Early Life Microbiota Colonization at Six Months of Age: A Transitional Time Point

Abstract

Background: Early life gut microbiota is involved in several biological processes, particularly metabolism, immunity, and cognitive neurodevelopment. Perturbation in the infant's gut microbiota increases the risk for diseases in early and later life, highlighting the importance of understanding the connections between perinatal factors with early life microbial composition. The present research paper is aimed at exploring the prenatal and postnatal factors influencing the infant gut microbiota composition at six months of age.

Methods: Gut microbiota of infants enrolled in the longitudinal, prospective, observational study "A.MA.MI" (Alimentazione MAmma e bambino nei primi MIlle giorni) was analyzed. We collected and analyzed 61 fecal samples at baseline (meconium, T0); at six months of age (T2), we collected and analyzed 53 fecal samples. Samples were grouped based on maternal and gestational weight factors, type of delivery, type of feeding, time of weaning, and presence/absence of older siblings. Alpha and beta diversities were evaluated to describe microbiota composition. Multivariate analyses were performed to understand the impact of the aforementioned factors on the infant's microbiota composition at six months of age.

Results: Different clustering hypotheses have been tested to evaluate the impact of known metadata factors on the infant microbiota. Neither maternal body mass index nor gestational weight gain was able to determine significant differences in infant microbiota composition six months of age. Concerning the type of feeding, we observed a low alpha diversity in exclusive breastfed infants; conversely, non-exclusively breastfed infants reported an overgrowth of Ruminococcaceae and Flavonifractor. Furthermore, we did not find any statistically significant difference resulting from an early introduction of solid foods (before 4 months of age). Lastly, our sample showed a higher abundance of clostridial patterns in firstborn babies when compared to infants with older siblings in the family.

Conclusion: Our findings showed that, at this stage of life, there is not a single factor able to affect in a distinct way the infants' gut microbiota development. Rather, there seems to be a complex multifactorial interaction between maternal and neonatal factors determining a unique microbial niche in the gastrointestinal tract.

Keywords: breast milk; delivery; maternal factors; neonatal microbiota; newborn; older siblings; weaning.

Figure 1

Box plots of the alpha diversity (Shannon index and number of operational taxonomic units (OTUs) identified) among T2 fecal samples (infants at 6 months of age) grouped according to different factors influencing early microbiota composition. Maternal factors: maternal pre-pregnancy body mass index (BMI) and gestational weight gain (WG). NW [n.37]: normal pre-pregnancy BMI (BMI < 25 kg/m²), OW [n.12]: excessive pre-pregnancy BMI (BMI ≥ 25 kg/m²); EWG [n.20]: excessive gestational WG, NWG [n.29]: optimal gestational WG). Food-related factors: Feeding (_EBF [n.31]: exclusive breast-feeding, NeBF [n.21]: not exclusively breast fed, mixed fed or exclusively formula fed) and Weaning (≤4 [n.41]: before 4 months of age, >4 [n.9]: after 4 months of age). Environmental factors: Delivery mode (CS [n.7]: cesarean section, VD [n.46]: vaginal delivery) and presence of Older siblings (FB [n.30]: first-born, nFB [n.22]: not first-born). *P = p-value.

Figure 2

Influence of different factors on early microbiota composition at the phylum level (16S rRNA gene amplicon sequencing) in T2 fecal samples (infants at 6 months of age). Starting from the left: i) and ii) Maternal factors: maternal pre-pregnancy body mass index (BMI) and gestational weight gain (WG), respectively. NW [n.37]: normal pre-pregnancy BMI (BMI < 25 kg/m²), OW [n.12]: excessive pre-pregnancy BMI (BMI ≥ 25 kg/m²); EWG [n.20]: excessive gestational WG, NWG [n.29]: optimal gestational WG). iii) Delivery mode (CS [n.7]: caesarean section, VD [n.46]: vaginal delivery). iv) Feeding (_EBF [n.31]: exclusive breast-feeding, NeBF [n.21]: not exclusively breast fed, mixed fed or exclusively formula fed). v) Weaning (≤4 [n.41]: before 4 months of age, >4 [n.9: after 4 months of age). vi) Presence of older siblings (FB [n.30]: first-born, nFB [n.22]: not first-born).

Figure 3

MaAsLin2 significant results and associations between postnatal factors (delivery, feeding, weaning, and presence of older siblings) and gut microbiota composition at the family level of infants at six months of age (T2). Based on normalized obtained significant results, the color scale-bar showed a positive relationship (red) and a negative one (blue) between taxa and factors, ranging from the highest positive normalization (+3) to the lowest one (−3).

Figure 4

MaAsLin2 significant results and associations between postnatal factors (delivery, feeding, weaning, and presence of older siblings) and gut microbiota composition at the genus level of infants at six months of age (T2). Based on normalized obtained significant results, the color scale-bar showed a positive relationship (red) and a negative one (blue) between taxa and factors, ranging from the highest positive normalization (+2) to the lowest one (−2).

Figure 5

Principal component analysis (PCA) of bacterial genera with a median abundance >0.1% (16S rRNA gene amplicon sequencing) of infants’ T0 (meconium) and T2 (six months of age). Infants were grouped based on the relative metadata: (A) maternal pre-pregnancy BMI (NW, normal BMI, or OW, mothers with overweight/obesity), (B) maternal gestational weight gain (NWG, normal WG, or EWG, excessive WG), and (C) type of delivery (VD, vaginally delivered, or CS, by caesarean section).

Figure 6

Discriminant analysis of principal components (DAPC) and relative score-plots. (A–F) report the DAPC score plots based on the relative abundances (16S rRNA gene amplicon) of genera with median values >0.1% found in infants at 6 months. Panel: (A) NW, normal BMI (<25 kg/m²); OW, women overweight/obesity (BMI ≥ 25 kg/m²); (B) NWG, optimal weight gain; EWG, excessive weight gain; (C) VD, vaginally delivered; CS, delivered by cesarean section; (D) BF, exclusive breast-feeding; FF, combined or exclusive formula-feeding; (E) ≤4, infants weaned before 4 months of age; >4, infants weaned after 4 months of age. Panel (F) FB, first-born; nFB, not first-born. (A1–F1) respectively indicate whether the infants (rows) could be assigned based on discriminant functions by K-means analysis. The cell colors represent the membership probabilities (K-means analysis) to belong to each cluster (red = 1, orange = 0.75, yellow = 0.25, white = 0) and blue crosses indicate the originally belonging cluster.