Early life gut microbiome and its impact on childhood health and chronic conditions

HMO degradation

Breast milk contains a diverse array of nutrients and bioactive compounds contributing to infant development.⁵⁸ Among its components are HMOs, complex carbohydrates that constitute the third most abundant component of breast milk, after lactose and fats.⁵⁸ More than 200 different HMOs have been identified,⁵⁹ with structural variations in their core structure, length, size, and glycosidic linkages.⁶⁰ HMOs are indigestible by the human digestive system and thus serve directly to shape the developing gut microbiome. HMOs exert direct effects on the host or act indirectly by affecting bacteria species or functions.⁶¹

HMOs act directly through their antimicrobial properties against pathogens, inhibiting pathogen colonization by functioning as a soluble decoy receptor. Experiments conducted in vitro have shown that they stimulate dendritic cell function, promote the production of interleukins (e.g., IL-10, IL-27, and IL-6), and can modulate toll-like receptor (TLR) expression; influencing immune recognition and response to pathogens.⁶² For instance Sodhi et al. showed that HMOs 2’-fucosyllactose (2’−FL) and 6’-sialyllactose (6’−SL) reduce inflammation by inhibiting TLR4 signaling in vivo.⁶³

Beyond their direct role, HMOs exert indirect effects through the metabolites produced by their fermentation by the gut microbiome, specifically short-chain fatty acids (SCFAs), and the cross-feeding interactions these metabolites facilitate within the gut microbiota. HMOs undergo saccharolytic fermentation by gut microbes, resulting in the production of SCFAs such as acetate, butyrate, and propionate; whose production varies depending on the infant’s age.⁶⁴ These SCFAs serve as energy sources for colonocytes, promote anti-inflammatory cytokine production, and inhibit pathogen growth.⁶⁵ In the healthy early-life gut microbiome, specific bacterial taxa such as Bifidobacterium and Bacteroides species play a key role in HMO breakdown. These bacteria possess a diverse array of genes encoding glycoside hydrolase (GH) enzymes, collectively enabling the degradation of a wide spectrum of HMO structures.⁶⁶ The location of GHs can vary by species, being either intracellular or cell-wall anchored. Intracellular GHs hydrolyze intact HMOs that are transported into the cell into monosaccharides that can be used for energy metabolism. In contrast, cell-wall anchored GHs break down HMOs extracellularly into mono- and disaccharides that are then transported into the cell for further metabolism, and can engage in cross-feeding interactions.⁶⁷ Among Bifidobacterium species, B. infantis is known to degrade all HMOs present in breast milk, a capability attributed to its wide genetic repertoire of GH enzymes, which work intracellularly.⁶⁸ In contrast, B. bifidum strains are moderately efficient at HMO degradation and typically employ extracellular degradation.^69–72 B. breve strains employ intracellular degradation, and their HMO-degrading capacity is strain-specific, with most strains being able to degrade only non-fucosylated HMOs.^71,73 A similar strain variability has been described for B. longum,^74,75 with most strains not being able to degrade fucosylated or sialylated HMOs.^69,71,75

Bacteroides (e.g., B. fragilis, B. thetaiotaomicron) and Phocaeicola species (e.g., P. vulgatus, P. dorei), metabolize HMOs through a mucin-glycan degradation pathway.^76–78 Unlike infant-type bifidobacteria, which specialize in efficient HMO utilization but not mucin glycans, these species are considered generalists in HMO utilization. The specificity of bifidobacteria confers them a selective advantage over Bacteroides species when directly competing in HMO degradation.

Nutrition

The gut microbiome plays an essential role in nutrition, predominantly providing energy and micronutrients locally to the host tissue of the GI tract. The most abundant products of microbial metabolism are SCFAs, which are produced from bacterial fermentation of non-digestible dietary substrates, also known as microbiota-accessible carbohydrates.⁷⁹ Concentration of each SCFA varies widely between individuals, depending on the diet and age.⁶⁴ SCFAs are hallmarks of gut health because of their various mechanisms, such as regulating intestinal pH to inhibit the proliferation of pH-sensitive pathogenic bacteria, supplying energy to intestinal epithelial cells, fortifying gut barrier integrity, and mitigating inflammatory responses.^80–83 The most abundant SCFA is acetate, typically produced in a 3:1:1 ratio relative to propionate and butyrate, respectively. During the early months, infant-related bifidobacteria that metabolize HMOs, such as B. infantis, B. longum and B. breve, are the major producers of acetate.^64,84 Butyrate and propionate levels start to increase at 8 and 10 months of age, respectively.⁶⁴ Tusukuda et al. hypothesize that this increase is driven by breastfeeding cessation, as reduced lactoferrin and increased free iron may drive these changes, though this was not explored further.⁶⁴ This increase in butyrate levels is correlated with an increase in butyrate-producing bacteria such as Faecalibacterium prausnitzii and Roseburia species.⁶⁴ Gut microbes also synthesize B vitamins and vitamin K2, which are essential for various physiological processes, including energy metabolism, nerve function, and bone health.^85,86 Compared to 4- and 12-month-olds, the newborn’s gut microbiome has a higher capacity for production of vitamins K2, B6, B7, and B9 (folate). On the other hand, genes for synthesis of vitamins B1, B5, and B12 increase with age.¹³ Vitamin K2 is exclusively produced by microbes where different isoforms are produced by Escherichia coli, Eggerthella lenta, Veillonella spp., and Bacteroides spp.⁸⁶ Its production in the infant gut (measured via stool) is influenced by factors such as delivery mode and feeding method, as these directly affect the composition and proportion of K2-producing bacteria in the gut.⁸⁷

Colonization resistance

The gut microbiota plays an important role in resisting the invasion of foreign pathogens and controlling the proliferation of resident pathobionts, a critical function known as colonization resistance. This resistance is vitally important in newborns who have immature immune systems.⁸⁸ In the healthy infant gut microbiome, colonization resistance is primarily orchestrated by Bifidobacterium species and pH regulation. Specifically, B. infantis has demonstrated remarkable efficacy in metabolizing HMOs from breast milk, leading to the production of lactate and acetate.⁸⁹ These SCFAs play a pivotal role in lowering the gut pH, creating an environment unfavorable for the growth of pathogenic bacteria.⁹⁰ For example, in vitro experiments have shown that acetate production by Bifidobacterium contributes to the prevention of translocation of E. coli O157:H7 Shiga toxin from the gut lumen into the bloodstream.⁹¹ Moreover, cross sectional studies have shown that supplementation of breastfed infants with B. infantis significantly reduces the gut pH, the levels of pathogenic bacteria (e.g. Haemophilus influenzae, Escherichia coli, Streptococcus mitis), and the number of genes coding for virulence factors and antibiotic resistance.^89,92–95 Supplementation of preterm infants with Bifidobacterium animalis subsp. lactis and with different combinations of Bifidobacterium and Lactobacillus species reduces severe necrotizing enterocolitis, which is caused by invasion of opportunistic pathogens into host tissue.⁹⁶ Beyond pH regulation, other mechanisms of colonization resistance mediated by Bifidobacterium involve the secretion of bacteriocins and transformation of primary into secondary bile acids which inhibit the growth of pathogenic bacteria.^97–99

Immune education

Recent studies highlight a critical early-life period where microbe-host interactions are key to optimal immune cell development and education. In a longitudinal study comparing preterm and full-term infants, Olin et al. observed that immune phenotypes diverged in the first months of life but converged by three months.⁸⁸ During this window of time, notable changes occurred in immune cell populations, cytokines profiles, and gene expression in both preterm and full-term infants, suggesting that microbial interactions at the gut mucosa are important in shaping this development. Further, the study indicates that the subset of infants experiencing early gut irregularity in their gut microbiome – defined by an extremely low level of Shannon diversity and near complete-dominance of Gammaproteobacteria or other atypical groups – display perturbations of immune system development. Specifically, these included increased expression of the microbial-induced Fc receptor PIgR, expansion of CD8+ T cells that interact with mucosal bacteria, and greater variability in immune cell populations⁸⁸ This suggests that early imbalances in the gut microbiome disrupt the standard and ideal development of the immune system. Giving further mechanistic evidence for this, Henrick et al. studied a large longitudinal cohort and found that the abundance of HMO-degrading Bifidobacteriaceae in early life significantly affects immune development.¹⁰⁰ Low levels of Bifidobacterium correlated with increased immune activation and higher levels of pro-inflammatory cytokines, whereas higher levels were associated with anti-inflammatory and regulatory immune cells and cytokines. Specifically, an exclusively microbial metabolite, indole-3-lactic acid, produced by select bifidobacteria was identified that influences immune-microbe interactions by dampening inflammatory responses, in particular Th2- and Th17-type responses in favor of Th1 and regulatory T cells. Other microbial metabolites like butyrate also promote regulatory T cell (Treg) generation, creating an immune environment conducive to tolerance and decreasing the likelihood of allergic responses.¹⁰¹

Delivery mode

The mode of delivery has a profound impact on the initial colonization and subsequent development of the infant gut microbiome. Vaginal delivery exposes the newborn to the mother’s vaginal and gut microbiota, primarily colonizing the infant’s gut with beneficial bacteria such as Bifidobacterium, Bacteroides, Lactobacillus, and Clostridium species.¹⁰ In contrast, infants delivered by C-section are more likely to be colonized by bacteria from the mother’s skin and the hospital environment, including Staphylococcus, Streptococcus, and Clostridioides difficile, as well as other opportunistic pathogens from the Enterobacteriaceae family.^10,11

Cesarean delivery has been associated with a delayed colonization of the gut by beneficial bacteria, particularly those in the Bacteroidota phylum, and a lower overall microbial diversity.^{48,49,102–104} While some studies have shown that these differences can persist for up to two years,^48,49 others show partial or almost complete recovery of the gut microbiome by 6 months of age.^102–104 This disturbance in early microbiome development has been suggested to contribute to the increased risk of immune-mediated conditions such as asthma, allergies, obesity, and other chronic diseases seen in C-section born children later in life.^33,105,106 Even though the gut microbiome of C-section-born infants may eventually resemble that of vaginally delivered infants, the existence of a critical early-life window of immune system maturation suggests that initial microbial imbalances may have long-term consequences“s.^4,41

Antibiotic exposure

Antibiotic exposure during the prenatal and neonatal periods can significantly impact the infant’s microbiome. Use of intrapartum antibiotics have been shown to reduce the amount of species transmitted from mothers to infants,¹⁰⁷ and prenatal and intrapartum antibiotic use has been associated with a marked decrease in the abundance of Bifidobacterium and Bacteroides, and an increase in the abundance of members of the Enterobacteriaceae family, and Enterococcus and Clostridium species.^12,108–110 Antibiotics have also shown to increase the abundance of antibiotic resistance genes in the infant’s gut microbiome.^109,111

Neonatal antibiotic use can delay the maturation of the gut microbiome and cause a potential regression relative to its expected developmental trajectory, with a marked decrease in Bifidobacterium species and an increase in members of the Enterobacteriaceae family.^31,48,112 In addition, the gut microbiota of antibiotic-exposed infants exhibits less strain diversity compared to non-exposed infants,¹¹³ which could impact the functional capacity of their gut microbiome. In some infants, these microbiome imbalances can persist long-term.²² Finally, antibiotic exposure has been linked to an increased likelihood of adverse health outcomes, including higher odds of atopic dermatitis (AD), asthma, obesity, and food allergies.^114,115

Infant diet

As discussed above, breastfeeding profoundly affects the development of the infant gut microbiome.^13,116–118 It promotes the dominance of beneficial Bifidobacterium species, such as B. infantis, B. breve, B. bifidum, and B. longum, which can degrade HMOs.^7,12,13,119 These HMOs, along with other bioactive components in breast milk like IgA and lactoferrin, enhance mucosal immunity and inhibit pathogen colonization.^58,120,121 In many Cesarean-delivered infants breastfeeding also counteracts imbalances in the gut microbiota by replenishing levels of bifidobacteria and promoting a microbial composition more similar to that of vaginally-delivered infants.^13,117 In contrast, formula-fed infants often have lower levels of Bifidobacterium species^13,116 and higher levels of opportunistic pathogens^117,122 potentially due to absence of key bioactive components present in breast milk.^116,118,123

The introduction of solid foods, typically around 6 months, marks a transformative phase in the development of the infant gut microbiome, diversifying the microbial composition and enhancing the functional capacity to metabolize new substrates like carbohydrates, fibers, and proteins.^55,64 Dietary diversity positively correlates with Bifidobacterium abundance,⁵⁴ but the timing of solid food introduction also shapes microbiota composition. Parkin et al. found that breastfed infants introduced to solids before 5 months had increased microbiome diversity, including higher levels of B. infantis.¹²⁴ Similarly, Differding et al. reported that early complementary feeding (≤3 months) increased diversity and butyrate-producing taxa such as Lachnospiraceae and Roseburia by 12 months, alongside elevated SCFA concentrations.¹²⁵

Maternal diet

The overall impact of maternal diet on the infant microbiome is an emerging topic of study. However, there is clear evidence linking maternal consumption of fruits and vegetables during pregnancy to the composition of the infant gut microbiome. At one month, infants born to mothers who ate more vegetables during pregnancy tended to have a lower Shannon diversity and a higher relative abundance of Bifidobacterium.¹⁹ Conversely, a lower intake of these foods was associated with an increase in microbial taxa such as Sutterella, Prevotella, Lachnoclostridium, and Flavonifractor at two months.²¹ Additionally, a diet high in fat during pregnancy resulted in lower levels of Bacteroides at six weeks of age.²⁰ This reduction appears to stem from a decreased Bacteroides abundance in the maternal gut microbiota, limiting vertical transmission during delivery, or due to an increased likelihood of cesarean delivery, associated with high-fat diets. Maternal diet also influences breast milk composition, including HMOs and other bioactive molecules that shape the infant gut microbiome.¹²⁶ For instance, Neu5Gc from red meat is incorporated into HMOs and associated with increased Bacteroides abundance in infant stool.¹²⁶

Maternal health and gestational age

Conditions like gestational diabetes (GDM) and obesity are linked to the maternal microbiome, and in turn also linked to microbial composition of the infant and its future health risks.^127–129 Infants born to mothers with GDM are more likely to develop obesity and metabolic diseases in later life.^129,130 Maternal obesity has also been associated with differences in the infant microbiome, including higher levels of Bacteroides.^131–133 However, disentangling direct microbiome effects from confounding lifestyle factors – such as shared dietary patterns, activity levels, breastfeeding duration, dietary transitions, and the introduction of complementary foods – is challenging.^131,133

In addition, maternal mood disorders, asthma, human immunodeficiency virus, Group B Streptococcus (GBS), eczema, and inflammatory bowel disease (IBD), have all been linked to variations in the infant’s gut microbiome.^36,134 For example, maternal prenatal stress was associated with an increased abundance of Proteobacteria in infants which may be linked to inflammation.¹³⁴ Reduced levels of beneficial bacteria Lactobacillus and Bifidobacterium in infants was associated with maternal stress¹³⁴ as well as maternal asthma.³⁶

Finally, gestational age plays a role in affecting the microbial landscape of the neonatal gut. Premature infants, defined as those born before 35 weeks of gestation, typically exhibit a microbiota characterized by less diversity than term infants, fewer bacterial species, and a distinct profile that can persist for up to four years.^15,135 These infants often experience delayed colonization by beneficial microbes such as Bifidobacterium, which dominate vaginally delivered term infants, and display an increased presence of opportunistic pathogens such as Staphylococcus, Enterococcus and Enterobacter.¹⁶ One study also observed markedly reduced SCFAs concentrations in preterm infants compared to full term infants, primarily due to lower acetate levels.¹⁴ These reduced levels reflect gut immaturity, with limited SCFA-producing bacteria potentially due to reduced exposure to maternal microbiota and HMOs and/or increased antibiotic use in preterm infants¹³⁶

Prebiotics and probiotics

Prebiotic and probiotic supplementation during and after pregnancy may contribute to better outcomes in certain conditions or specific instances. For example, supplementation with Lactobacillus rhamnosus GG strain during pregnancy was reported to promote the colonization of Bifidobacterium in the infant gut.^137,138 Supplementing infants with different strains of Bifidobacterium infantis or a mix of Bifidobacterium and Lactobacillus is effective in increasing gut bifidobacteria levels when paired with breastfeeding to supply the HMOs that feed the bifidobacteria. This supplementation also helps lower gut pH and enhance the production of SCFAs.^92,95,139

Probiotics have shown success in restoring the microbial and functional profile of infants born by C-section to one more similar to those born vaginally. A study by Gong et al. found that a two-week supplementation of infants born by C-section with a probiotic mix containing B. longum, L. acidophilus, and E. faecalis promoted the growth of beneficial bacteria such as Bacteroides, Veillonella, and Faecalibacterium while reducing the levels of Klebsiella.¹⁴⁰ Korpela et al. found that supplementing with a mixture of B. breve, Propionibacterium freudenreichii, and L. rhamnosus during pregnancy and to babies up to 6 months, increased the abundance of genes associated with HMO degradation in both vaginally and C-section-born infants.¹⁴¹ This effect may be exclusively attributed to B. breve, which is a specialized HMO degrader.¹⁴¹ Probiotics have also shown promising effects on the gut microbiome of preterm infants. Supplementing with a mixture of Bifidobacterium and Lactobacillus strains lead to a community that resembles the gut microbiome of full-term infants.^142,143

Supplementing infant formula with HMOs has been shown to shift the gut microbiome toward a composition similar to that of breastfed infants by promoting the growth of Bifidobacterium and reducing levels of opportunistic pathogens like Clostridioides difficile.^144–146 Similar benefits have been observed when supplementing infant formula with probiotics or symbiotics.^147–149 Lagkouvardos et al. showed that a formula supplemented with Lactobacillus fermentum and galactooligosaccharides increased the levels of Bifidobacterium and Lactobacillaceae, while decreasing Blautia, Ruminococcus gnavus, and butyrate levels at 4 months of age.¹⁴⁸ However, the study did not provide species-level data for Bifidobacterium, making it unclear whether the increases were specific to infant-associated species or included other non-infant-associated species.

Studies have also focused on the effects of probiotics in preventing chronic conditions in high-risk infants. Prenatal supplementation of women with obesity using a Bifidobacterium and Lactobacillus probiotic has been shown to reduce Collinsella —a genus associated with obesity¹⁵⁰— and increase Akkermansia in their infants, potentially redirecting their gut microbiome to a profile not associated with obesity.¹⁵¹ Additionally, probiotics – alone or in combination with prebiotics – given to both pregnant mothers and infants have been linked to a reduced risk of developing eczema.^152,153

These findings suggest that prebiotics and probiotics, specifically particular strains, can be a promising synergistic approach to fostering the growth of beneficial gut bacteria, supporting microbiome development in preterm infants, and restoring the gut microbiome of infants born by C-section or vaginally born infants that have not been exposed to beneficial microbes. Thus, early supplementation could help alleviate a range of pediatric health issues, including metabolic disorders, allergies, and autoimmune diseases.

Allergic disease and asthma

The rising prevalence of allergies among children has propelled research pointing to a complex relationship between genetic predispositions and multiple environmental factors.¹⁵⁵ Notably, many of these environmental influences – including antibiotic use, Cesarean delivery, and dietary habits – can modulate the gut microbiome. Thus, the gut microbiome may contribute to the development of allergic conditions, which often follow a trajectory from atopic dermatitis (AD) and food allergy to asthma and rhinitis, a progression commonly referred to as the atopic march.¹⁵⁵

AD often emerges in infancy, affecting an estimated 16.5% of children in the US.¹⁵⁶ Although AD may resolve with age, children with a persistent phenotype have a 4-fold and 7-fold higher risk of developing asthma and allergic rhinitis, respectively.¹⁵⁷ The gut microbiome has been associated with the development of AD, though a causal relationship has not been firmly established. Infants younger than 6 months with AD often exhibit high levels of Akkermansia.^158,159 However, it is unclear if Akkermansia species contribute to AD pathogenesis or serve as disease-associated biomarker, as A. muciniphila has been described to help maintain intestinal integrity^160,161 and has been linked to protective effects against metabolic syndrome.^162–164 High levels of A. muciniphila may represent a host immune response to an inflammatory event rather than a direct causal factor in AD pathogenesis.¹⁶⁵ Additionally, high levels of Enterobacteriaceae, particularly Klebsiella pneumoniae, are found in infants with AD from birth to 3 months.¹⁶⁶ At one week of age, infants born by C-section who later develop eczema, have higher levels of Escherichia, Citrobacter, and Enterobacter than infants who do not develop the condition.¹⁶⁷ Conversely, infants from 3 weeks to 12 months with AD show low levels of the Erysipelotrichaceae family^166,168 and butyrate-producing Faecalibacterium,^158,166,169 a taxa associated with anti-inflammatory properties. Related to this, infants with lower levels of fecal butyrate at 3 and 6 months had an increased risk of developing AD up to 8 years of age.¹⁷⁰ These patterns suggest a potential causal role for the gut microbiome in AD, but whether these taxa are contributors to the disease or predictive biomarkers requires intervention studies to address.

Food allergies affect approximately 6% of children¹⁷¹ and 8% of adults in the US.¹⁷² IgE-mediated food allergies often follow eczema and can manifest within an infant’s first year, typically around 6 months when solid foods are introduced. Food allergies are driven by a complex interplay of genetics and environmental factors.¹⁷³ However, recent research also uncovered a direct link between the early gut microbiome composition and IgE-mediated food allergies.^174,175 For example, one study showed that mice that were colonized by bacteria from infants with cow’s milk protein allergy (CMPA) exhibited severe allergic reactions to cow’s milk, while those colonized by bacteria from healthy infants did not.¹⁷⁵ Supporting this, a longitudinal study found that infants with elevated levels of Clostridia and Firmicutes at 3 and 6 months of age were more likely to outgrow milk allergy later in childhood.¹⁷⁶ The gut microbiome also regulates food allergy-related immune responses through the induction of Tregs, which are essential for suppressing hypersensitive immune reactions. In murine models, introducing a defined consortia of Clostridia species induced Tregs expressing the transcription factor ROR-γt in a MyD88-dependent manner, effectively mitigating food allergy symptoms.¹⁷⁴

Human infants born via C-section are at increased risk of developing food allergies later in life,^{105,177–179} perhaps because they lack the diverse protective bacteria acquired during vaginal birth.^{13,42,48,106,180} While distinct gut microbiome changes have been observed in cohorts with various food allergies (such as egg, peanut, soy, wheat, and milk), inconsistency across studies complicates interpretation.^{174,176,179,181}

Asthma affects approximately 8.1% children in the US,¹⁸² and the gut microbiome in early infancy has been closely linked to its development. Children at high risk were found to exhibit delayed gut microbiome diversification in the first year of life.¹⁸³ Delayed microbiome maturation – the process of the microbiome changing with age – at 1 year of age has been associated with asthma development at age 5.¹⁰⁶ Compared to vaginally-born babies, those born via C-section face twice the risk of developing asthma,¹⁰⁵ but this risk can be mitigated if their gut microbiome reaches appropriate maturation by age 1.¹⁸⁴ However, those children retaining a C-section gut microbiome signature at age 1 have three times the asthma risk by age 6 compared to C-section babies without the signature. The main involved taxa are again bifidobacteria as low levels of Bifidobacterium species at 1 month, 3 months, and 1 year of age have been associated with an increased risk of developing asthma by 5 years of age.^29,106,185 In animal models of asthma, all three main SCFAs^186,187 have been shown to protect against airway inflammation, and this is attributed to the stimulation of specific immune cell subsets capable of preventing T helper 2 immune responses.¹⁰⁰

Metabolic diseases

Children born to mothers with obesity have a higher risk of developing obesity later in life.^127,133 This connection is further supported by the description of microbes connected to obesity passed down from mother to child at birth.^127,133 Additionally, a reduction in butyrate and other SCFA-producing bacteria in newborns from mothers with obesity has been linked to an increased risk of obesity but the mechanism here is not clear.^131,133

Autoimmune disease

The early-life development of the gut microbiome has also been linked with Type 1 diabetes (T1D).^31,188,189 SCFA-producing bacteria, such as Bifidobacterium and Lachnospiraceae, are consistently reduced in T1D patients. A decrease in SCFAs may contribute to increased intestinal permeability and bacterial antigen exposure, potentially contributing to an undesired immune response that may lead to autoimmunity.

Inflammatory Bowel Disease (IBD), encompassing Crohn’s disease and ulcerative colitis, can already manifest in childhood. Studies have shown a reduction in bacterial diversity and alterations of specific bacterial taxa in the gut of children with IBD, but this could be driven by the high level of existing inflammation.^9,118 As in T1D, the microbial imbalance associated with triggering IBD is thought to involve a compromised intestinal barrier, which allows bacteria to penetrate the mucosal layer and activate damaging immune responses, thereby triggering an underlying genetic predisposition. Longitudinal microbiome studies following infants from early life until developing IBD do not exist and will be required to establish whether early life microbiome disruptions predispose to it.

Sample methodology

Standardized sample collection and storage are essential for reliable microbiome research. Variability in protocols, including differences in storage conditions and processing times, can obscure biological differences. Prompt stabilization, through immediate cooling and storage at − 80°C, or using validated preservation methods such as desiccation or stabilization buffers are crucial to maintain sample integrity,¹⁹⁰ as rapid compositional changes occur at room temperature.¹⁹¹ In addition, using DNA extraction methods with minimal levels of bias is crucial to perform accurate microbiome analysis.

Larger and more diverse cohorts

Few studies on the infant microbiome involve larger cohorts and many studies are based on small to medium-sized cohorts (fewer than 400 participants), which limits statistical power and the ability to detect subtle differences and meaningful associations.¹⁹² Moreover, larger cohorts can better capture the diversity of the human population, addressing the disparity in microbiome research by encompassing various ethnicities and their genetic backgrounds, environmental variables, and lifestyle factors.¹⁹³ In the context of the early life gut microbiome, capturing such diversity is crucial, as we have seen early microbial colonization and development are highly influenced by these factors.

Longitudinal sampling

Increased sampling frequency enables researchers to better understand the temporal dynamics of the gut microbiome. Individuals seem to differ in the rate of change of their gut microbiomes, even over short time scales, suggesting that characterizing temporal variability is crucial for understanding individual microbiomes.^194,195 In the context of the infant gut microbiome, high-frequency sampling is particularly valuable for tracking microbial development and maturation through critical early life stages.⁷ Understanding these temporal dynamics simplifies the identification of connections between microbial species and health outcomes, facilitating the interpretation of correlations among taxonomic groups.¹⁹⁶ However, longitudinal microbiome data analysis remains challenging and the field could benefit from improved statistical methodologies.

Multiple samples per family

Collecting microbiome samples from multiple family members offers the opportunity to dissect the contributions of genetic and environmental factors to the gut microbiome.^197,198 Tavalire et al. demonstrated that while environmental factors account for most of the variance in microbial composition among family members, genetic similarity more frequently explains variance in microbial abundance than does a shared home environment.¹⁹⁹ Similarly, Xie et al. found that microbial profiles of twin pairs living in different regions differed more than those of twins living in the same region, suggesting a significant environmental influence.²⁰⁰ However, their study also revealed that genetic factors play an important role in determining microbial abundance. Notably, they observed that the variation in the abundance of certain bacterial groups, such as Dorea, Prevotella, and Oxalobacter, is predominantly influenced by genetics rather than environmental factors.

Detailed survey data on health and lifestyle

Integrating detailed survey data on infants’ health, diet, medication use, and lifestyle provides valuable context to microbiome data. Such comprehensive metadata enables more nuanced analyses, helping to identify specific factors that influence the gut microbiome and facilitate the removal of microbiome variability that is due to confounding factors such as diet, medication use, other diseases, etc. A recent large-scale study by Gacesa et al. illustrates the potential of gathering extensive metadata. Their analysis of the gut microbiome of 8,208 Dutch individuals from a three-generational cohort, encompassing 2,756 families, included more than 200 host and environmental factors.²⁰¹ These factors include detailed information on physical and mental health, medication use, dietary habits, socioeconomic status, and both childhood and current exposome. This comprehensive metadata collection enabled researchers to identify more than 2,500 associations between the microbiome and health, providing a thorough analysis of how lifestyle, diet, and health parameters influence the gut microbiome.

Sampling before disease onset

Collecting microbiome samples before the onset of disease offers significant methodological advantages for gut microbiome research by allowing researchers to establish temporal relationships between microbiome changes and disease development. It also removes variability that is an indirect effect of the disease rather than contributing to the disease. The hallmark TEDDY study exemplifies this approach, focusing on the onset and development of T1D and its relationship with the gut microbiome.³¹ By collecting stool samples monthly from children starting at three months of age until T1D diagnosis, researchers showed the potential protective effect of SCFAs in early-onset T1D and identified microbial biomarkers of disease development. Healthy controls had higher levels of Streptococcus thermophilus and Lactococcus lactis while cases had more Bifidobacterium pseudocatenulatum, Roseburia hominis, and Alistipes shahii.³¹ These biomarkers can identify individuals at risk of developing T1D before clinical symptoms appear, facilitating early intervention and potentially preventing disease onset.

Comprehensive functional analysis

While much of the current research focuses on the taxonomic composition of the gut microbiome, it is increasingly evident that microbial functions are equally, if not more, crucial for understanding the microbiome’s role in health and disease.²⁰² To embrace the idea that the microbiome is better described through its functions than its composition, we advocate for the widespread adoption of shotgun metagenomics in microbiome studies. Unlike 16S rRNA sequencing, which has limited taxonomic resolution and cannot perform functional analysis, shotgun metagenomics assesses the functional capacity of microbial communities. This approach enables researchers to infer how microbial communities may contribute to host metabolism, immune function, and disease.²⁰³ This approach offers a more comprehensive understanding of the microbiome’s role in health, particularly in identifying the functional capacity that may affect host physiology. However, to determine what microbes are actively doing, additional techniques like transcriptomics and metabolomics should be used, but these come with additional requirements on sample collection. By integrating functional analysis into microbiome research, we can move beyond simply identifying “who is there” to understanding “what they are doing,” ultimately leading to more targeted and effective interventions for promoting health and preventing disease.