Table 2.

Metaomics technologies in neonatal microbiome studies (02/2021–09/2021).

Approach	Study title	Samples	Bioinformatics tools	Outcomes
16S rRNA gene sequencing	Fetal meconium does not have a detectable microbiota before birth (80)	Meconium (n=14) and infant stool (n=25)	DADA2 (Taxonomic profiling)	▪ Fetal gut colonization of healthy term infants occurs at and after delivery, not before. ▪ Positive aerobic and anaerobic fetal meconium clinical cultures were detected as probable skin contaminants, most often Staphylococcus epidermidis, however, they were not discovered by sequencing in most samples.
16S rRNA gene sequencing Metabolomics	Distinct gut microbiota and metabolite profiles induced by delivery mode in healthy Chinese infants (81)	Stool samples from 60 infants	Tax4Fun (Microbial Functional profiling) OSI/SMMS (Metabolic profiling)	▪ Vaginally delivered infants had the highest abundance of Bifidobacterium, Lactobacillus genera, Bacteroides and Parabacteroides phyla, while the cesarean section delivered infants that had a high level of Klebsiella ▪ Vaginally delivered infants were associated with a high abundance of DL-norvaline and DL-citrulline. In contrast, cesarean section delivered infants were enriched in trans-vaccenic acid and cis-aconitic acid. ▪ Feces of vaginally delivered infants was positively correlated with tryptophan metabolism and pyruvate metabolism. However, feces of CS delivered infants was positively correlated with ABC transporters
Metagenomics	Human milk virome analysis: changing pattern regarding mode of delivery, birth weight, and lactational stage (82)	Transient HM sample (TMS; Postpartum (7–15 days) and mature HM samples (MMS; postpartum 45–90 days).	QIIME (Taxonomic profiling) PRINSEQ-lite (Filter and trim metagenomic data)	▪ HM virome may influence the composition of an infant's gut microbiome early in life, which might have short- and long-term health effects. ▪ Most prevalent virus family in the transitory HM of the regular spontaneous vaginal delivery group was Podoviridae. ▪ Myoviridae was predominant in both transient and mature HM in the premature group (all C-section), and Podoviridae was predominant in transient HM. At the same time, Siphoviridae and Herpesviridae were predominant in mature HM.
16S rRNA gene sequencing	Antibiotic treatments during infancy, changes in nasal microbiota, and asthma development: population-based cohort study (83)	Nasal samples from 697 children	USEARCH (Processing metagenomics data) UPARSE (Generating OTUs clusters)	▪ Exposure to ≥2 antibiotic treatments between the ages of 0 and 11 months was linked to an increased chance of developing asthma. ▪ Infants with more antibiotic treatments were more likely to have a profile with early Moraxella sparsity.
16S rRNA gene sequencing Metabolomics	Effects of antibiotic treatment and probiotics on the gut microbiome of 40 infants delivered before term by cesarean section analyzed by using 16S rRNA quantitative polymerase chain reaction sequencing (84)	Fecal samples of 40 premature infants delivered by cesarean section	QIIME Mothur (Taxonomic profiling) Tax4Fun BugBase (Measuring phenotypes in microbiome) iPath (Metabolics pathways)	▪ Antibiotics increase the prevalence of pathogenic bacteria while probiotics increase the prevalence of beneficial bacteria and the cellular community prokaryote function and contribute to the Bifidobacteria biofilm formation. ▪ Probiotics reduce the adverse effects of antibiotics on the composition and function of the gut microbiota ▪ Albumin was most factors influencing the composition of the gut microbiota at the genus level, and Sphingomonas was negatively correlated with Albumin.
16S rRNA gene sequencing	Maternal diet during pregnancy and intestinal markers are associated with early gut microbiota (85)	116 Maternal, neonatal fecal swabs	DADA2	▪ Maternal diet during gestation was associated with the diversity and richness of neonatal microbiota. ▪ Mothers having high lipids intake and consumers of SFA had lower Proteobacteria relative abundance in their microbiota. ▪ Total lipids were negatively associated with Escherichia/Shigella genus and positively linked to Firmicutes phylum, including genera from the Ruminococcaceae groups and the Blautia, Roseburia, Rombustia, and Faecalibacterium genera. These genera also showed a negative correlation with fiber and vegetable source proteins. ▪ Fat-related nutrient intake by mothers, including total lipids, SFA, and MUFA, showed enrichment in Firmicutes phylum genera and reduction in Proteobacteria phylum genera in the offspring microbiota
16S rRNA gene sequencing	Breastfeeding promotes early neonatal regulatory T-cell expansion and immune tolerance non-inherited maternal antigens (86)	Stool and blood samples of 38 term neonates born by cesarean section grouped according to feeding method (breast milk versus formula)	QIIME2	▪ Proportion of regulatory T cells (Tregs) increases at birth and 3 weeks of age. It is nearly 2 fold higher in exclusively breastfed neonates than those who only received formula milk. ▪ Breastfed neonates have a specific and Treg-dependent decrease in proliferative T-cell responses to non-inherited maternal antigens (NIMA), which is associated with a reduction in inflammatory cytokine production enrichment of short-chain fatty acid-producing taxa (Veillonella and Gemella) in stool samples of exclusively breastfed neonates.
16S rRNA gene sequencing	Transient effect of infant formula supplementation on the intestinal microbiota (87)	Stool and blood samples of 24 infants	DADA2	▪ Firmicutes, Proteobacteria, and Actinobacteria were the most frequent group found in all samples, bacterial genera Bifidobacterium, Bacteroides, and Parabacteroides had significantly higher relative abundance in vaginally delivered infants ▪ Supplementation caused transitory microbiota alterations, including increases in Campylobacter, Dermabacter, Peptoniphilus, Prevotella, and S24-7, as well as a decrease in Eggerthella. However, these differences diminished by the age of 6 months
16S rRNA gene sequencing	Maternal diet shapes the breast milk microbiota composition and diversity: impact of mode of delivery and antibiotics exposure (88)	120 Breast milk samples from healthy mothers	DADA2	▪ Maternal diet influences the composition and diversity of breast milk microbiota, with the most important contributions coming from dietary fiber and plant and animal protein intakes. ▪ Lower levels of Lactobacillus, Bacteroides, and Sediminibacterium genera were observed in Cluster II (high intake of animal proteins & lipids)/C-section/antibiotics exposure compared with the other groups
16S rRNA gene sequencing Metabolomics	Association of the birth mode of delivery with infant fecal microbiota, potential pathobionts, and short-chain fatty acids: a longitudinal study over the first year of life (89)	fecal Samples from 70 infants	DADA2	▪ CS infants had a higher abundance of the pathobionts Clostridium neonatale and Clostridium perfringens and a lower abundance of potentially beneficial Bifidobacterium and Bacteroides spp. ▪ a higher fecal butyrate concentration at 3 months.
16S rRNA gene sequencing	Influence of human milk on very preterms' gut microbiota and alkaline phosphatase activity (90)	117 preterm infants (≤32 gestational weeks).	QIIME	▪ HM was positively associated with beneficial bacteria, such as Bifidobacterium, Bacteroides ovatus, and Akkermancia muciniphila, as well as bacterial diversity. ▪ Neonates fed with HM during the first week of life had a higher abundance of Bifidobacterium content and fecal ALP activity on the 26th postnatal day.
16S rRNA gene sequencing Metabolomics	The Effects of Different Modes of Delivery on the Structure and Predicted Function of Intestinal Microbiota in Neonates and Early Infants (91)	A stool sample from 82 healthy newborns (39 boys and 43 girls), .	QIIME PICRUSt (Functional profiling) (Pathway profiling) KEGG	▪ The genera Bifidobacterium, Lactobacillus, and Bacteroides were more prevalent in the vaginal delivery group than in the CS group, which exhibited a predominance of Staphylococcus, Streptococcus, and Corynebacterium in the 3-day-old infants' samples. ▪ In the samples from 30- to 42-days, Bifidobacterium, Lactobacillus, Escherichia-Shigella, and Bacteroides were the frequent genera present in the vaginal delivery group, while in the CS delivery group; the predominant genera were Escherichia-Shigella, Bifidobacterium, Bacteroides, and Staphylococcus. ▪ Predicted functions of the vaginal delivery group revealed higher metabolic and biodegradation rates of carbohydrates, vitamins, and xenobiotics than those in the CS group, which led to the stability of the microbiota.
16S rRNA gene sequencing	Maternal Vegetable and Fruit Consumption during Pregnancy and Its Effects on Infant Gut Microbiome (92)	39 infant stool samples were obtained at 2 months postpartum	DADA2	▪ The amount of fruits and vegetables consumed during pregnancy is linked to different alterations in the newborn gut microbiota at 2 months of age. ▪ Abundance of unhealthy infant gut microbiomes, such as Erysipelatoclostridium, Betaproteobacteria, and Lachnospiraceae, was negatively linked with higher maternal nutritional intake of fructose, dietary fiber, folic acid, and ascorbic acid.
16S rRNA gene sequencing	Gut microbiota development during infancy: Impact of introducing allergenic foods (93)	Fecal samples of 288 exclusively breast-fed infants	DADA2	▪ Exclusively breastfed infant at 3 months, gut microbiota was highly heterogeneous, forming three distinct groups: Bifidobacterium-rich, Bacteroides-rich, and Escherichia/Shigella-rich. ▪ Increased abundance of Clostridium sensu stricto at 3 months was linked to the presence of atopic dermatitis on examination at age 3 and 12 month. ▪ Introduction of allergenic solids promoted a significant increase in Shannon diversity and representation of particular microbes, such as Prevotellaceae and Proteobacteria (e.g., Escherichia/Shigella) as compared with infants exclusively breast-feed.