**4. Type of infant feeding**

Another important factor modulating microbial colonization in infants is the type of feeding. The diet during early life will influence on the establishment and composition of the gut microbiota during childhood and even adult life [51]. Breast milk meets the infant's needs by providing nutrients appropriate to the infant's developmental stage, as well as growth factors, antimicrobial peptides, and proteins to support their developing immune system. Even though breast milk provides all the necessary nutrients for a suitable development of the baby, many babies cannot take it for several reasons and they are fed with infant formulas. Infant formulas provide a greater weight gain and increase the risk of obesity, hypertension, and diabetes [52]. Therefore, it is necessary to continue studying the composition and the positive effects of breast milk versus milk infant formula in order to better understand the beneficial role of breast milk on offspring's health to improve the outcomes in the formula-fed infants.

Breast-feeding brings clear short-term benefits for child health by reducing mortality and morbidity from infectious diseases. There is evidence on the effects on child health and growth of exclusive breast-feeding for 6 months. Kramer et al. showed that infants who were exclusively breast-fed for 6 months experienced lower morbidity from gastrointestinal and allergic diseases, while showing nondeficits in growth rates to non–breast-fed children [53]. Based on such evidence, WHO and UNICEF recommend that every infant should be exclusively breast-fed for the first 6 months of their life; continued breast-feeding for up to 2 years or longer is also recommended [54]. Also, there is evidence of long-term benefits of breast-feeding such as increased school achievement and performance in intelligence tests, reduced mean blood pressure, lower total cholesterol, and a lower prevalence of overweight and obesity leading to lower incidence of inflammatory bowel diseases, type 2 diabetes, and obesity later in life [54, 55].

Regarding the mode of delivery, epidemiological studies suggest that caesarean delivery is associated with increased risk of overweight and obesity later in life [44]. A study has found that caesarean section delivery was associated with adiposity at 6 weeks of age, being this association stronger in children born from obese mothers and having higher risk of obesity and overweight at 11 years old [45]. Although the mode of delivery may affect the colonization of the intestinal microbiota in the baby and will increase the risk for later obesity development, it has been found that perinatal exposition of the infant born by caesarean section respect to the vaginal discharge, can partially restore its gut microbiota and resembles to

The microbiota of the babies by the end of the first year of life presents a different microbial profile in comparison to adults. The initial gut composition of the infant is simple, dynamic, and very unstable and undergoes marked fluctuations influenced by external factors [47]. At the beginning, the gut environment is aerobic, but through colonization, the oxygen level is reduced generating a suitable environment for the growth of anaerobes [48]. The intestinal microbiota of neonates is characterized by low diversity and a relative dominance of facultative anaerobes of the phyla *Proteobacteria* and *Actinobacteria* [49]. After birth, the phyla *Firmicutes* and *Bacteroidetes* increase their diversity and dominance, reaching over 3 years old a total resemblance to the adult in terms of composition and diversity [50]. These results indicate that dietary intake during the first 1500 days of life is a critical factor in the establishment of gut microbiota community and its role in the development of obesity is a matter of research

Another important factor modulating microbial colonization in infants is the type of feeding. The diet during early life will influence on the establishment and composition of the gut microbiota during childhood and even adult life [51]. Breast milk meets the infant's needs by providing nutrients appropriate to the infant's developmental stage, as well as growth factors, antimicrobial peptides, and proteins to support their developing immune system. Even though breast milk provides all the necessary nutrients for a suitable development of the baby, many babies cannot take it for several reasons and they are fed with infant formulas. Infant formulas provide a greater weight gain and increase the risk of obesity, hypertension, and diabetes [52]. Therefore, it is necessary to continue studying the composition and the positive effects of breast milk versus milk infant formula in order to better understand the beneficial role of breast milk on offspring's health to improve the outcomes in the formula-fed

Breast-feeding brings clear short-term benefits for child health by reducing mortality and morbidity from infectious diseases. There is evidence on the effects on child health and growth of exclusive breast-feeding for 6 months. Kramer et al. showed that infants who were exclusively breast-fed for 6 months experienced lower morbidity from gastrointestinal and allergic diseases, while showing nondeficits in growth rates to non–breast-fed children [53]. Based on such evidence, WHO and UNICEF recommend that every infant should be

babies born by vaginal delivery avoiding the problems that this entails [46].

and discussion.

infants.

**4. Type of infant feeding**

270 Adiposity - Omics and Molecular Understanding

Human milk is a dynamic fluid that contains many hundreds to thousands of distinct bioactive molecules that confers beneficial properties for infants. Human milk changes in composition from colostrum to late lactation, and varies within feeds, diurnally, and between mothers [56].

The composition of this complex mixture differs also during the lactation period, from colostrum through transitional to mature milk. Colostrum is produced during the first days of postpartum, it contains high amounts of secretory IgA, lactoferrin, leukocytes, and epidermal growth factor. Transitional milk typically occurs from 5 days to 2 weeks postpartum, it shares some of the characteristics of colostrum but there is an increase in milk production to support the nutritional and developmental needs of the rapidly growing infant. By 4–6 weeks postpartum, human milk is considered fully mature and it remains stable in composition over the course of lactation [57–59]. Thus, infant formula should adapt to different physiological and nutritional needs of the growing baby.

Regarding the gut microbiota acquisition, the first colonizers of the infant gut are facultative anaerobes including *Staphylococcus, Streptococcus, Escherichia coli*, and *Enterobacteria* that will be later replaced by strict anaerobes that dominate the gastrointestinal tract, primarily *Clostridium, Bifidobacterium spp*., and *Bacteroides* [60]. This change in dominant taxa representation can be attributed to the introduction of breast milk or formula-feeding, signifying the first diet-related colonization event in the infant gut microbiome [61, 62]. Breast milk has been shown to be an excellent and continuous source of potentially beneficial and commensal bacteria, including *Staphylococci, Streptococci, lactic acid bacteria*, and *Bifidobacteria*, with bacterial cell numbers reaching 103–105 ml–1 of breast milk. Although the commensals' origin is unknown, it is inevitable that bacterial from mother's skin are transferred to the baby during breast-feeding, but there is also other hypothesis wherein bacteria from the maternal gut may reach the mammary glands via maternal dendritic cells and macrophages [63]. More than 700 species of bacteria have now been identified in human colostrum and breast milk, including multiple species of lactic acid bacteria as well as species typically colonizing the oral cavity of infants [64].

The presence of *Bifidobacteria* in breast milk is important for the colonization of the infant gut, since it mediates the activation of IgA-producing plasma cells in the human neonatal intestine. It is well established that a gut microbiota dominated by *Bifidobacteria* typifies that of the healthy breast-fed infant [65]. There are conflicting results regarding differences in the relative abundance of these bacteria between breast- and formula-fed infants. Many studies have reported that formula-fed infants display dominance of *Bifidobacterium spp*. similar to what has been observed in breast-fed infants [61, 66]. However, another study reported approximately double the count of *Bifidobacterium* in breast-fed infants compared to those formula-fed [67].

Comparisons between breast-fed and formula-fed infants show that breast-fed infants tend to contain a more uniform population of gut microbes dominated by *Bifidobacteria* and *Lactobacillus* [67], whereas formula-fed infants exhibit higher proportions of *Bacteroides, Clostridium, Streptococcus, Enterobacteria*, and *Veillonella spp*. [66–69].

Although infant formulas have evolved greatly during last years, a formula providing exactly the same benefits than human milk has not yet been developed. Among others, human milk contains substantial quantities of complex nondigestible oligosaccharides (known as human milk oligosaccharides, HMOs). HMOs are considered a type of prebiotics as they promote the growth and proliferation of beneficial commensals and, consequently, prevent pathogen colonization of the infant gut and exert positive health effects [70]. Thus, the chemical composition of breast milk does influence the gut microbiome through supplying oligosaccharides that are selectively utilized by specific bacteria in the gut [60].

Another way to modify the gut microbiome is by the administration of probiotics. Probiotics are defined as "live microorganisms which when administered in adequate amounts, confer a health benefit to the host" [71]. *Lactobacillus* and *Bifidobacterium* species isolated from human milk are the most commonly used probiotic strains. They exert beneficial properties in the gut by suppressing the proliferation of pathogenic microbes, has been extensively studied [72]. For this reason, another area of research regarding formula enrichment is in HMOs and probiotics and their effects on the infant gut microbiota.

Certain gut-associated bacterial populations such as *Bifidobacterium spp*. possess gene clusters dedicated to the metabolism of HMOs [73, 74]. Degradation of these compounds produces lactate and short-chain fatty acids (SCFA), which in turn generates an acidic environment that prevents pathogen invasion [75]. Besides *Bifidobacteria*, HMOs may be consumed by other species such as *Bacteroides spp*. (e.g., *Bacteroides* fragilis and *Bacteroides vulgatus*) that consumes a broad range of HMO glycans [76]. Thus, HMOs play an important role in the gut colonization of the infants.

Among the most common prebiotics are fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), inulin, and lactulose. The prebiotic mixture of 90% GOS plus 10% FOS has been assessed to be safe when added to infant formula [77]. Several randomized controlled trials have been made to evaluate the efficacy and safety of prebiotic supplementation in infant formulas [78, 79]. After compiling data of these trials into a meta-analysis, weight gain [weighted mean difference 1.07 g/day] was significantly higher among formula-fed infants supplemented with prebiotics compared to the placebo group [80]. In addition, a large number of clinical trials in term of infants have shown controversial results related to the increase in *Bifidobacteria* in feces due to supplementation of infant formula with GOS and FOS. A systematic review published by Rao et al. [78] reported that some of the randomized controlled trials (RCTs) showed a trend of increasing *Bifidobacteria* counts in formula supplemented fed infants, and another systematic review published by Mugambi et al. [81] failed to show the increase in *Bifidobacteria* or *Lactobacillus* or the decrease of pathogens in infants fed with prebiotic supplemented formula.

Nonetheless, there are promising results from studies which have assessed the effect of prebiotic supplemented formulas on the gut microbiota of infants. Prebiotics are able to change gut metabolic activity, bring stool consistency, and defecation frequency closer to that of breastfed infants. Other outcomes included better weight gain and softer stools, and a significant reduction in stool pH for infants who received prebiotic supplementation [78, 81]. Moreover, prebiotics have been used to prevent or treat obesity. Compared to probiotics, human studies with prebiotics have shown more promising results in obesity management, with reductions in body weight and fat mass in adults [82–84] in contrast with results from meta-analysis mentioned above, where the supplementation with prebiotics was significantly associated to a higher weight gain [80].

In the last years there is a growing interest in the simultaneous administration of prebiotics and probiotics, what is termed "symbiotic." There are a few recent studies which have assessed the effect of symbiotic supplementation on the infant health. The ESPGHAN Committee on Nutrition showed an increase in stool frequency for three types of symbiotic (*B. longum* BL999 plus GOS/FOS, *B. longum* BL999 plus *L. rhamnosus* LPR plus GOS/FOS, and *L. paracasei* subsp. paracasei plus *B. animalis subsp. lactis* plus GOS) [79]. Also, Ringel-Kulka et al. showed that a yogurt with the probiotic bacteria *Bifidobacterium* animalis subspecies lactis (BB-12) and the prebiotic inulin significantly reduced days of fever, improved social and school functioning, and increased frequency of bowel movements in healthy children attending child care centers [85]. Regarding to obesity interventions with symbiotic, Safavi et al. [86] found that treatment of overweight children with a symbiotic mixture of the prebiotic, FOS, in combination with seven probiotic strains was associated with a decreased BMI *z*-score compared to placebo.

Studies suggest that pre-, probiotic, and symbiotic supplementation may be beneficial in the prevention and management of disease where the gut microbiota has a key role (e.g., necrotizing enterocolitis, gastroenteritis, or obesity). Although these studies show promising beneficial effects, the long-term risks or health benefits of pre- and probiotic supplementation are not clear as results from single studies need to be replicated in well-defined RCTs. Nonetheless, there is active research on functional food that contains pre-, probiotics, and symbiotics supplementation because they can influence not only the microbiota favoring the growth of beneficial microorganisms, but also the mucosal immune system associated to the gut [87].
