatives of Bacteroides fragilis
and Lactobacillus sakei were reduced in CS-born children.
The results suggest that birth mode effects on the gut micro-
biota may in some cases persist even into adulthood. Notably,
the latter 2 studies employed quantitative measures (FISH and
qPCR, respectively) that can estimate the absolute abundance
of the bacteria, in contrast to sequencing which only reveals
relative abundances. Clearly, future studies need to employ
methods that allow the absolute abundances to be estimated
[28].
Impact of Intrapartum Antibiotics on Infant Gut
Microbiota
In most hospitals, antibiotic treatment given to the mother is
an integral part of CS delivery. Therefore, there has been some
unclarity as to whether the effect of CS birth is exclusively due
to the lack of exposure to maternal faecal microbes or partly
due to the antibiotic treatment. Considering that regardless of
maternal microbiota composition, CS eliminates the contact
between maternal faecal microbes and infant, thus preventing
colonization, and any additional microbiota-affecting treat-
ments seem trivial. If a microbe has no contact with the infant,
it cannot colonize the infant regardless of exposure to antibi-
otics. This view was recently confirmed in a study comparing
the effects of pre- versus post-CS antibiotic prophylaxis on
the infants’ gut microbiota composition [29]. No significant
differences in gut microbiota composition were found be-
tween infants exposed and not exposed to maternal antibi-
otic prophylaxis, demonstrating that antibiotic exposure is not
a driver of the CS-induced microbiota imbalance. Along the
same lines, in an analysis of 700 infants, stratification by intra-
partum antibiotic exposure did not alter the results regarding
the effect of CS birth on infant gut microbiota [18].
The effect of antibiotic treatment during vaginal birth is an
important issue in itself, since a large proportion of infants are
exposed to intrapartum antibiotics either due to prophylaxis
or treatment of maternal infection. In some countries, all
mothers who carry group B Streptococcus are given antibi-
otic prophylaxis during labour. The aim of intrapartum antibi-
otic prophylaxis is to prevent transmission of pathogenic bac-
14 Reprint with permission from:
Ann Nutr Metab 2021;77(suppl 3):11–19
DOI: 10.1159/000518498
Table 2. Consistent gut microbiota patterns observed in vaginally
born infants exposed to intrapartum antibiotics compared to those
not exposed to antibiotics
Increased Decreased Reference
in exposed in exposed
Bifidobacteria x [30–34]
Clostridia x [20, 30, 32]
Enterobacteria x [20, 30, 32]
Streptococci x [30, 32]
Gram-negative pathogens x [18]
Gram-positive pathogens x [18]
teria from mother to infant. Antibiotics are not selective be-
tween pathogens and non-pathogens, so if the transmission
of pathogens is prevented, so must be the transmission of
non-pathogenic bacteria with a similar antibiotic sensitivity
profile.
In studies investigating the impact of intrapartum antibiot-
ics in vaginally born infants, bifidobacteria appear to be par-
ticularly strongly negatively affected by the antibiotic expo-
sure (Table 2). There is discrepancy regarding the impact of
intrapartum antibiotic exposure on Bacteroides, with effects
varying from negative [17, 20, 30] to neutral [20, 31–33] to
positive [30]. Importantly, the impact of antibiotic exposure is
modified by feeding practice, with formula-fed infants more
severely affected [34–36]. Overall, the emerging evidence
suggests that antibiotic exposure during birth can be likened
to a chemical CS from the microbial perspective, eliminating
the natural transmission of gut microbiota, especially bifido-
bacteria, from mother to infant.
Consequences of Disrupted Vertical Microbial
Transfer
Due to the age-associated microbiota succession, the host
receives specific microbial signals at specific age windows
(Fig. 1). If these signals are altered or missing due to abnormal
microbiota development, the host’s immune and metabolic
development may be affected. Deviations from normal mi-
crobiota succession in infants and young children usually oc-
cur without overt symptoms, but can increase the susceptibil-
ity to later health problems, such as allergic disease [37–39],
autoimmune diseases [40], and overweight [41].
Bifidobacteria normally form the dominant taxon in healthy
vaginally born breastfed infants, representing up to 90% of the
total gut microbiota. Due to their abundance and metabolic
capacities, they have important effects on gut microbiota
Korpela
 Increased risk of allergic disease and overweight in later childhood

Altered immune and metabolic programming

Pain, gut symptoms

Altered microbial Reduced colonisation

signals to the host resistance against
pathogens Inflammation

Altered SCFA and

bile acid compositions Increased pH Weakened gut barrier

Low abundance of Bifidobacteria

Fig. 2. Effects of low abundance of bifido-
bacteria in infants.

composition and function and host health (Fig. 2). Unfortu-
nately, bifidobacteria are perhaps the most vulnerable bacte-
rial group in infants, affected negatively by all unnatural prac-
tices such as CS birth, antibiotics, formula feeding, early
weaning, and even maternal stress. Bifidobacteria are the
dominant HMO-utilizing group in the infant gut.
As a consequence of the reduced abundance of dominant
HMO fermenters and lower fermentation activity, an increased
intestinal pH and decreased levels of the short-chain fatty acid
propionate has been observed in CS-born infants [42]. As
HMOs constitute approximately 20% of all carbohydrates in
breast milk [43], and the majority of them are degraded by gut
bacteria into short-chain fatty acids [44], HMO fermentation
represents a significant energy source for the infant. This indi-
cates that CS-born infants do not receive the full nutritional
value of breast milk. Furthermore, the altered gut pH has been
shown to be permissive for pathogen colonization [42], and
indeed several studies have observed increased abundances
of pathogens in CS-born infants (Table 1). Bifidobacteria have
anti-streptococcal activity, indicating that a low abundance of
bifidobacteria in the gut may increase the growth of patho-
genic streptococci [31]. In addition to the increase in patho-
genic microbes, there is evidence that a microbiota with re-
duced capacity for oligosaccharide utilization degrades the
intestinal mucus, potentially leading to weakened intestinal
barrier [45]. This together with the increase in pathogenic mi-
crobes is likely to stimulate inflammation both systemically
and locally. Indeed, signs of pain and distress in infants are cor-
related with low abundance of bifidobacteria [46, 47].
Not only are bifidobacteria important for early-life gut
health but also have widespread immunological effects,
which is particularly important during the time of immune
system development. Indeed, numerous studies have shown
that a low abundance of bifidobacteria, especially B. longum,
and a high abundance of Proteobacteria in infancy is associ-
ated with an increased risk of allergic diseases in later child-
hood [48–51]. Several studies have shown that low early
abundance of bifidobacteria is predictive of later adiposity, as
well [41, 52, 53].
As a demonstration of the clinical consequences of the
disrupted microbial colonization, several large cohort studies
and meta-analyses have shown that birth by CS is associated
with long-term health effects, including increased risk of
chronic immune diseases and obesity [54–56], with some of
the negative health associations reaching into adulthood [54].
While some associations may be caused by confounding ef-
fects, the clear alterations in gut microbiota and the impor-
tance of the gut microbiota for host physiological develop-
ment support the causal attribution.
Correction of Infant Microbiota after Disrupted
Colonization
CS deliveries are increasing worldwide, affecting over 50% of
infants in certain regions [57]. Additionally, over 30% of infants
are exposed to antibiotics during vaginal birth in developed
countries [58, 59]. These figures are alarmingly high, suggest-

Impact of Delivery Mode Reprint with permission from: 15

Ann Nutr Metab 2021;77(suppl 3):11–19
DOI: 10.1159/000518498
 Benefits Disadvantages

• The cheapest and least invasive • Efficacy dependent on

method maternal genotype
• Promotes normal microbiota • Ineffective at full microbiota
composition and function restoration
• Additional health benefits for
Breastfeeding both infant and mother

• Can be effective at normalising • Ineffective in formula-fed

bifidobacterial abundance
when combined with
breastfeeding or prebiotics
• Safe and widely available
Probiotics
infants without added prebiotics
• Effect is strain-specific
• Ineffective at full microbiota
restoration

• The most effective method • Requires careful screening of

• Full microbiota restoration the mother and monitoring of
• Restores vertical transmission the infant
of microbes from mother • Only in hospital
to infant • Not suitable if mother carries
Fig. 3. Studied methods of microbiota • One-time application potential pathogens
normalization in CS-born infants. CS, C-
FMT
section; FMT, faecal microbiota trans-
plantation.

ing that natural microbial colonization at birth is currently dis-
rupted in up to half of infants even in regions with low CS rates
and good antibiotic stewardship. Therefore, there is a pressing
need to restore normal gut microbiota composition in CS-
born and antibiotic-exposed infants. Only a few different
methods have been studied in terms of their efficacy in restor-
ing normal gut microbiota in CS-born infants (Fig. 3).
A study comparing the microbiota found on different in-
fant body sites within minutes after birth and meconium sam-
ples taken within the first 24 h to maternal skin and vaginal
samples showed, unsurprisingly, that vaginally born infants,
still covered with vaginal fluids at the time of sampling, har-
boured vaginal microbes, and CS-born infants harboured skin
microbes [60]. These results were interpreted to mean that
the infant gut is colonized by maternal vaginal microbes at
birth, although faecal microbiota was not sampled in the
study. However, the samples were taken too early to distin-
guish colonizers from transient passers-by. In fact, it was
shown already in 1990 that vaginal lactobacilli do not colonize
the infant gut [9], and later research has confirmed this [7, 10].
The adult vagina is a very different environment compared to
infant body sites, and consequently microbes adapted to the
vaginal environment, predominantly specific Lactobacillus
species, are unlikely to find suitable habitats on the infant
body. Human vaginal lactobacilli are highly adapted to that
environment and rarely observed in other body sites [61].
Therefore, vaginal microbes are not useful in terms of infant
gut microbiota restoration [8]. The infant gut does harbour a
low abundance of lactobacilli, but those are gut adapted or
food derived, rather than of vaginal origin [61]. Whether the
transient exposure to vaginal microbes during birth plays a
role in, for example, immune stimulation, is currently un-
known.
A few studies have tested the efficacy of probiotics as a
means of microbiota restoration in CS-born infants. A
6-month daily supplementation with a probiotic product
containing several Lactobacillus and Bifidobacterium strains
has been shown to alleviate the CS- and antibiotic-associated
microbiota disturbance in breastfed infants [62]. Importantly,
the treatment reduced the incidence of allergic diseases in
CS-born infants, demonstrating that gut microbiota-targeting
interventions have beneficial health effects [63]. However, the
effect of the probiotic intervention on the gut microbiota was
dependent on breastfeeding and failed to increase bifidobac-
teria in the exclusively formula-fed infants [62]. In another tri-
al, Lactobacillus reuteri-containing formula was fed to vagi-
nally born and CS-born infants for 6 months, with beneficial
effects on the abundances of bifidobacteria and lactobacilli in
the CS-born group [64]. These results are very promising,
demonstrating the effectiveness of such simple, safe, and
cost-effective interventions. However, probiotics represent a
small fraction of the total microbial diversity that normally
colonize infants and cannot fully restore normal gut micro-
biota. Importantly, probiotics are not maternally derived. It is
likely that maternal strains are adapted to the specific mother-
infant dyad and have been preselected by maternal immune

16 Reprint with permission from: Korpela

Ann Nutr Metab 2021;77(suppl 3):11–19
DOI: 10.1159/000518498
system and gut secretions to suit her infant and her breast milk
composition.
Breast milk is highly bifidogenic, so breastfeeding could be
expected to restore the low abundance of bifidobacteria in
CS-born infants. Exclusive breastfeeding has been found to
have a beneficial impact on the gut microbiota of CS-born
infants, especially regarding bifidobacteria [65, 66]. However,
the effect of breastfeeding is dependent on breast milk oligo-
saccharide composition [67]. The oligosaccharide 2′fucosyl-
lactose in breast milk alleviates the effect of CS birth on the
gut microbiota, but not all mothers secrete 2′fucosyllactose
[67]. The presence of fucosylated oligosaccharides in breast
milk, the so-called secretor status, is determined by the moth-
er’s FUT2 genotype. The results indicate that infants of non-
secretor mothers and formula-fed infants are at a particular
risk of gut dysbiosis if born by CS. Overall, the results show
that breastfeeding should be considered essential for infants
with disrupted gut colonization.
Due to the fact that infants are normally colonized by ma-
ternal faecal microbes during birth, birth can be seen as a fae-
cal microbiota transplantation (FMT) event from mother to
infant. In a recent pilot study, CS-born infants were given an
oral FMT, a few grams of faecal matter mixed with breast milk,
from their own mothers within hours after delivery [8]. The
treatment fully restored normal gut microbiota composition,
validating that maternal faecal microbes are the natural colo-
nizers of the infant gut. While all procedures transferring a
community of microbes from 1 person to another are inher-
ently risky due to the potential of transferring pathogens, FMT
from carefully screened healthy mothers was shown to be
safe [8].
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Conclusion
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Conflict of Interest Statement
The writing of this article was supported by Nestlé Nutrition Institute
and the author declares no other conflicts of interest.
Funding Sources
The writing of this article was supported by funding from the Univer-
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 Focus
Early childhood development of a mature and
diverse adult-like gut microbiota, which is
stable and resilient towards perturbation,
appears to be an important asset of health
Reprinted with permission from: Ann Nutr Metab 2021;77(suppl 3):21–34
Gut Microbiota Development: Influence of Diet from Infancy to
Toddlerhood
Martin Frederik Laursen
Key Insight
Upon birth, the infant gastrointestinal tract becomes colonized
with a diversity of microbes, which gradually develop into the
complex microbial community known as the gut microbiota.
The gut microbiota interacts with the gut epithelium, immune
and nervous systems and influences metabolism. Aberrations
in the gut microbiota have been associated with a range of
inflammatory, infectious, autoimmune, neurological, and
metabolic conditions. Thus, understanding the processes that
govern the initial colonization and establishment of microbes
in our gastrointestinal tract is of great importance.
Current knowledge
A key factor influencing gut microbiota development in early life
is diet. The succession of microbes from infancy to early tod-
dlerhood is driven by the type of milk-feeding and complemen-
tary feeding. Breastfeeding maintains the gut microbiota in a
state of low diversity, enabling Bifidobacterium species to pre-
dominate and favoring saccharolytic fermentation. Formula-fed
infants have a more diverse gut microbiota, characterized by
higher prevalence of potentially pathogenic taxa, such as Clos-
tridium and Enterobacteriaceae, and a higher degree of proteo-
lytic fermentation. The gradual progression towards comple-
mentary feeding results in further diversification and maturation
of the gut microbial community, as well as expansion of bacte-
rial products from saccharolytic and proteolytic metabolism.
Practical implications
Due to the abundance of human milk oligosaccharides, breast-
feeding promotes the growth of beneficial Bifidobacterium
karger@karger.com © 2021 Nestlé Nutrition Institute, Switzerland/
S. Karger AG, Basel
Birth mode
Furred
animals
Maternal
Gut microbiota
diet Antibiotics
Infant feeding
Contact with and diet
siblings
Geographic
Rural/urban location
environment
Infant feeding and diet are key forces that drive the development of the
gut microbiota from infancy and throughout childhood.
species in the infant gut. The metabolites of Bifidobacterium
species lower intestinal pH, creating an environment hostile to
pathogenic bacteria. In addition, they exert anti-inflammatory
effects, protect the gut epithelium, and regulate the immune
system. These properties may result in reduced risk of infec-
tious and immune-related diseases for breastfed infants har-
boring these species. The addition of human milk oligosac-
charides to infant formulas may constitute an avenue for im-
proving the gut microbiota in infants that cannot be breastfed.
Lack of diet-promoted maturation of the gut microbiota dur-
ing complementary feeding is associated with poor growth
and neuro-, bone, and immune development. Further studies
disentangling the complex interactions between infant diet,
gut microbiota and health are warranted in order to approach
clinical implication.
Recommended reading
Laursen MF, Bahl MI, Licht TR. Settlers of our inner surface:
factors shaping the gut microbiota from birth to toddlerhood.
FEMS Microbiol Rev. 2021;1:1–14.
 Review Article
Reprint with permission from:
Ann Nutr Metab 2021;77(suppl 3):21–34
DOI: 10.1159/000517912
Gut Microbiota Development:
Influence of Diet from Infancy to
Toddlerhood
Martin Frederik Laursen  
National Food Institute, Technical University of Denmark, Kongens Lyngby, Denmark
Key Messages
• The gut microbiota develops drastically during the first year of
life and is influenced by a range of external factors, with diet
being a major player.
• Breastfeeding versus formula feeding strongly affects gut mi-
crobiota composition and metabolism during early infancy.
Breastfeeding promotes the dominance of specific human milk
oligosaccharide-degrading Bifidobacterium species in the gut,
which may contribute to protection against infectious and im-
mune-related diseases. Formula feeding results in a more di-
verse gut microbiota with a higher prevalence of opportunistic
pathogenic bacterial taxa and proteolytic metabolism in the gut.
• Complementary feeding and the increasing dietary fiber and
protein intake induces a shift from the milk-adapted gut micro-
biota and metabolism toward one with vastly increased diver-
sity and proportions of the fiber-degrading bacterial families
Lachnospiraceae, Ruminococcaceae, and Bacteroidaceae, and
their major metabolic end products short chain fatty acids. In-
adequate maturation of the gut microbiota during comple-
mentary feeding is associated with poor growth and develop-
ment in early life.
Keywords
Gut microbiota · Diet · Breastfeeding · Complementary
feeding
karger@karger.com © 2021 Nestlé Nutrition Institute, Switzerland/
S. Karger AG, Basel
Abstract
Early life is a critical period as our gut microbiota establishes
here and may impact both current and future health. Thus, it
is of importance to understand how different factors govern
the complex microbial colonization patterns in this period.
The gut microbiota changes substantially during infancy and
toddlerhood in terms of both taxonomic composition and
diversity. This developmental trajectory differs by a variety of
factors, including term of birth, mode of birth, intake of anti-
biotics, presence of furred pets, siblings and family members,
host genetics, local environment, geographical location, and
maternal and infant/toddler diet. The type of milk feeding and
complementary feeding is particularly important in early and
late infancy/toddlerhood, respectively. Breastfeeding, due to
the supply of human milk oligosaccharide into the gut, pro-
motes the growth of specific human milk oligosaccharide
(HMO)-utilizing Bifidobacterium species that dominate the
ecosystem as long as the infant is primarily breastfed. These
species perform saccharolytic fermentation in the gut and
produce metabolites with physiological effects that may con-
tribute to protection against infectious and immune-related
diseases. Formula feeding, due to its lack of HMOs and high-
er protein content, give rise to a more diverse gut microbiota
that contains more opportunistic pathogens and results in a
Correspondence to:
Martin Frederik Laursen, mfrla @ food.dtu.dk
more proteolytic metabolism in the gut. Complementary
feeding, due to the introduction of dietary fibers and new
protein sources, induces a shift in the gut microbiota and me-
tabolism away from the milk-adapted and toward a more ma-
ture and diverse adult-like community with increased abun-
dances of short chain fatty acid-producing bacterial taxa.
While the physiological implication of these complementary
diet-induced changes remains to be established, a few recent
studies indicate that an inadequately matured gut microbiota
may be causally related to poor growth and development.
Further studies are required to expand our knowledge on in-
teractions between diet, gut microbiota, and health in the
early life setting. © 2021 Nestlé Nutrition Institute, Switzerland/
S. Karger AG, Basel
Key Insight
The healthy fetus is devoid of microbial organisms, but upon
birth our gastrointestinal (GI) tract becomes colonized with
a multitude of microbes, gradually developing into a com-
plex microbial community during the first year of life. This
community, termed the gut microbiota, interacts with our
gut epithelium, immune, and nervous systems and influenc-
es our metabolism. During the last decades, the gut micro-
biota has been linked with a range of gut inflammatory, in-
fectious, allergic, autoimmune, neurological, and metabolic
diseases. Thus, understanding the processes that govern ini-
tial colonization and establishment of microbes in our GI
tract is of great importance. A key factor influencing the gut
microbiota development in early life is diet. This article re-
view our current knowledge of how the type of milk feeding
and complementary feeding affects the microbial ecosys-
tem in the gut and how this may impact current and future
health.
Current Knowledge
Type of milk-feeding and complementary feeding dictates the
microbial succession throughout infancy and into early tod-
dlerhood. Breastfeeding keeps the gut microbiota in a state of
low diversity dominated by human milk oligosaccharide-uti-
lizing Bifidobacterium species that produce various metabo-
lites characteristic of saccharolytic fermentation. Formula
feeding allows for a somewhat more diverse gut microbiota
to colonize and is characterized by higher prevalence of po-
tential pathogenic taxa such as Clostridium and Enterobacte-
riaceae and a higher degree of proteolytic fermentation. The
gradual cessation of milk-based feeding and progression in
22 Reprint with permission from:
Ann Nutr Metab 2021;77(suppl 3):21–34
DOI: 10.1159/000517912
complementary feeding, characterized by increased con-
sumption of dietary fibers and proteins, results in a further
diversification and maturation of the microbial community in
the gut with increased abundance of the bacterial families
Lachnospiraceae, Ruminococcaceae, and Bacteroidaceae, as
well as expansion of bacterial products from saccharolytic
and proteolytic metabolism.
Practical Implications
The breastfeeding promoted Bifidobacterium species pro-
duce metabolites that lower intestinal pH and thus creates an
environment hostile to pathogenic bacteria, they exert anti-
inflammatory properties, protect the gut epithelium and reg-
ulate the immune system. These properties may result in re-
duced the risk of infectious and immune-related diseases for
breastfed infants harboring these species. Formula fed infant
are more frequently colonized with opportunistic pathogenic
and toxin-carrying bacterial species and exhibit a mostly pro-
teolytic metabolism in the gut, which may influence the risk
of infectious, but also metabolic and immune-related diseas-
es. Lack of diet-promoted maturation of the gut microbiota
during complementary feeding is associated with poor growth
and neuro, bone and immune development, although the ev-
idence base for this is extremely limited at present. Further
studies disentangling the complex interactions between in-
fant diet, gut microbiota and health are warranted in order to
approach clinical implication.
Recommended Reading
Laursen MF, Bahl MI, Licht TR. Settlers of our inner surface:
factors shaping the gut microbiota from birth to toddlerhood.
FEMS Microbiol Rev. 2021;1:1–14 [1].
Gut Microbiota Development in Early Life
Despite recent controversies, the general consensus is that
the healthy fetus is devoid of microbial organisms [2]. During
and following birth the neonatal external body surfaces and
gastrointestinal (GI) tract rapidly becomes colonized with a
multitude of microorganisms, reaching >108 microorganisms
per gram feces in a matter of hours to days [3] and over weeks
to months increase up to 1011–1012 microorganisms per gram
feces [4, 5], comparable to the microbial densities found in the
adult gut [6, 7]. The infant gut microbiota undergoes drastic
changes during the first years of life in terms of taxonomic
Laursen
 Within individuals
(alpha diversity)
Microbiota diversity
Between individuals
(beta diversity)
1 2 3 4+
a Age, years
Fig. 1. Development of the gut microbiota in early life. a Develop-
ment of gut microbiota diversity with age [97]. b Development of the
composition and diversity (Fig. 1). As a function of age, the in-
fant becomes exposed to and colonized by a range of new
microbes, increasing diversity within the individual (termed al-
pha diversity). However, differences in gut microbiota com-
position between individuals (termed beta diversity), is initial-
ly high, resulting from individual exposures to various different
environmental sources of microbes, but these differences
gradually decrease as selective forces, such as diet, converge
microbiota compositions (Fig. 1a). Thus, the individual infant
continuously acquires new microbial taxa with age, but when
comparing across infants the gut microbiota gradually be-
comes taxonomically more similar. Aerotolerant and faculta-
tive anaerobes, such as lactic acid bacteria (Lactobacillales
genera, such as Streptococcus, Enterococcus and Lactoba-
cillus) and Enterobacteriaceae are among the major initial
colonizers of the neonatal gut, but their proportions rapidly
decrease within days to weeks and the gut microbiota in
breastfed (BF) infants becomes dominated by anaerobic
breast milk-promoted Bifidobacteriaceae. As the gut micro-
biota develops during infancy and early childhood, abun-
dance of other anaerobic bacterial families, such as Bacteroi-
daceae, Lachnospiraceae, and Ruminococcaceae increase
(Fig. 1b). In early life, the gut microbiota is unstable and less
resilient to perturbation than the adult gut microbiota [8], but
the ecosystem develops in alpha diversity until around 3–5
years of age, where a stable adult-like microbiota has estab-
lished [9]. However, before this state is reached, the gut mi-
crobiota is more susceptible to modulation by external fac-
tors. Factors influencing the infant gut microbiota develop-
ment include (as reviewed in [10]); term of birth, mode of birth,
intake of antibiotics, presence of furred pets, host genetics,
Diet and Infant Gut Microbiota
Bacteroidaceae
Relative abundance
Lachnospiraceae
Ruminococcaceae
Bifidobacteriaceae
Lactobacillales Enterobacteriaceae
1 2 3 4+
b Age, years
average gut microbiota composition with age, showing the relative
abundance of the major microbial families/orders [11, 13].
siblings and family members, geographical location/popula-
tion, growing up environment (i.e., urban vs. rural), maternal
diet (through breast milk), and infant diet (Fig. 2). While all of
these individually have shown impact, infant diet has been
identified as a major contributor to gut microbiota develop-
ment in early life [11, 12]. In this regard, early infancy diet, such
as formula versus breast milk has been relatively well studied,
whereas much less is known about the effects complemen-
tary feeding on infant gut microbiota composition [13]. Given
the dominating influence of breastfeeding on gut microbial
composition as well as its protective effects against infectious
and some metabolic diseases [14], and the involvement of gut
bacteria in regulation of host immune system and metabolism
[15], it is of great importance to obtain a deep understanding
of the potential microbe-mediated host effects of feeding
mode in early infancy. Also, the complementary feeding pe-
riod (6–24 months of life) coincides with a critical period in
gut microbiota development, transitioning away from the in-
fluence of milk-based diet. As early childhood development
of a mature and diverse adult-like gut microbiota, which is
stable and resilient toward perturbation, appears to be an im-
portant asset of health [16], assessing how dietary changes in
early life affect gut microbiota trajectory is of great value.
Early Infancy Feeding and Gut Microbiota
Breast milk is the recommended first nutrition for the infant,
providing all necessary nutrients to support growth and de-
velopment, as well as passive immunity to protect against in-
fectious diseases during infancy [17]. After lactose and lipids,
Reprint with permission from: 23
Ann Nutr Metab 2021;77(suppl 3):21–34
DOI: 10.1159/000517912
 Mode of feeding
(early infancy diet)

Antibiotics Maternal diet

Term of birth
Mode of birth Furret pets

Host genetics

Geographical location/population Gut microbiota

Siblings and family members

Urban vs. rural

Complementary diet
(late infancy/toddler diet)

Fig. 2. Factors influencing the gut microbiota in early life. Gut micro- environment, such as urban versus rural and geographical location/
biota vary by term of birth, mode of birth, oral antibiotics, mode of population [10]. Mode of milk feeding and complementary diet has
feeding, maternal diet, presence of furred pets, host genetics, pres- been identified as the strongest determinants of infant gut micro-
ence of siblings and family members, complementary diet, and local biota composition [11, 12, 89] as indicated by the arrow sizes.

human milk oligosaccharides (HMOs) represent the third
most abundant component of breast milk, reaching 20–25
g/L in colostrum and ranging between 5 and 15 g/L in mature
milk [18]. HMOs are short saccharides composed of 5 mono-
meric building blocks (Glucose, Galactose, N-acetylglycos-
amine, Fucose, and Sialic acid). From these 5 building blocks,
>200 different HMOs structures have been identified [19].
Whereas lactose, protein, and lipid serve as important macro-
nutrients for the BF infant, HMOs are virtually ingestible by the
infant’s own GI saccharolytic enzymes. Therefore, HMOs pass
through the upper GI tract and reach the colon largely intact.
Throughout the GI tract, they can exert direct biological func-
tions, such as antiadhesion of pathogens and modulation of
epithelial cell responses [18], but in the colon they also serve
as metabolic substrates for gut bifidobacteria. Some of these
bifidobacteria harbor an arsenal of membrane transporters
and saccharolytic enzymes which cleave and internalize
HMOs [20] to implement the monomers into the central cat-
abolic pathways, leading to the main end products lactate,
acetate, formate, and 1,2-propandiol [21–24] (Fig.  3a). Al-
though other bacteria (e.g., Bacteroides, Lactobacillus, and
recently the Roseburia/Eubacterium group) exhibit some de-

24 Reprint with permission from: Laursen

Ann Nutr Metab 2021;77(suppl 3):21–34
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 Breastfed Formulafed

HMOs Breast milk Formula milk Proteins Peptides Amino acids

5–15 g/L 100–150 g/L
>200 different structures
Some proteins (and peptides) escape digestion and
HMOs are not digested in upper GI tract and reach the absorption in upper GI tract and reach colon were they are
colon were they are degraded into monosaccharides degraded into amino acids and fermented by gut microbes
and fermented by gut microbes

C. perfringens C. difficile

B. breve
B. bifidum
B. longum subsp. infantis
E. coli
HMO degrading Bifidobacterium species Protein degrading species
(e.g., B. bifidum and B. breve or B. longum subsp. infantis) (e.g., Clostridium, Enterobacteriaceae)

HMO catabolites
Acetate BCAA catabolites
Lactate lsovalerate
Formate Isobutyrate
1,2-Propanediol AAA catabolites
AAA-derived Co-HMO metabolism products p-cresol
ILA Phenylacatate
PLA Tryptamine
4-OH-PLA
Potential benefical health effects
Fewer opportunistic pathogens Potential detrimental health effects
Fewer antibiotic resistance genes More opportunistic pathogens
Lower gut inflammation More antibiotics resistance genes
Protect gut epithelium Higher gut inflammation
Immune system regulation Increased risk of metabolic and
Decreased risk of immune-related immune-related diseases?
a diseases? b

Fig. 3. Mode of feeding influences early infancy microbiota and sac-
charolytic versus proteolytic fermentation in the gut with potential
implications for health. a Breast milk contains HMOs, which upon
ingestion by the infant pass undigested through the upper GI tract
until they reach the colon. Here, they are digested by specific HMO-
degrading Bifidobacterium species into various metabolites with po-
tential beneficial health effects. b Formula milk contain excess pro-
teins, some of which, upon ingestion, is incompletely digested and
absorbed in the upper GI tract and reaches the colon. Partly digest-
ed protein, peptides, and individual amino acids are metabolized by
gut microbes, including opportunistic pathogenic bacteria, such as
Clostridium and Enterobacteriaceae species, yielding various amino
acid catabolites, some of which have potential detrimental health
effects. HMO, human milk oligosaccharide; AAA, aromatic amino
acid; BCAA, branched chain amino acid; GI tract, gastrointestinal
tract; ILA, indolelactate; PLA, phenyllactate; 4-OH-PLA, 4-hy-
droxypheyllactate.

gree of HMO utilization [25–27], the extensive arsenal of en-
zymes and transporters is restricted to specific species within
the Bifidobacterium genus. Whereas some species, such as B.
bifidum, employ extracellular saccharolytic enzymes to de-
grade HMOs and internalize mono- and disaccharides, other
species such and B. breve and especially B. longum subsp.
infantis transport the intact HMO structures and degrades
them inside the cell [20]. The ability of these species to effi-
ciently utilize HMOs makes them dominant members of the
gut of BF infants [11, 28, 29]. Importantly, other Bifidobacte-
rium species commonly found in the adult gut (e.g., B. ado-
lescentis, B. catenulatum, and B. angulatum) cannot utilize
HMOs and therefore are less abundant and/or infrequent
members of the gut microbiota of BF infants [20]. Thus, due
to the HMO content, breast milk represents a strong selective
factor for shaping the early infancy microbiota, dominated by
specific Bifidobacterium species (Fig. 3a). Through HMO ca-
tabolism, these Bifidobacterium species produce metabolites
with potential host-health effects. Acetate and lactate are 2
main end products of bifidobacterial HMO metabolism and
are responsible for the low pH found in feces from BF infants
[24, 30, 31], which is likely to suppress the growth of oppor-

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Ann Nutr Metab 2021;77(suppl 3):21–34
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tunistic pathogenic species within Clostridiaceae, Enterobac-
teriaceae, and Staphylococcaceae [30, 32]. Further, Bifido-
bacterium-produced acetate was found to inhibit entero-
pathogenic E. coli infection in a mouse model, by maintaining
the epithelial barrier function upon insult [33]. HMO-utilizing
Bifidobacterium species are also main producers of a limited
set of the aromatic amino acid catabolites, namely indolelac-
tate (ILA), phenyllactate, and 4-hydroxypheyllactate (4-OH-
PLA), in the gut of BF infants [29]. These metabolites are pro-
duced when the bacteria grow on HMOs (and have access to
the aromatic amino acids tryptophan, tyrosine, and phenyl-
alanine, also contained in breast milk), and interact with vari-
ous receptors expressed in immune cells, exhibit immuno-
modulatory potential and protect the gut epithelium [29, 34–
36]. This may contribute to the anti-inflammatory status
observed in the GI tract of BF infants with Bifidobacterium-
rich communities [37]. However, despite being BF, some indi-
viduals (e.g., infants born preterm, by C-section or in popula-
tion with high consumption of antibiotics which disrupts ver-
tical transmission from the maternal gut) lack Bifidobacterium
species, such as B. longum, B. breve, and B. bifidum [38, 39].
Lack of Bifidobacterium is associated with immune dysregu-
lation [40] and development of asthma [41] and autoimmune
diseases [42], but early life intervention with a HMO-degrad-
ing B. longum subsp. infantis strain in BF infants modulates
immune responses away from the allergy and autoimmunity
associated immune-phenotypes [40]. In both full term [31]
and preterm [43, 44] BF infants lacking bifidobacteria, oral ad-
ministration of HMO-utilizing Bifidobacterium spp. were
found to decrease the abundance of opportunistic patho-
gens, antibiotic resistance genes, and reduce intestinal in-
flammation, demonstrating the importance of this group of
bacteria for infant health (Fig. 3a).
On the contrary, exclusively formula-fed (FF) infants har-
bor a more diverse microbiota with lower abundances of
HMO-utilizing Bifidobacterium species, often with increased
abundances of Clostridium species (C. difficile and C. perfrin-
gens) and Enterobacteriaceae species (e.g., E. coli) [45–47]
(Fig. 3b). The lack of HMOs and higher protein content in for-
mula is likely to explain these observations. Although many
infant formula products are supplemented with fructo-oligo-
saccharides and/or galacto-oligosaccharides, these are not
as selective since they can be utilized by most Bifidobacte-
rium species (including the adult-associated B. adolescentis
and B. catenulatum) [48, 49] and additional can stimulate
growth of various Lactobacillus, Streptococcus, and Bacte-
roides species [50, 51]. Studies of the fecal metabolome in
exclusively FF (even when formula is containing galacto-oli-
gosaccharides) versus exclusively BF infants, suggest that
proteolytic rather than saccharolytic metabolism dominates
26 Reprint with permission from:
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in the FF gut [52, 53]. Whereas remnants of HMO metabolism
(e.g., fucose), their direct catabolic end products (e.g., lactate
and 1,2 propanediol) and aromatic amino acid derived co-
HMO metabolism products [29, 35] (e.g., indolelactate and
4-hydroxypheyllactate) dominate in BF infants, various differ-
ent protein fermentation products such as isovalerate, isobu-
tyrate, phenylacetate, p-cresol and tryptamine dominate in
FF infants [52, 53] (Fig.  3). Some of these metabolites (e.g.,
p-cresol and phenylacetate) are converted in the liver to me-
tabolites with detrimental effects, such as p-cresol-sulfate,
which is a uremic toxin [54] and phenylacetateglutamine
which may contribute to development cardiovascular diseas-
es [55]. Phenylacetate has also been shown to promote ac-
cumulation of fat in the liver [56]. Further, bacterially pro-
duced tryptamine in FF infant gut may shift tryptophan me-
tabolism away from serotonin and influence immune system
development [57]. Biogenic amines produced by bacterial
amino acid metabolism (such as tryptamine and histamine)
can cause GI symptoms and allergic reactions [58, 59]. In ad-
dition C. perfringens and C. difficile that often colonize the
gut of FF infants are potential toxin-carrying opportunistic
pathogens [60, 61] and the gut microbiota of FF infants has a
higher abundance of antimicrobial resistance genes [62, 63].
Last, FF infant have higher levels of inflammation markers
than BF infants [64].
In summary, breastfeeding, due to the presence of HMOs,
promotes growth of beneficial bifidobacteria in the infant gut,
which produces metabolites that may contribute to preven-
tion of GI infections and support immune system develop-
ment. By contrast, formula feeding leads to a gut microbiota
with higher abundances of opportunistic pathogens and a
mostly proteolytic gut metabolism, leading to potentially det-
rimental health effects (Fig. 3). However, it has recently be-
come technically and legally possible to add synthetically
produced HMOs (e.g., 2′FL) to infant formula in order to stim-
ulate a BF-like gut microbiota and metabolism [65]. A recent
double-blinded randomized controlled trial investigated how
addition of 2 HMOs, namely 2′FL and LNnT, to infant formula
influence the gut microbiota, and compared HMO-supple-
mented and unsupplemented formula groups with a refer-
ence group of BF infants [66]. The addition of HMOs to the
formula resulted in a microbiota configuration that was more
similar to that of the BF infants. Infants receiving HMO-sup-
plemented formula, similar to the BF infants, had higher rela-
tive abundances of Bifidobacterium and lower relative abun-
dances of Enterobacteriaceae and Peptostreptococcaceae
(the bacterial family that includes C. difficile) compared to in-
fants receiving unsupplemented formula. Although promis-
ing, this study is the first of its kind in investigating the influ-
ence on the gut microbiota and confirmatory studies are re-
Laursen
quired. Nonetheless, this strategy may constitute an avenue
to improve the gut microbiota in infants that for some reason
cannot be BF.
Complementary Feeding and Gut Microbiota
At some point during infancy milk-based feeding is no longer
adequate to cover the nutritional requirements of the infant.
Therefore, supplementation with additional foods, alongside
milk-feeding, is needed. The period of transition from exclu-
sive milk-feeding toward eating family foods is termed com-
plementary feeding and usually covers age 6–24 months [67,
68]. It is recommended by WHO to start complementary
feeding around 6 months of age [67]; however, in some coun-
tries/populations is it not uncommon to introduce other
foods as early as 2–3 months of age [69]. Early complemen-
tary feeding (before 3 months of age) has been linked to in-
creased risk of GI and respiratory infections, obesity, and al-
lergies, but this may rather be attributed to shorter duration of
breastfeeding [70]. However, late introduction to comple-
mentary feeding can also be problematic as it may result in
feeding problems, inadequate nutrition and growth [70], and
failure to induce oral tolerance [71]. Recent evidence suggests
that the infant gut microbiota development may be causally
linked to healthy growth [72, 73] and protection against food
allergies [74, 75].
Despite extensive progression, during the last decade, in
our understanding of gut microbial succession in early life, and
our continuously expanding knowledge about microbial ca-
pacity to consume various dietary compounds, we know sur-
prisingly little about causal effects of diet on gut microbiota
during complementary feeding. As outlined above, the com-
plementary feeding period coincides with a phase of drastic
changes in the gut microbiota (Fig. 1), including a rapid decline
in HMO-degrading Bifidobacterium species. The prominent
increase in alpha diversity and appearance/bloom in Bacteroi-
daceae, Lachnospiraceae, and Ruminococcaceae species,
mirror the increased complexity of the diet with the introduc-
tion of fibers from various fruits, vegetables, cereals/porridge,
and breads as well as new protein sources that is, in the form
of meats, dairy products, and legumes/lentils. As dietary fibers
and secondly (incompletely digested) proteins/peptides are
the main sources of energy for gut microbes [76] these mac-
ronutrients would be expected to have most impact on the
microbial composition. Carbohydrates (dietary fibers) are pre-
ferred energy-sources for microbes, but in shortage of these
a higher degree of proteolytic fermentation occurs in the gut
(as illustrated in the example of metabolism in the gut of FF
infants mentioned above). This balance not only depends on
Diet and Infant Gut Microbiota
diet but will also vary with passage of luminal content through
the colon, as dietary fibers will gradually become depleted [76].
The main catabolic end products of dietary fiber metabolism
are the short chain fatty acids (SCFAs) acetate, butyrate, and
propionate [77]. Whereas the former (together with lactate,
formate, and succinate) is produced in high quantities in early
infancy (e.g., by Bifidobacterium, Lactobacillus, and Entero-
bacteriaceae spp.), butyrate and propionate concentrations
are initially very low but increase with infant age [24]. In addi-
tion, products of protein fermentation, such as branched chain
fatty acids (BCFAs) are almost undetectable during breastfeed-
ing, but follow a similar pattern of increase with age [24]. These
changes in SCFAs and BCFAs are coinciding with the introduc-
tion of solid foods and cessation of breastfeeding [24]. Con-
sistent with the typical gut microbiota developmental pattern,
some key Lachnospiraceae (Anaerostipes, Roseburia, and Eu-
bacterium) and Ruminococcaceae (Faecalibacterium, Gem-
minger, and Subdoligranulum) species are butyrate producers
[24, 78], whereas Bacteroides are common propionate pro-
ducers [79]. These species harbor an extensive catalog of en-
zymes (glycosyl hydrolases) for degradation of dietary fibers
[80] into these SCFAs [79]. In addition, some Bacteroides and
Clostridium species may utilize various amino acids from di-
etary proteins to form BCFAs [81]. Thus, complementary feed-
ing is very likely to causally affect microbiota composition and
metabolism. As SCFAs have a range of physiological effects,
including influence on intestinal barrier function, host metab-
olism, immune system, and nervous system [82], these are
plausible mediators of microbiota-host interactions during
complementary feeding affecting host health (Fig.  4). Much
less is known about the physiological effects of BCFAs [83].
Longitudinal studies, designed with multiple samplings
around the period of first introduction to solid foods, have
demonstrated increased alpha diversity and abundance of
Lachnospiraceae genera (such as Blautia) after introduction of
solid foods [84, 85]. Supporting this, earlier introduction to
complementary foods is associated with higher microbial al-
pha diversity throughout infancy, including higher abundance
of the butyrate producing Lachnospiraceae genera Roseburia
[86]. In a randomized controlled trial [87] the authors com-
pared the gut microbiota in infants that were weaned tradi-
tionally (with spoon feeding) to infants weaned with a baby-
led approach (no spoon feeding, but only complementary
“finger foods” consumed), with complementary diet assessed
by 3-day dietary records. The baby-led weaned infants were
introduced to solid foods roughly 3 weeks later (6 months of
age) and consumed significantly less fruits and vegetables and
total dietary fiber at 7 months of age, which was associated
with a reduced alpha diversity and lower abundance of spe-
cific Lachnospiraceae (Roseburia facies and Eubacterium rec-
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 Meats and dairy
Fruits and
vegetables Breads and cereals

Complementary diet

Fig. 4. Complementary feeding increases Protein

gut microbial diversity and production of Dietary fiber
BCFAs and SCFAs with potential implica-
BCFAs (protein)
tion for growth, neuro, bone, and immune
Isovalerate
development. As complementary feeding Aplha diversity Isobutyrate
progresses, the milk-based component is Lachnospiraceae
gradually replaced by other foods such as Ruminococcaceae
SCFAs (dietary fiber)
Bacteroidaceae Butyrate
meats and dairy, fruits and vegetables,
Propionate
and bread and cereals, which are directly Acetate
and possibly indirectly (through the gut Gut microbiota
microbiota) impacting growth and devel-
opment. These foods contain protein and
dietary fibers that modulate the infant gut ?
microbiota, increasing alpha diversity and
?
the abundance of key bacterial families
that produce SCFAs (note that acetate is
also produced in high amount during ear-
ly infancy) and BCFAs during comple-
Neuro
mentary feeding. This “natural” develop- Growth
development
ment of the gut microbiota and its me-
tabolites is associated with healthy
growth, neuro, bone development, and
Bone
appropriate immune system regulation. Immune system regulation
development
BCFAs, branched chain fatty acids; SCFAs,
short chain fatty acids.

tale) and Ruminococcaceae (Faecalibacterium prausnitzii)
species during complementary feeding [87]. In addition, inde-
pendent of the feeding groups, consumption of breads and
cereals, as well as meat products at 7 months of age were
positively associated with alpha diversity at 12 months of age
[87].
A study including 9 months old Danish infants assessed the
complementary feeding diet by 7-days dietary records and a
used multivariate statistical analysis to generate the dietary
pattern “family foods” [88, 89], describing the infant’s progres-
sion in complementary feeding (from milk-based toward
family foods). Progression in complementary feeding, char-
acterized by higher dietary fiber and protein intake, correlated
significantly with gut microbial alpha diversity [89]. Specifi-
cally, consumption of rye bread and cheese and meat prod-
ucts were main food groups driving these associations [89],
suggesting that these complementary foods are contributing
to the diversification of the infant gut microbiota, at least in
this cohort. Importantly, these associations were not solely
mediated by cessation of breastfeeding, as they were found
in both partially BF and partially FF infants [13]. Progression in
complementary feeding also correlated positively to the
abundance of several Lachnospiraceae and Ruminococcace-
ae spp., but negatively to Bifidobacterium [13], thus marking
the transition of the breast milk promoted Bifidobacterium-
rich gut community toward the fiber and protein promoted
(more diverse) gut microbial community, characterized by
butyrate, propionate, and BCFA-producing bacteria (Fig. 4).
Indeed, interventions with meat as a main complemen-
tary food compared to dairy [90] or cereals [91] has revealed

28 Reprint with permission from: Laursen

Ann Nutr Metab 2021;77(suppl 3):21–34
DOI: 10.1159/000517912
significantly increased abundance of butyrate-producing
Lachnospiraceae genera. A recent 7-week intervention
study comparing a refined grain cereal product to a whole
grain cereal product as first complementary food, demon-
strated a significant increase in Bacteroides and Lachno-
clostridium (Lachnospiraceae family) and a decrease in
Escherichia (Enterobacteriaceae family) over time only in
the whole grain cereal group. However, other intervention
with specific complementary foods such as legumes versus
corn-soy flour [92] or meat versus cereals [93] show limited
differential impact on the gut microbiota, possibly due to
these infants still receiving a high proportion of breast milk
in their diet or other study participant characteristics. Ulti-
mately, more randomized controlled trials are needed to
establish direct causal proof of links between specific com-
plementary foods and specific microbial taxa. Nonetheless,
as outlined below, a few recent studies indicate that spe-
cific Lachnospiraceae species affected by complementary
foods may influence growth and development and protect
against food allergies (Fig. 4).
Stunted undernourished Bangladeshi infants/toddlers
have an immature gut microbiota compared to same age
healthy well-nourished counterparts [94], likely as a result of
inadequate and/or delayed complementary feeding. Trans-
plantation of the gut microbiota, using fecal samples from
stunted, malnourished versus healthy infants, into germ-free
mice (devoid of microbes) has revealed differential growth
patterns, recapitulating the growth phenotypes observed in
these infants [73]. Oral introduction of some of the Lachno-
spiraceae species (such as Ruminococcus gnavus and Clos-
tridium symbiosum) that appear during complementary feed-
ing in healthy infants was shown to promote growth and bone
development in mice previously transplanted with the mal-
nourished gut microbiota [73]. Furthermore, complementary
food intervention in stunted malnourished Bangladeshi tod-
dlers rationally designed to repair the immature gut microbi-
ota suggest that a combination of banana, chickpea, soy, and
peanut matures the gut microbiota (increase abundance of
some Lachnospiraceae and Ruminococcaceae species) and
influence blood markers of healthy growth, bone, immune,
and neurodevelopment [72, 95]. However, despite having a
similar energy, macronutrient and fiber content, other ratio-
nally designed microbiota-directed complementary food
combinations were not effective, illustrating the need for a
deeper understanding of the effects of specific food combi-
nations.
Recent studies have demonstrated that blood concentra-
tions of food specific immunoglobulin E (IgE), an important
mediator in food allergy, is elevated in germ-free mice com-
pared to conventional (colonized) counterparts after weaning
Diet and Infant Gut Microbiota
onto an antigen-containing diet [75]. In addition, colonization
of previously germ-free mice with gut commensal microbes
inhibit the increase in food-specific IgE and disruption of the
gut microbiota by oral administration of antibiotics to con-
ventional mice increases food-specific IgE concentrations in
blood [75]. Thus, gut microbes play a causal role in regulation
of IgE levels during the food antigen exposure and may affect
the risk of developing food allergy. Another study performed
gut microbiota transplantation from healthy or cow’s milk al-
lergic 6-month old infants into germ-free mice and chal-
lenged them with the cow’s milk allergen β-lactoglobulin [74].
In contrast to the mice colonized with the cow’s milk allergic
infant gut microbiota, mice colonized with the healthy infant
gut microbiota were protected against allergic responses. In
the healthy infant gut microbiota, Anaerostipes coccae, a
Lachnospiraceae species that increases during complemen-
tary feeding and associates with consumption of vegetables
and fruits [96], was identified as a bacterial taxon that medi-
ated this protection. By comparing mice colonized with cow’s
milk allergic microbiota to mice mono-colonized with A. cac-
cae it was demonstrated that this species protected the mice
against allergic food response [74]. While results from these
studies are encouraging, it must be emphasized that our cur-
rent knowledge on the effects of different complementary
foods on gut microbiota and thereof potential health impact
is still extremely limited.
Future Directions
Undoubtedly, diet plays a major role for the succession of the
infant gut microbiota, yet we still have a very incomplete un-
derstanding of the details. There is a need for well-designed
and well-powered interventions in various different popula-
tions in order to assess the impact of formula supplemented
with different HMOs on gut microbiota and metabolome in
early infancy, including relevant measures of host impact,
such as prevalence of infectious and immune-related diseas-
es, during and after intervention. There is a lack of studies with
detailed dietary recordings coupled to measurements of the
gut microbiota at a finer resolution scale. In example, to im-
prove our knowledge on dietary factors shaping the infant gut
microbiota, we need prospective cohort studies with detailed
information about infant diet, obtained from multiple con-
secutive days of dietary recordings throughout periods of
complementary feeding, combined with dense fecal sampling
and state of the art microbiome (obtaining species and strain
level resolution) and metabolome profiling, as well as deep
genomic and phenotypic characterization of fecal microbial
isolates to decipher molecular mechanisms. These studies
Reprint with permission from: 29
Ann Nutr Metab 2021;77(suppl 3):21–34
DOI: 10.1159/000517912
should be accompanied by intervention studies, in relevant
study populations, designed to introduce specific comple-
mentary foods using frequent longitudinal sampling and the
state-of-the-art methods of microbiome and metabolome
profiling. Hopefully, this will lead to identification of key diet-
microbe-metabolite interactions that beneficially impact as-
pects of host physiology, such as metabolism and immunol-
ogy. Ultimately, if evidence is adequate, specific dietary inter-
vention should be tested in disposed/susceptible study
populations (e.g., disposed to allergic or autoimmune diseas-
es or individuals at risk of malnourishment and stunting) to
provide proof of applicability.
Summary and Conclusions
The infant gut microbiota undergoes significant temporal de-
velopment during the first years of life. A multitude of factors
such term of birth, mode of birth, antibiotics, contact with
furred animals and siblings/family members, local environ-
ment, geographical location, and maternal and infant diet af-
fect the trajectory of this development. Of these, infant/tod-
dler diet has been identified as one of the most influential
factors. The neonatal gut microbiota is initially characterized
by random colonization of environmental microbes, but in a
matter of days to weeks the gut ecosystem and the type milk-
feeding selects for microbes adapted to the GI environment
and equipped to consume the indigestible (or incompletely
digested) constituents of breast or formula milk. Breast milk
feeding selects for specific HMO-degrading Bifidobacterium
species that vastly dominate the gut ecosystem and produce
metabolites with beneficial effects likely contributing to the
protection against GI infections and supporting immune sys-
tem development. On the contrary, formula-milk feeding is
less selective and the gut microbiota of FF infants is more di-
verse with a higher frequency and proportion of protein de-
grading opportunistic pathogenic species. This may increase
risk of GI infections and the apparent mostly proteolytic me-
tabolism is possibly detrimental to health as some of the me-
tabolites are associated with kidney, liver, and cardiovascular
diseases in adults.
As the infant transitions into complementary feeding the
gut microbiota diversifies along with the increasing amounts
of dietary fiber and proteins in the diet. Specifically, members
of the bacterial families Lachnospiraceae, Ruminococcace-
ae, and Bacteroidaceae capable of consuming various dietary
fibers from fruits, vegetables, breads and cereals, and possibly
proteins from meat and dairy sources, increase in abundance
during complementary feeding. This leads to a shift in gut
metabolism with the appearance or increase of the SCFAs
30 Reprint with permission from:
Ann Nutr Metab 2021;77(suppl 3):21–34
DOI: 10.1159/000517912
butyrate and propionate as well as the BCFAs. While the phys-
iological relevance of these bacterial taxa and metabolites
remains to be established in this early life context, recent re-
search suggests that they may be important for healthy
growth and development and protection against food aller-
gies.
Clearly, the question of when to start introducing various
complementary foods in order to achieve optimal health and
gut microbiota progression is central. Early introduction,
combined with reduced or lack of breastfeeding is presum-
ably not beneficial, as it will disrupt the Bifidobacterium dom-
inated gut community, associated with protection against in-
fectious and immune-related diseases. On the other hand,
very late introduction as observed in populations where food
is scare and breastfeeding of the infant is prolonged without
adequate complementary foods would also be expected to
be adverse, given the inability of the gut microbiota to mature
when deprived of key nutrients and the obvious negative im-
plications for growth and development. Undoubtedly, further
studies are required to improve our understanding of the in-
triguing links between complementary feeding, gut microbi-
ota, and health.
Conflict of Interest Statement
The writing of this article was supported by Nestlé Nutrition
Institute and the author declares no other conflicts of interest.
Funding Sources
The writing of the manuscript was supported by Nestlé Nutri-
tion Institute.
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34 Reprint with permission from: Laursen

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 Focus
When possible, studies should be conducted in more than one
location so that we can better understand microbe-health
relationships that are specific to one culture/region versus those
that are common to all

Reprinted with permission from: Ann Nutr Metab 2021;77(suppl 3):36–48

Microbiomes and Childhood Malnutrition: What Is the Evidence?

Michelle K. McGuire and Mark A. McGuire

Key insights
Feeding practices,
genetics, gender,
Malnutrition is a complex state that goes beyond the nutrient status, lifestyle,
deprivation or excess of nutrients. There is growing evidence endemic microbial
exposure, location
that dysbiosis of the enteric microbial community may play
an important role in predisposing an individual to both
extremes of malnutrition. Healthy and malnourished infants Healthy children Malnourished children
(Microbiome) (Microbiome)
have different fecal microbiomes, and these differences may
precede the onset of physiological symptoms. This highlights
the importance of the gut microbial community as a
modulator of risk or resilience towards the effects of
ࣼ Higher diversity ࣼ Lower diversity
malnutrition. However, other factors are also involved in ࣼ Prevalence of certain strains ࣼ Different microbial profile
predisposing an individual towards nutrition-related disorders, (Bacteroidetes, Bifidobacteria) from that of healthy children
ࣼ Steadily evolves over time ࣼ Age-delayed
including genetic background, gender, infant feeding
practices, and nutritional status.
Many factors influence the development of the gastrointestinal micro-
biome, resulting in key differences in the microbiomes of healthy and
malnourished children.
Current knowledge
The greatest opportunity for altering the enteric microbiome have a higher diversity of bacterial species, as well as a greater
to optimize lifelong health lies within the first 1,000 days of life. prevalence of certain strains, such as Bacteroidetes and bifido-
Underpinning this is the concept that variations in fetal and bacteria. Healthy fecal microbiomes also evolve with age. An-
infant programming through environmental exposures can other relevant finding is that microbial communities vary along
impact health in later life. This also fits with the hypotheses that the gastrointestinal tract, suggesting that the small intestinal mi-
(1) early life exposure to microorganisms protects against al- crobiome should also be taken into account when performing
lergic disease (hygiene hypothesis) and (2) early life exposure studies. Finally, there is no one-size-fits-all definition of a
to those microorganisms with which we have co-evolved is “healthy” microbiome. Future studies should account for these
needed to develop tolerance and avoid unhealthy inflamma- and other confounding variables in the study design and data
tory responses (“related old friends” hypothesis). However, the analyses. Going forwards, we should aim to advance beyond the
current state of knowledge is not sufficient to enable us to cataloging of microbes towards understanding their functions
conclude how variations in the early life microbiome contrib- within the organism as a whole.
ute towards the systemic manifestations of health or disease.

Recommended reading
Practical implications Robertson RC, Manges AR, Finlay BB, Prendergast AJ. The hu-
Studies comparing the gastrointestinal microbiomes of healthy man microbiome and child growth – first 1000 days and be-
and malnourished children have revealed that healthy children yond. Trends Microbiol. 2019;27(2):131–47.

karger@karger.com © 2021 Nestlé Nutrition Institute, Switzerland/

S. Karger AG, Basel
 Review Article

Reprint with permission from:

Ann Nutr Metab 2021;77(suppl 3):36–48
DOI: 10.1159/000519001

Microbiomes and Childhood

Malnutrition: What Is the Evidence?
Michelle K. McGuire a Mark A. McGuire b    

aMargaret Ritchie School of Family and Consumer Sciences, University of Idaho, Moscow, ID, USA;
bDepartment of Animal, Veterinary, and Food Sciences, University of Idaho, Moscow, ID, USA

Key Messages in individuals and populations (referred to as the double bur-

• Healthy and malnourished infants have different fecal
microbiomes, and this difference can precede onset of
malnutrition suggesting that microbiome community
structure might impart resilience and/or risk for malnutrition.
• Early differences in fecal microbiomes can be associated with
later risk for overweight and obesity, but these differences
vary by population and sometimes by sex.
• Longitudinal studies designed to prospectively assess
variation among infant gastrointestinal microbiomes (and
their likely sources, such as the human milk microbiome),
infant feeding practices, nutritional status (including
micronutrients), and systemic and intestinal inflammation are
needed to tease apart these potentially important
relationships.
Keywords
Growth · Health · Human milk · Infant · Malnutrition ·
Microbiome · Human nutrition · Obesity
Abstract
Both undernutrition and overnutrition continue to represent
enduring global health crises, and with the growing implica-
tions of both forms of malnutrition occurring simultaneously
den of malnutrition), understanding their biological and envi-
ronmental causes is a primary research and humanitarian ne-
cessity. There is growing evidence of a bidirectional associa-
tion between variation in the gastrointestinal (GI) microbiome
and risk of/resilience to malnutrition during early life. For ex-
ample, studies of siblings who discordantly do or do not de-
velop severe malnutrition show clear differences in the diver-
sity and composition of fecal microbiomes. These differenc-
es are transiently lessened during refeeding but re-emerge
thereafter. These findings have been somewhat recapitulated
using animal models, but small sample sizes and limited range
complicate interpretation of results and applicability to hu-
mans. Mechanisms driving these differences are currently un-
known but likely involve a combination of inflammatory path-
ways (and perhaps antioxidant status of the host) and effects
on nutrient availability, requirements, and utilization by both
host and microbe. A less robust literature also suggests that
variation in GI microbiome is associated with risk for obesity
during childhood. The putative impact of GI microbiomes on
malnutrition is likely modified by a variety of important vari-
ables such as genetics (likely driven, in part, by evolution),
environmental pathogen exposure and its timing, dietary fac-
tors, and cultural/societal pattern (e.g., use of antibiotics).
Given the growing double burden of malnutrition, this topic

karger@karger.com © 2021 Nestlé Nutrition Institute, Switzerland/ Correspondence to:

S. Karger AG, Basel Michelle K. McGuire, smcguire @ uidaho.edu
demands a focused interdisciplinary approach that expands
from merely characterizing differences and longitudinal
changes in fecal microbes to examining their functionality
during early life. Understanding the complex composition of
human milk and how its components impact establishment
and maintenance of the recipient infant’s GI microbiome will
also undoubtedly shed important light on this topic.
© 2021 Nestlé Nutrition Institute, Switzerland/
S. Karger AG, Basel
Introduction
Preventing and treating malnutrition, particularly in children,
remain a global health priority [1]. Preventing undernutrition is
important because inadequate food intake increases risks for
nutritional deficiencies, impaired immune function, and de-
layed cognitive and physical development. Similarly, being
overweight increases one’s risk for a variety of diet-related
noncommunicable diseases such as heart disease, stroke, di-
abetes, and cancer. Undernutrition and overnutrition have, in
the past, been considered somewhat separate health chal-
lenges affecting different populations. However, with indus-
trialization has come the simultaneous double burden of mal-
nutrition (DBM), even within the same household or popula-
tion [2]. Indeed, the prevalence of obesity continues to rise on
a global basis while nutrient deficiencies remain common [2,
3].
Whereas the drivers of this somewhat new nutrition reality
of DBM are clearly complex, malnutrition is often not as sim-
ple as nutrient deprivation or excess [4, 5]. For example, there
is mounting evidence that a dysbiosis of enteric microbes
might play a role in predisposing an individual to both ex-
tremes of the nutrient status continuum (see Table  1 for a
summary of common measures of malnutrition in children).
The opportunity to alter the enteric microbiome for optimiz-
ing lifelong health is likely greatest in the first 1,000 days of life
[6], dovetailing nicely the concept of the developmental ori-
gins of health and disease (DOHaD), which proposes that vari-
ation in fetal and infant programming through environmental
exposures impact lifelong health [7], and the hygiene hypoth-
esis and related old friends hypothesis which posit that (1)
early life exposure to microorganisms protects against allergic
disease [8] and (2) early life exposure to microorganisms with
which we have co-evolved is needed to develop tolerance so
they do not evoke unhealthy inflammatory responses [9], re-
spectively. Another important concept is eco-homeorhesis
which posits that what is “normal” and “healthy” in 1 ecosys-
tem may not confer optimal fitness in another [10]. Indeed, it
is possible that there exists no 1-size-fits-all construct when
it comes to a healthy GI microbiome in early life. Rather, as-
Infant Nutrition, Microbiomes, and Health
sembly of an “optimal” GI microbiome likely differs depending
on a multitude of factors such as genetics, endemic microbes,
chronic food availability, and cultural practices. Relationships
among these concepts, nutrition, the enteric microbiome,
and short- and long-term health are illustrated in Figure 1.
The focus of this study is to, largely through the lenses of
these various overlapping concepts and hypotheses, review
the complex interactions among early life nutrition, establish-
ment and variation in GI microbiomes, and overall health and
nutritional status during childhood. We mainly discuss human
studies of general malnutrition, but also briefly consider indi-
vidual nutrients (particularly iron) and selected animal studies.
Factors Impacting Early Establishment of GI
Microbiome
Whereas it has long been held that the healthy infant is born
with a sterile GI tract, some evidence suggests that initial mi-
crobial colonization may occur in utero [11, 12], although
skepticism remains (e.g., [13]). Nonetheless, the origin of a
large proportion (if not all) of the pioneering bacteria is re-
lated to exposure during birth. Azad and colleagues estimated
that ∼70% of colonizing GI bacteria originate from maternal
feces in vaginally delivered infants, whereas a smaller propor-
tion (∼40%) originate from this source in infants born via Cae-
sarean section [14]; whether the Caesarean section is planned
or emergency as well as antibiotic use also appear to impact
early colonization [15, 16]. However, not all studies have re-
ported an enduring impact of delivery mode on GI microbi-
ome [17].
After initial colonization, feeding mode directs establish-
ment and stabilization of the GI microbiome – likely driven by
differences between the components found in human milk
(HM) and formula. The fecal microbiota of breastfed infants is
not only different in composition than that of formula-fed in-
fants, but it is also more stable and less diverse [15, 18]. There
are likely many reasons for these differences including HM
oligosaccharides (HMOs) indigestible by human enzymes but
selectively metabolized by microbes [19]. Even within exclu-
sively breastfed infants, different HMO profiles have been as-
sociated with variation in infant fecal microbiomes [20, 21].
Because HMO profiles are largely driven by maternal genetics
[22], it is possible that variation in HMOs and the resulting in-
fant GI microbiome represent eco-homeorhetic adjustments
to “normal” to support optimal health in a particular environ-
ment and may also support the “old friends” hypothesis in that
these microbial profiles nurture endemic, nonpathogenic
bacterial taxa in such a way that they are tolerated by and per-
haps even benefit the host. HMO concentrations and profiles
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Table 1. Common measures of malnutrition in children

Condition Commonly used metric and/or sign

Low birth weight, g <2,500

MAM Weight-for-height Z score between −2 and −3 without bilateral pitting edema
SAM Weight-for-height Z score <−3, or mid-upper arm circumference <115 mm, or bilateral pitting edema
Kwashiorkor SAM with bilateral pitting edema
Marasmus SAM without bilateral pitting edema
Underweight Weight-for-age Z score <−2
Stunting Height-for-age Z score <−2
Wasting Weight-for-height Z score <−2
Overweight Body mass index Z score >2 or weight-for-length Z score >97th percentile
At risk of overweight Weight-for-length Z score >85th percentile

MAM, moderate acute malnutrition; SAM, severe acute malnutrition.

Hygiene and Old Friends Hypotheses: Safe exposure to endemic, environmental

microbes in early life is important to development of tolerance.

Feeding choices

Eco-homeorhesis: Genetic, Genetics

behavioral, and Emerging
environmental factors Environmental evidence
likely customize microbes Overweight suggests that
microbiome for optimal microbial
fitness for a given location dysbiosis can
and culture. The thrifty Bacterial dysbiosis increase risk of
gene hypothesis fits within and/or resilience
this eco-homeorhetic to malnutrition.
construct. A B1 B2 B3

Well nourished
B5 B6 B7 C

B9 B12 D Fe

E Protein Se K

Nutrient intake

Undernourished

Developmental Origins of Health and Disease: Establishment of the

gastrointestinal microbiome in the “1st 1,000 days” impacts lifelong health.

Fig. 1. Putative relationships among early-life microbial exposures, nutrition GI microbiomes, and malnutrition. GI, gastrointestinal.

can also vary by season, suggesting that environment (e.g., of milk’s own inherent microbiome is likely one of the most
food availability, pathogens) can impact their synthesis [23]. important [24]. Like HMO profiles [25], those of the HM mi-
Many additional components (e.g., lysozyme and lactofer- crobiome (HMM) vary greatly among women and globally
rin) in milk are known to affect GI microbes, but the presence [26]. Although milk and infant fecal microbiomes differ sub-

38 Reprint with permission from: McGuire/McGuire

Ann Nutr Metab 2021;77(suppl 3):36–48
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stantially in composition, variation in one is highly correlated
with variation in the other [27, 28]. The origins of the HMM are
thought to be a combination of maternal skin and GI tract,
also fitting nicely into the old friend hypothesis and highlight-
ing the likely importance of a tempered introduction of envi-
ronmental microbes via breastfeeding/HM to develop toler-
ance in the infant. It is also noteworthy that this microbial
communication between mother and infant is likely bidirec-
tional, with continuous inoculation of the mammary gland via
the infant’s mouth [29].
Introduction of complementary foods also impacts the in-
fant’s GI microbiome [30, 31] although cessation of breast-
feeding [32] might be more important suggesting that HM
helps stabilize the infant’s GI microbiome. Changes that occur
around weaning are associated with shifts in the microbial
community’s capacity to metabolize typically consumed
foods [32]. In addition, there are shifts in species that produce
short-chain fatty acids and B vitamins (e.g., folate and vitamin
B12), as well as taxa involved in amino acid metabolism [30,
33–35]. To our knowledge, there have been no studies de-
signed to understand whether these longitudinal changes in
GI microbiomes and their functional capacities are custom-
ized to best suit the future dietary landscape, but this has been
hypothesized [34].
In summary, many factors impact the diversity and com-
position of the GI microbiome during early life, and there ex-
ists substantial variation in GI microbiomes (and their func-
tional capacities) among infants. Central to the aim of this
manuscript, we ask whether this variability predisposes an in-
fant to secondary malnutrition – be it undernutrition or over-
nutrition – and if so, is this a one-size-fits-all construct or is
this effect modified by environment? Related, can nutritional
status (e.g., iron) of an infant impact GI microbiome to predis-
pose the infant to acute and/or long-term health outcomes,
and if so, is this relationship context specific?
GI Microbiomes and Childhood Undernutrition
It is well established that fecal microbiomes differ between
lean and obese adults and that – although these differences
are clearly due to a complex constellation of genetics, diet,
and other biological and behavioral patterns – there might be
particular GI microbiomes that impart resilience to or risk of
both under- and overnutrition. For instance, some taxa are
genetically equipped to harvest a greater proportion of the
energy in our foods than others, supplying volatile short-
chained fatty acids that can serve as ATP sources to entero-
cytes and colonocytes [36]. Individuals hosting a greater per-
centage of these taxa likely garner more calories from a given
Infant Nutrition, Microbiomes, and Health
amount of food (example of the thrifty gene concept). None-
theless, it is important to remember that only a small propor-
tion (∼5–10%) of energy obtained from the diet comes
through microbial action in the large intestine. Rather, most is
obtained via digestion by host enzymes and absorption in the
stomach and small intestine. Some taxa can also synthesize
essential vitamins (e.g., vitamin B12), therefore supplying these
nutrients to the host and possibly shielding him/her from de-
ficiencies. While the absorption of vitamin B12 synthesized in
the colon or cecum is unlikely [37], some bacteria may ben-
efit from increased levels. As such, variation in early-life GI
microbiome might also predispose an infant/child to malnu-
trition and, conversely, variation in early life nutritional status
may have acute and/or enduring impacts on the GI microbi-
ome. It is also possible that variation in the GI microbiome
plays a role in environmental enteric dysfunction, a subclinical
and chronic disorder characterized by poor growth, blunting
of the small intestine villi, nutrient malabsorption, and chron-
ic inflammation [38].
To our knowledge, the first study comparing GI microbi-
ome between healthy and malnourished children was con-
ducted by Monira et al. [39] and included 7 Bangladeshi chil-
dren with low weight-for-height and 7 healthy children of
study staff. There were substantial differences in both diver-
sity and composition of the GI microbiome: compared to
the malnourished children, samples from healthy children
had higher bacterial diversity and greater relative abun-
dance of Bacteroidetes, whereas Klebsiella and Escherichia
were 174- and 9-fold lower. Interpretation, however, was
severely limited by the fact that the study was small, cross-
sectional, and the 2 cohorts were from different socioeco-
nomic groups.
This study was followed by another [40] which compared
GI microbiome of Malawian twins discordant for kwashiorkor
– a form of severe acute malnutrition (SAM) characterized by
nutritional edema. GI microbiomes of 9 healthy, same-gender
twins were compared with those of 13 same-gender twin
pairs who were discordant for kwashiorkor. Principal coordi-
nates analysis was used to visualize composite variation in
inferred fecal microbiome functionality with age. Overall met-
abolic functions of the healthy twin pairs’ fecal microbiomes
were found to steadily change over time as would be expect-
ed; the same was found for the healthy co-twins from discor-
dant twin pairs. Conversely, metabolic functions of feces col-
lected from children with kwashiorkor were age-delayed, and
although they transiently became more like healthy children
of similar ages during feeding this regressed when feeding
was ceased. To assess causality, this research group then
treated gnotobiotic mice with feces collected from twins in
this study [41]. Results from mice (5 per single stool inoculum)
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Ann Nutr Metab 2021;77(suppl 3):36–48
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in which transplantation efficiency was >50% demonstrated
reduced growth and lean mass in mice receiving stool from 5
stunted/underweight donors compared to mice receiving
stool from 3 healthy donors. It is unclear if this reduction in
replicates from the total donor population allowed for com-
parison between the mice inoculated with feces from twin
cohorts or from a similar age cohort. A direct comparison
within twin pairs would strengthen the confidence in the re-
sults. Further, transplant efficiency was defined based upon
operational taxonomic unit (OTU) diversity relative to the un-
cultured donor sample, which suggests that relative abun-
dance of bacterial taxa is not as important as mere presence
in the community. Finally, clarity on food intake of the mice
would be helpful in understanding the changes in growth. The
authors [41] report that food intake was not different for 4 days
during the 35-day period, but it is not clear what days intake
was measured. Random forest-based modeling identified Bi-
fidobacterium longum in discriminating weight and lean mass
gain when utilizing feces from children at 6 months of age
while Faecalibacterium prausnitzii was more important when
utilizing feces of children at 18 months of age. The potential
impact of age-related GI microbiome could be critical for po-
tential success of interventions impacting malnutrition and
emphasizing the importance of clear comparisons within an
age group.
The same group then characterized what it considered
to be healthy GI microbiome projections during the first 2
years of life in 12 healthy Bangladeshi infants and estab-
lished a “microbiome-for-age Z score” (MAZ) to assess GI
microbiome “maturity” [42]. This metric was applied to 64
Bangladeshi infants with SAM before and after nutritional
treatment; SAM was associated with microbiome immatu-
rity, and although MAZ score improved upon treatment,
there was regression to immaturity afterward. In a subset of
malnourished infants who provided samples before antibi-
otic administration (common to SAM treatment), lower mi-
crobial diversity prior to feeding was not attributable to an-
tibiotics. The authors concluded that microbiome immatu-
rity may play a role in predisposing young children to
malnutrition. However, it should be noted that feeding
mode and duration of exclusive breastfeeding were not
controlled for, even though the authors provided some ev-
idence that intake of infant formula was associated with
higher maturity values. This weakness in experimental de-
sign makes its results difficult to interpret.
This study was followed by another that (1) assessed GI
microbiome in 27 twin pairs and 2 sets of triplets (from the
same population) with healthy growth patterns, (2) compared
findings with those published previously by Subramanian et al.
[42] in Bangladesh (they were similar), (3) and related MAZ to
40 Reprint with permission from:
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subsequent growth in the Malawian cohort [43]. They found
that MAZ at 12 months predicted future ponderal (but not lin-
ear) growth at 18 months, but again myriad confounding and
possibly interacting factors (e.g., maternal nutritional status,
variation in milk composition, and infant feeding practices)
cannot be ruled out. In fact, lack of controlling for these and
other confounding factors either in experimental design or
statistical analysis is a major weakness of most of the research
conducted on this topic and the reader will notice that this
theme represents a common thread running through this
study. A subsequent study [44] also suggested an expected
maturation in the GI virome among the Malawian twin cohort;
this trajectory was impaired in SAM and not repaired with
refeeding.
The researchers then used a defined 25-bacterium com-
munity isolated from a single fecal sample collected from 1
stunted Malawian infant transplanted into germ-free mice fed
a diet representative of that which is consumed in Malawi to
test whether the addition of bovine milk oligosaccharides
(BMOs) could rescue growth [43]. Mice (4–5 per group) fed
the BMO-fortified diet had increased body weight compared
to mice fed the control diet with or without inulin. Unfortu-
nately, comparison to mice transplanted with microbiota
present in healthy Malawi infants was absent in the experi-
mental design. Mice not fed BMO had sustained body weight
losses for 30 days after colonization differing from the work
of Blanton et al. [41] where only temporary body weight (<8
days) losses were noted in any group. Further, while food con-
sumption was not statistically different, the mice fed the
BMO-fortified diet consumed 25% more than controls. Similar
effects were found with gnotobiotic piglets. Additional studies
utilizing larger sample sizes and appropriate controls are
needed to further understand these findings.
It is interesting to speculate what causes or prevents “mat-
uration” of the fecal microbiota, but Vonaesch and colleagues
[45] provide strong evidence that stunted children may have
small intestine bacterial overgrowth dominated by taxa typi-
cally found in the oropharyngeal cavity. Their data suggest a
“decompartmentalization” of the GI tract and are not only in-
teresting from a mechanistic point of view but also remind us
that microbial communities vary along the GI tract, and that
the fecal microbiome likely provides a poor representation of
what is likely more important for nutrition, that is, the small
intestine microbiome.
In another related study utilizing data from the Bangladeshi
and Malawi cohorts described above, Gough and colleagues
[46] used a case-control approach to investigate whether
particular fecal microbial community structures or individual
taxa are associated with linear growth faltering. A total of 10
cases and 8 controls from Malawi, and 6 cases and 5 controls
McGuire/McGuire
from Bangladesh, where cases were defined as HAZ ≤−3 and
controls as −3 < HAZ ≤−2. They found that reduced fecal mi-
crobiome diversity and increased relative abundance of Acid-
aminococcus sp. predicted more severe later growth deficits
and hypothesized that microbial fermentation of glutamate
might play a role in this association. To test this, they used
KEGG enzyme abundance data for the Malawi cohort and
found that the abundance of genes encoding key glutamate-
metabolizing enzymes was negatively associated with future
height-for-age Z score. However, cases in both cohorts were
younger and relatively shorter (HAZ) than controls making it
impossible to disentangle whether the differences in fecal mi-
crobiome are related to age-related factors (e.g., exclusive
breastfeeding status) or health status. In addition, because the
controls were also stunted, the data cannot be used to infer
differences between well growing and growth-stunted in-
fants. Future studies should better account for these impor-
tant variables.
In a set of studies carried out in Senegal and Niger, Million
and colleagues [47] studied 69 children with varying nutri-
tional status. Location was found to be the major driver of
overall fecal microbial community structure when all children
were considered together, whereas age, sex, and nutritional
status did not explain much of the overall taxonomic variation.
However, the researchers found lower levels of obligate an-
aerobes associated with malnutrition. This approach was fol-
lowed up with an individual patient data meta-analysis which
included 108 children with SAM and 77 well-nourished chil-
dren from 5 African and Asian countries. Results indicate that
members of the Bacteroidaceae, Eubacteriaceae, Lachnospi-
raceae, and Ruminococceae families were depleted while En-
terococcus faecalis, Escherichia coli, and Staphylococcus au-
reus were consistently more abundant in feces collected from
children with SAM. Depletion of obligate anaerobes, de-
creased total bacterial number, and lower relative abundance
of Methanobrevibacter smithii (a member of the Archaea do-
main) were also associated with malnutrition. M. smithii is
thought to enhance GI bacterial polysaccharide digestion by
optimizing hydrogen levels; its potential role in increasing
metabolizable energy has been discussed as has its use as a
therapeutic for modulating energy balance in humans [48].
The authors suggest that maternal antioxidant intake (and
thus, milk antioxidant composition) might modify the impor-
tance of fecal redox capacity to infant nutritional status. If cor-
rect, then it is likely that the presence of GI microbiota with
redox capacity might be particularly important in cases of ma-
ternal antioxidant deficiency and/or nonexclusive breastfeed-
ing and inadequate infant antioxidant consumption – again,
suggesting that what would be considered “optimal” microbi-
omes in various contexts differs.
Infant Nutrition, Microbiomes, and Health
In a subsequent study [49], the same group compared GI
microbiomes of 5 healthy infants and 10 infants with kwashi-
orkor; they also found a depletion in oxygen-sensitive pro-
karyotes including M. smithii. Diversity of anaerobic taxa was
lower in the malnourished infants, and much of this difference
was due to species not previously identified in human stool
samples. The authors posited that there is a “loss” of anaerobic
species in patients with kwashiorkor. It is worth noting, how-
ever, that although ages were not statistically different be-
tween the groups the malnourished cohort was substantially
younger than the controls (13.4 vs. 25.1 months). As such, dif-
ferences could be explained by myriad other factors such as
breastfeeding status. In addition, the small sample sizes com-
bined with the fact that subjects were recruited from 2 distinct
locations (Senegal and Niger) make the results difficult to in-
terpret. Future studies should endeavor to recruit healthy and
malnourished subjects at similar ages living in the same loca-
tion.
In the only study to our knowledge to compare infants with
kwashiorkor (n = 54) to those with marasmus (n = 33), Kris-
tensen and colleagues [50] studied Ugandan children aged
6–24 months. After adjusting for sex and diarrhea at admis-
sion, they found higher diversity in samples collected from
infants with kwashiorkor. When severity of wasting, mid-up-
per arm circumference, and stunting were considered sepa-
rate from type of SAM, no relationships were found with over-
all GI microbiome. There were also no differences in individ-
ual taxa. Unfortunately, markers of inflammation and
differences in microbial community function were not as-
sessed. In addition, children with kwashiorkor were older than
those with marasmus (17 vs. 15 months, respectively; p =
0.04), and age and infant feeding patterns were not controlled
for in the analyses. This is particularly unfortunate given the
fact that these authors had previously published findings [51]
that children with kwashiorkor in this population were less
likely to be breastfed and that household dietary diversity
scores were also lower in these children.
In contrast, in a longitudinal study of 78 Peruvian infants
between 5 and 12 months of age [52], those who became
stunted during the study had higher bacterial diversity and al-
tered GI microbiome coupled with elevated biomarkers of in-
flammation that preceded growth faltering. Differences in fe-
cal microbial diversity also preceded growth faltering. Impor-
tantly, the researchers took care to document infant feeding
practices and reported no differences in duration of exclusive
breastfeeding and age at introduction of solid foods; this rep-
resents an important strength of this study compared to al-
most all others on the topic. The authors concluded that the
origin of immune activation in stunted children may be sus-
tained, low-grade microbial translocation across the GI mu-
Reprint with permission from: 41
Ann Nutr Metab 2021;77(suppl 3):36–48
DOI: 10.1159/000519001
cosa. However, because differences in GI microbiome were
also present prior to growth stunting it is also possible that
microbially derived inflammatory metabolites might drive
chronic local irritation. Metagenomic analysis to examine mi-
crobial function would be helpful to tease apart these inter-
twining possibilities.
In another study [53] characterizing longitudinal relation-
ships among GI microbiome, growth, and inflammation in 691
Malawian infants, MAZ and GI microbial diversity were associ-
ated with weight gain from 6 to 12 months, but associations
with systemic inflammation were inconsistent. Although in-
fant feeding status was not reported, the analyses were ad-
justed for infant age on day of fecal collection. Other studies
(e.g., [54]), have also provided weak evidence for a link be-
tween an “inflammatory” GI microbiome and growth, but
these studies lack evaluation of inflammatory status in the in-
fant.
GI Microbiome Functionality and Childhood
Undernutrition
Highlighting the potential importance of GI microbiome
function rather than composition, Mayneris-Perxachs et al.
[55] utilized a case-control study in malnourished versus well-
nourished Brazilian infants (n = 326) 6–24 months of age.
Importantly, cases (n = 158) and controls (n = 168) were
matched for age, although matching was within 6 months – a
relatively long period of time in this context – and cases were
older than controls (14.8 vs. 12.3 months, p < 0.001). In addi-
tion, birth weight was lower in cases than controls (−1.7 vs.
−0.52 WAZ, p < 0.001). Whether the infant was breastfed was
apparently similar between groups, although it is unclear from
the report if this refers to ever having been breastfed or breast-
fed at the time of sample/data collection. Whereas they did
not directly assess GI microbiome, they evaluated urinary
metabolic phenotypes and indirectly inferred source (host vs.
microbe). They found that both stunting and wasting were as-
sociated with increased proteolytic activity of the infants’ GI
microbiomes as well as dysregulated choline metabolism and
metabolic adaptations to reduce energy expenditure. Accel-
erated catch-up growth was seen in children with the most
pronounced energy-saving metabolic adaptations. The au-
thors posit that alterations in methyl donor capacity might
trigger early-life epigenetic programming toward a “thrifty”
phenotype. Whereas a favoring of GI microbiota that is able
to utilize protein as an energy source seems like a reasonable
and complementary response to inadequate nutrient avail-
ability, it remains unclear how this would occur. Also, will in-
creased proteolytic activity reduce availability of essential
42 Reprint with permission from:
Ann Nutr Metab 2021;77(suppl 3):36–48
DOI: 10.1159/000519001
amino acids to the infant and lead to decreased growth and
synthesis of related compounds such as serotonin? Answer-
ing these questions will require studies coupling longitudinal
assessment of infant growth, GI microbiomes and metabo-
lites, possibly stably labelled amino acids, and circulating me-
tabolomics.
To help in this regard, Giallourou et al. [56] evaluated lon-
gitudinally collected urine samples and growth in malnour-
ished Peruvian, Bangladeshi, and Tanzanian infants (n = 281,
249, and 249, respectively) during the first 24 months of life.
Infants from each location (n = 22, 28, and 13 for Peru, Ban-
gladesh, and Tanzania, respectively) with LAZ ≥−0.75 at birth
and ≥−1.25 at 24 months of age were classified as a “healthy”
reference group. Both plasma and urinary metabolomes were
assessed, with the latter (in healthy infants) being used to ex-
plore the existence of age-discriminatory metabolites com-
mon across cohorts (phenome-for-age Z score, PAZ). PAZ
scores in malnourished children were lower than those of
their healthy counterparts, and low PAZ predicted later linear
and ponderal growth faltering. After controlling for cofound-
ers such as breastfeeding and diarrhea (none of which were
found to be important), several urinary metabolites arising
from microbial-host co-metabolism were positively correlat-
ed with age and/or nutritional status – many related to protein
and amino acid metabolism. Importantly, these generalized
findings were consistent among locations suggesting that
they might represent relationships that might be applicable to
other populations. Whether these associations between PAZ
and nutritional status are causal in nature is unknown, as is
whether differences in GI microbiomes represent mediating
factors.
Micronutrients (Particularly Iron), Microbiomes,
and Malnutrition
Although most research regarding GI microbiome and early-
life malnutrition deals with generalized undernutrition (e.g.,
kwashiorkor), micronutrient intake might also be important.
Although many microbes synthesize vitamins that can be
used both by themselves and their hosts, they also rely on the
presence of some nutrients (e.g., some vitamins, essential
minerals) for survival. It is plausible, therefore, that endemic
nutrient deficiencies might evolutionarily select for GI mi-
crobes that produce those nutrients – another possible ex-
ample of eco-homeorhesis. Yet, aside from iron, very little
research has been conducted on this topic resulting in a weak
knowledge base in this regard. As such, here we focus mainly
on studies relating infant iron consumption and status on the
GI microbiome.
McGuire/McGuire
 In a double-blind, controlled intervention trial carried
out in Côte d’Ivoire, iron supplementation of 6- to 14-year-
old children (n = 139) increased fecal enterobacteria and
decreased lactobacilli and bifidobacteria [57]. This shift was
accompanied by increased calprotectin, a marker of GI in-
flammation. Additional negative outcomes of iron supple-
mentation were reported in 6-month-old Kenyan infants
(n = 115) [58] and 1-year-old Peruvian infants (n = 95) [59],
with the latter study revealing sex-specific responses. These
findings are bolstered by an extensive literature relating iron
status (and supplementation) to risk of infection, particu-
larly by pathogens with substantial requirements for this
mineral – the most studied being those that cause malaria
[60]. Indeed, Plasmodium falciparum has evolved elaborate
mechanisms to sequester iron, and supplementing the host
with iron can enhance this pathogen’s survival and repro-
duction. This finding has led the WHO to recommend that
iron interventions only be administered in areas of endemic
malaria when diagnosis, prevention, and treatment schemes
are employed [61].
Because most microbes have a requirement for iron, hu-
mans have evolved a constellation of mechanisms whereby
iron is withheld from pathogens during infection, ultimately
impairing bacterial growth while increasing risk for host iron
deficiency. Interestingly, HM is a poor source of iron, and its
iron content is relatively refractory to maternal iron status –
suggesting an adaptive advantage to low levels of iron con-
sumption in early life. Several studies have provided evidence
that, in addition to iron supplementation increasing risk for
certain infections including those causing diarrhea, it can also
impact the GI microbiome of children. For instance, several
have found that iron interventions increase Enterobacteria-
ceae and decrease Lactobacilli and Bifidobacteria [57, 58, 62,
63]. This finding is particularly interesting because members
of the Lactobacillaceae family, common in milk and dairy
products and considered to be health-promoting probiotics,
have an extremely low requirement for iron. As such, when
iron is abundant in the environment, these taxa are likely out-
competed by iron-requiring taxa. It is noteworthy that the im-
pact of iron supplementation on GI microbiome appears to
be context specific, likely impacted by typical core GI micro-
bial community structures, chronic dietary intake, and en-
demic pathogen exposure. For example, Dostal and col-
leagues [64] found no effect of high-dose iron supplements
on fecal microbiomes in iron-deficient children with low en-
teropathogen burden living in South Africa. This discrepancy
in findings across locations highlights the need to study these
interactions in each location of interest and the critical impor-
tance of not extending findings and conclusions drawn from
one locale to another.
Infant Nutrition, Microbiomes, and Health
GI Microbiomes and Childhood Obesity
There is also substantial interest in the complex interactions
among GI microbiome, obesity risk, and inflammation in ear-
ly life. One of the first studies to examine this was a case-
control design in Finland; 24 normal-weight children were
matched for gestational age and BMI at birth, delivery mode,
probiotic intervention, duration of breastfeeding, and antibi-
otic use [65]. GI microbiome was assessed at 6 and 12 months
and relationship to risk for obesity during the first 7 years of
life assessed. Bifidobacterial numbers in stool samples during
infancy were higher in children who remained healthy weight
than those who became overweight; S. aureus was higher in
those who became overweight. The researchers hypothe-
sized that S. aureus may trigger low-grade inflammation, con-
tributing to the development of obesity and link their findings
to an extended concept of the hygiene hypothesis. In a com-
panion case-control study involving 15 overweight children at
10 years of age and 15 healthy-weight children matched for
sex, gestational age and BMI at birth, mode of delivery, and
duration of breastfeeding, Luoto et al. [66] found that bifido-
bacterial numbers tended to be lower in fecal samples col-
lected at 3 months of age in the overweight children. The
same research team later reported that Staphylococcus was
elevated in feces of infants’ overweight mothers compared to
healthy-weight mothers during pregnancy [67], supporting
the possibility that the infants’ fecal microbiomes were shaped,
at least in part, either during delivery, via their mothers’ HMM,
or through other physical contacts in the shared environment.
Evidence of intergenerational transmission of the overweight
phenotype from mother to offspring via microbiome transfer
has also been provided by Tun and colleagues [68], who uti-
lized multiple mediator path modeling to show that infant fe-
cal microbiome (particularly Lachnospiraceae) mediated the
association between maternal prepregnancy overweight and
risk of overweight in children at 1 and 3 years of age.
In a larger study of 330 healthy Danish infants, Bergstrom
and colleagues [69] found that increasing abundance of
several taxa (e.g., Firmicutes) and decreasing levels of M.
smithii in feces of infants between 9 and 18 months of age
were associated with greater BMI gains – a finding some-
what in conflict with that reported in malnourished children
and described previously suggesting that M. smithii might
facilitate microbial fermentation of polysaccharides in the
colon. The reason for this discrepancy is unknown, but
again highlights the importance of not assuming similar
findings in disparate locations. Results from the CHILD study
in Canada suggested that higher microbial richness and en-
richment of Lachnospiraceae at 3–4 months increased risk
of obesity at 12 months [70]. In a companion study [68],
Reprint with permission from: 43
Ann Nutr Metab 2021;77(suppl 3):36–48
DOI: 10.1159/000519001
abundance of Lachnospiraceae at 3–4 months was associ-
ated with more frequent use of household disinfectants, and
was found to statistically mediate an apparent association
between disinfectant use and increased odds of becoming
overweight or obese at 3 years. However, exclusive breast-
feeding was associated with lower use of disinfectant, com-
plicating interpretation. Dogra et al. [71] reported that in-
fants who acquire higher levels of Bifidobacterium relative-
ly later had lower adiposity at 18 months, but differences in
feeding practices were not accounted for.
Several studies have also considered this topic in prema-
ture infants. Scheepers et al. [72] studied 909 preterm infants
and found that higher levels of fecal B. fragilis at 1 month was
associated with higher BMI between 1 and 10 years, but only
when the infants consumed a low-fiber diet. This association
was modified by maternal lifestyle factors, making the results
challenging to interpret. Importantly, feeding practices in the
first month (exclusive breastfeeding, formula feeding, or a
combination) and duration of breastfeeding were controlled
for in the analysis. White and colleagues [73] found that early
detection of Bacteroides in stool samples from 218 preterm
and term infants at 1 month was associated with reduced
growth in the first 6 months in male infants; although the au-
thors controlled for whether or not the infant had received
formula they did not control for duration of breastfeeding.
Additional evidence exists that the association between GI mi-
crobiome and growth might be sex-specific [69, 74, 75].
It should be noted that, similar to the literature on antibiot-
ics and growth faltering, there is a relatively large literature
examining the association between antibiotic use in infancy
and risk of developing obesity. Findings from these studies,
however, are notably inconsistent [75–77]. Again, it is likely
that confounders (e.g., infant GI microbiome community
structure and feeding patterns) modify any effect that might
exist and that any impacts are geographically/culturally spe-
cific.
Conclusions and Major Research Gaps
Risk of and resilience to malnutrition as they relate to variation
in the early-life GIM warrants continued examination because
it is likely that, at least in some contexts, different GI microbi-
omes predispose (or program) individuals to acute and chron-
ic health trajectories (hygiene and old friends hypotheses,
DOHaD, 1st 1,000 days emphasis). Most studies to date are
understandably epidemiologic in nature, and the design of
most interventions has focused on describing members of the
fecal microbiome that could impact health through inflam-
mation and nutrient utilization. However, consumption and
44 Reprint with permission from:
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DOI: 10.1159/000519001
absorption of nutrients by the host and production and use of
nutrients by bacteria can create competitive scenarios that
are typically not considered in any of these studies. Impor-
tantly, what is a “healthy” GI microbiome might be modified
by genetics, nutrient availability, endemic microbe exposures,
feeding patterns, and sex (eco-homeorhesis), but these inter-
actions have not been studied using adequate prospective,
longitudinal experimental designs in at-risk populations. Be-
cause GI microbiomes are clearly different across locations
and these customized differences might be important for
acute and chronic health, researchers and public health ex-
perts should apply a healthy dose of restraint when consider-
ing extending findings from 1 location to another. In sum-
mary, the current state of the science is not adequate to con-
clude that variation in the early-life microbiome can lead to
either undernutrition or obesity, and additional rigorous stud-
ies are needed in this regard.
Research in this area must carefully take into account the
age and feeding history of study participants, or these impor-
tant factors might completely confound research results and
substantially complicate their interpretation. When possible,
studies should be conducted in more than one location so
that we can better understand microbe-health relationships
that are specific to one culture/region versus those that are
common to all. Scientific focus should expand well beyond
characterizing the association between generalized protein-
energy malnutrition, impaired growth, and energy imbalance
(e.g., kwashiorkor and marasmus) and GI microbiome. Rather,
and as highlighted by evidence that iron supplementation
during infancy and early childhood might negatively impact
early-life GI microbiome, micronutrients should also be con-
sidered. And, as always, studies should be powered sufficient-
ly to address the major hypotheses being tested.
Elegant and complex animal studies have examined the
effect of GI microbiome through inoculation from healthy or
stunted infants. These studies have examined an array of
-omics to identify potential markers or causative factors for
the phenotypes observed in the afflicted children. However,
relatively poor establishment of the bacterial community into
the germ-free models and limited numbers of replicates, both
from donors and recipient animals within a donor cohort, re-
strict our ability to interpret the results – let alone apply them
to the human condition. In addition, a lack of growth or even
body weight loss with the limited animal numbers is a concern
for validity of these models. Finally, these models which utilize
weaned animals do not include the potential contribution of
HM to the nutrient or health status of children, which would
be the normal feeding for the children studied. Thus, these
models fail to reflect the various food sources consumed by
children.
McGuire/McGuire
 Given the growing double burden of malnutrition, under- Conflict of Interest Statement
standing the complex relationships among infant nutrition,
The writing of this article was supported by Nestlé Nutrition Institute
nutritional status, and microbiomes should garner intense re-
and the authors declare no other conflicts of interest.
search activity and funding. Adequately describing this com-
plicated interaction will demand a focused interdisciplinary
approach among nutritionists, microbiologists, immunolo-
Author Contributions
gists, epidemiologists, and clinicians. For these studies to pro-
vide insight, they should expand from simply cataloging mi- Both authors contributed equally to the writing of this manuscript.
crobes to examining their functionality. Because HM contains
a constellation of components (e.g., antimicrobial factors, nu-
trients, and microbes) that can impact establishment of the GI
microbiome, understanding these biological compounds and
microorganisms is likely crucial to this type of research. The
expanded use of well-powered, mechanistic studies using
validated animal models is also clearly needed.

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