Discussion
Main Findings
A lower diversity in gut microbiota was observed in neonates born via cesarean delivery compared to those born through vaginal delivery.Bifidobacterium spp. were predominant in both groups, with the abundance higher in the CS group
The diversity of gut microbiomes in neonates, which is significantly influenced by the mode of delivery, is consistent with findings from previous studies (10, 14). A study conducted by Biasucci et al. in 2008, utilizing 16S rRNA methods and collecting fecal samples on the third day of life, indicated that the intestinal microbiota of neonates delivered by cesarean section appears less diverse regarding bacterial species. A later systematic review by Rutayisire et al. (2016) concluded that low total diversity of the gut microbiota during the first week of life was reported in infants delivered by cesarean section (15). However, a recent study by Chu et al. in 2017 showed no difference in microbiota community function regardless of the delivery mode (16). Studies on neonatal gut microbiomes exhibit variations such as timing of stool collection, sample size, and techniques used to identify bacterial genus/species. Furthermore, environmental and genetic factors also play a role in influencing individual gut microbiomes.
A significantly higher abundance of Bifidobacterium found in the CS group is consistent with a study by Azad et al (7). However, other studies have reported higher levels of Bifidobacterium in vaginal delivery groups (10, 17). Bifidobacterium is an essential bacterium providing the most common genera in the infant gut microbiome. Bifidobacterium breve (B. breve) andBifidobacterium longum (B. longum) are common species and are more prevalent during infancy than adulthood, especially during the first year of life. Bifidobacterium is involved in various physiological and immunological functions such as the digestion of human milk oligosaccharides, improving gut barrier function, and reducing intestinal permeability (18-20). Lower levels ofB. longum may be associated with allergic diseases such as atopic dermatitis (21, 22), but the outcome of such bacterial abundancy has not been well stated. Some studies have shown the benefit of high levels of Bifidobacterium; however, a decrease in Bifidobacterium colonization early in life may increase the risk of neonatal complications (23).
Consistent with previous studies, our study found lower levels of Bacteroides in the CS group (7, 24), and several studies have shown delayed colonization of Bacteroides(22, 24, 25). Bacteroides is the major genus found in adults, but these babies had lower Bacteroides levels since they lacked exposure to it during cesarean delivery. This may result in a negative impact on infant immune development, maintenance of intestinal homeostasis (26), and, later, food digestion(27).
In this study, Staphylococcus aureus (S. aureus ) was higher in the CS group, consistent with the study of Shao et al.(24). The proposed source of S. aureuscolonization was maternal skin flora (28). Intestinal colonization with Staphylococcus aureus is associated with higher levels of inflammation, which contribute to the development of inflammatory diseases such as asthma and allergies, including atopic dermatitis, or atopic eczema (29), and food allergies(30).
We identified higher levels of C. perfringens in the VG group, comprising 5.08% of all identified species, consistent with a previous study (7). This may be attributed to initial gut colonization from maternal vaginal and fecal microbiota. Association with a higher risk of developing necrotizing enterocolitis, especially in preterm infants (31, 32), and prolonged diarrhea(33) has been found.
Strengths and Limitations
Our study had several strengths. Firstly, it was the first to report data on the differences in gut microbiome between neonates delivered via vaginal delivery and those delivered by cesarean delivery in Thailand. Secondly, we collected all fecal samples from healthy neonates within 48 hours, and neonates whose mothers had used antibiotics were not included in the study, except when prophylactic antibiotics had been used for cesarean delivery. Thirdly, the adjusted DNA extraction protocol can be used as an alternative technique in subsequent studies to create the proper meconium-specific protocol for analyzing a neonate’s gut microbiome.
The limitation of our study was the potential confounding effect of breastfeeding practices on a neonate’s gut microbiome. Thus, collection of the first passing of meconium may represent an opportunity to access the neonatal gut microbiome directly after delivery(34). Second, we have not stated the details of cesarean delivery, such as presenting with membrane rupture before cesarean delivery. These variations may result in different neonatal gut microbiomes.
Interpretation
There were many explanations for this lower diversity. Firstly, there was a lack of transmission of gut microbiome from mother to child through the vaginal route. Secondly, there was a lack of or delayed colonization with Bacteroides (8, 35), which can be persistent (24) or delayed by up to 1 year in some infants (8). And finally, empirical antibiotics were used in cesarean deliveries (36).
The reasons behind elevated Bifidobacterium levels in the gut microbiomes of cesarean-delivered infants in some studies are not yet fully understood (7, 10). Although most studies associate vaginal delivery with higher Bifidobacterium levels, some studies present different findings (8, 27). Potential explanations include the use of antibiotic prophylaxis for cesarean delivery, which could lead to a decrease in overall gut microbial diversity or selectively eliminate certain bacterial species(7, 8). For instance, Bifidobacterium species may be sensitive only to penicillin administration, not cephalosporins(8, 27). Nonetheless, conflicting results in other studies were observed. For instance, Jakobsen et al.(8) did not observe a significant difference in infant gut microbiome between mothers who had received prophylactic antibiotics and those who did not. Yassour et al. demonstrated a lower level of Bifidobacterium species in infants born via cesarean section and exposed to antibiotics, including cefazolin, compared to infants not exposed to antibiotics (36). Our institutional protocol is to administer cefazolin before making a skin incision on the mother in the CS group. Cephalosporin does not affect maternal microbiomes(37); however, there has been limited research on the specific effects of cefazolin on Bifidobacterium levels in the infant gut microbiome. Therefore, we cannot draw a definite conclusion regarding the influence of empirical antibiotic use on the abundance of Bifidobacterium in our study.
Another hypothesis suggests that early breastfeeding may influence Bifidobacterium levels in infants, as breastfed infants are more likely to have higher levels due to the utilization of oligosaccharides as a food source (1). Cesarean-delivered infants are more likely to experience delays in breastfeeding initiation, potentially affecting Bifidobacterium abundance (38, 39). However, in our hospital, there is a policy of early breastfeeding for all mothers, making it uncertain whether this could confound our study. One of the challenges in obtaining information about breastfeeding practices was the reliance on subjective self-reporting by mothers, which could introduce informative bias on aspects such as timing, frequency, and the amount of breast milk.
In the future, exploring the impact of antibiotic prophylaxis on the gut microbiome of infants born through cesarean delivery and its long-term health effects is interesting. Moreover, considering that gut microbiota diversity tends to diminish after six months of life(15), efforts should be directed toward identifying factors that restore healthy gut microbiota.
Our study has limitations, with 47 (34.6%) and 96 (68.6%) samples in the VG and CS groups being unsuccessfully sequenced. This may be attributed to sterility or very low bacterial abundance, particularly in the CS group, where the tar-like meconium posed extraction difficulties due to its low biomass microbiome and high PCR inhibitor concentration(40). Meconium analysis lacks standardized sequencing techniques for microbial assessment of the gut. We adjusted our extraction protocol to enhance DNA yield, yet uninterpreted samples suggest a potentially minimal bacterial presence. Future research should employ additional techniques to achieve clearer conclusions.