Delivery effects on the infant fecal metabolome

It is already known, that the infant microbiota is impacted by the mode of delivery (Gronlund et al. 1999, Mackie et al. 1999, Penders et al. 2006, Dominguez-Bello et al. 2010). During vaginal birth, the neonate is mainly exposed to maternal bacteria through the birth canal, and the birth canal is occupied by a characteristic set of bacteria (Dominguez-Bello et al. 2010), which pass over and forms the infants microbiota. Further, after birth the GIT of infants get colonized immediately through environmental and skin exposure of the mother (Biasucci et al. 2008). The GIT shows distinct patterns of the microbial composition in infants born by C-section or by vaginal birth.

Knight et al. showed that the infants gut microbiota born by C-section is similar to the mother’s skin microbiota. The infants born vaginally showed similar patterns to the mother’s vaginal microbiota (Dominguez-Bello et al. 2010). It was observed, that the bacterial composition in infants born by C-section was less diverse, than those born vaginally with predominating groups of Bifidobacteria species (Biasucci et al. 2008). Those altered microbial compositions in either infant born by C-section or by vaginal birth may also result to a differently impacted fecal metabolome. Hereof, relatively few studies were available evaluating the possible relationship between the mode of delivery and the metabolome (Fanos et al. 2012).

However, in 2009, Hyde et al. detected a modified metabolomics profiles in liver samples of piglets due to the mode of delivery, mentioning higher levels of oxaloacetate, aspartate, α-ketoglutarate and glutamate and lower levels of glucose and succinate in the cesarean born piglets (Hyde et al. 2009).

Also Diaz et al. observed distinct urinary NMR metabolite signatures significantly (p<0.05) changed through the different mode of delivery, such as trigonelline and indoxyl sulfate (in vaginal delivery) or acetone and dimethylamine (in cesarean section) (Diaz et al. 2016). Nevertheless, studies investigating the mode of delivery into relation to the metabolome, especially considering human individuals and the fecal metabolome are rare.

In order to evaluate the impact of the mode of delivery on the metabolite profile, the fecal samples of either VD or CS were analyzed by month, without considering the type of feeding. In order to detect only mass signals significant for the different modes of delivery, mass signals impacted though feeding were eliminated. Through the application of the Mann-Whitney test (p-value > 0.01) and log2 fold change calculations, five metabolites were detected as significantly impacted by the mode of delivery

(Bazanella et al. 2017) in month 1, but which could not persist in the following months, namely metabolites with the nominal mass 344 (positive ionization mode) and 328, 280, 890 and 288 (negative ionization mode).

Figure 3.4-27: Differences in the metabolite profile due to cesarean or vaginal delivery in month 1.

Log2 fold change values for metabolites increased or decreased through vaginal delivery (yellow) or cesarean section (blue), analyzed in HPLC-(+)-ToF-MS (top) and UHPLC-(-)-ToF-MS (bottom) mode.

The mass signals were assigned to metabolites listed in databases but did not reveal useful information.

Nevertheless, this study showed that the fecal metabolome was impacted by the mode of delivery in early life stages, even if no further information was given on the origin and the classification of the metabolites. In respect thereof, MS/MS experiments may be helpful to characterize the metabolome or possibly identify those metabolites impacted by mode of delivery in early life stages. This may further lead to a better understanding of the impact of the mode of delivery, in relation to health-promoting effects and the developing microbiota and their corresponding metabolome.

-4 -2 0 2 4 6 8

m/z288.1123 m/z890.2705 m/z280.1149 m/z328.2379 m/z344.2823

LOG2 fold change Vaginal Delivery (VD) vs. Cesarean Section (CS)

Cesarean Section Vaginal Delivery VD

CS UHPLC-(+)-ToF-MS

UHPLC-(-)-ToF-MS

3.5 Summary and Conclusion

The aim of the study was to elucidate the impact of breast feeding and two different formulas, one without, the other with bifidobacteria-supplementation (probiotics) on the fecal metabolome of healthy infants by applying ultra-high performance liquid chromatography-mass spectrometry based metabolomics approach, in a double-blinded, randomized and placebo controlled intervention trial.

Additional 16S rRNA sequencing complemented the analysis in order to investigate the complex interplay between organisms and metabolites.

Probiotic supplementation of infant formula became popular, aiming at beneficially influencing the gut microbiome and infant well-being. However, only little is known about the effects of probiotic-supplemented formula on the development of the intestinal ecosystem. Therefore, during the first year of life, infants were fed a bifidobacteria-supplemented (intervention) or non-supplemented (placebo) formula, either from birth on or after weaning. The infants were divided into two main groups, including exclusively fed infants and mixed fed infants, whereas analyses were performed concentrating on the exclusively fed infants. This allowed a more precise evaluation of the impact of breast feeding and formula feeding with or without probiotics on the infants’ gut microbiome. Monthly fecal samples up to one year of age were collected, in order to evaluate the overall fecal metabolite and microbiota profile impacted through breast feeding, interventional formula and the placebo formula.

The metabolite profile showed a clear separation between breastfed infants and formula-fed ones in early life stages, which converged over time. The monthly evaluation of the data revealed slightly differences in the metabolite profiles between the interventional and placebo formula group in month 1, which identity remains unclear. However, one metabolite discriminative between both formula and increased in the placebo formula-fed infants could be identified as a lysophosphatidylethanolamines (LysoPE) 15:0/0:0. It was remarkable that several metabolites of the probiotics formula group leads to an approximation towards the breastfed infants. This finding leads to the assumption that probiotic supplementation may help to approximate breast milk and strengthened the use of probiotics. However, the occurrence of these similar effects remains still unknown and for the moment was suggestively an initial sign for the effects of probiotics. To investigate the impact of probiotics in contrast to breast milk, further research is strongly needed.

Additionally, the metabolomics approach revealed that through breast- and formula feeding, pathways of the lipid metabolism showed to be highly impacted, especially metabolites of the primary bile acid biosynthesis. The glycine-conjugated bile acids (GCDCA and GCA) were increased in the formula-fed infants over time. Several bile acids were affected differently in the two formula groups. While some bile acids in the probiotics group lead to an approximation towards the breastfed infants, they were increased in the non-probiotic group. Further, the sulfated bile acids (cyprinolsulfate and sulfocholic acid) were erratically increased and widely spread in breastfed infants over time, whereat the pattern in the formula-fed ones was quite lower and rather consistent. The fecal samples of the infants fed with formula showed increased levels of intermediates of the vitamin E biosynthesis, which were not only correlated with each other, but also highly correlated with the amount of formula the infants were fed with.

It was observed that different fatty acid classes, such as saturated, unsaturated and hydroxylated fatty acids were significantly different between the breast fed infants and the formula-fed ones. Further, SCFA were analyzed which revealed increased levels of lactic acid and pyruvic acid in the breast fed infants, whereas propionic acid, butyric acid, valeric acid and isovaleric acid were significantly different in the formula-fed infants. However, SCFA profiles were not different between the interventional and the placebo formula fed infants. The correlation between SCFA and OTUs revealed positive correlations between lactic acid and species of the Bifidobacterium, Streptococcus and Lactobacillus, all LAB bacteria in the gut. Moreover, the correlation of all metabolites and OTUs revealed 6 OTUs being involved in the feeding-specific shaping of the fecal ecosystem at month 1. Both, metabolomic and the 16S data revealed that the differences in bacterial and metabolite profiles between interventional and placebo groups disappeared over time. Further, the fecal samples of the infants were evaluated to detect differences in the metabolite profile due to cesarean section or vaginal delivery, which revealed some metabolites to be discriminating between both modes of delivery. In conclusion, this placebo-controlled intervention study clearly showed that bifidobacteria-supplemented formula modulates the infant fecal metabolome and microbiome at very early stages in life, with no detectable long-term consequences for gut microbiome assembly or function. The impact of sequentially changing bacterial and metabolite profiles on human health remained completely unclear and requires additional studies.

Chapter IV