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.