The results of this study indicated that the diversity of intestinal flora was influenced by AED, delivery method, and siblings, with significant effects on Bifidobacteria and Bacteroides populations, which remained dominant at a combined occupancy of 60–70% in the bacterial flora of infants aged up to 6 months. These findings confirm the results of an earlier pilot study  in 1-month-old infants.
AED to β-lactamase antibiotics has a major influence on Bifidobacteria population in early infants regardless of the use of ABPC or CEZ with the influence being especially marked in 1-month-old infants. The influence of ABPC persisted until 3 months, but then gradually weakened and mostly disappeared by the 6th month. These results also comply with those of the pilot study . Several previous studies have suggested a minor effect of IAP on the Bifidobacteria occupancy. However, these studies were limited to use of antibiotics for specific reasons, such as for GBS-positive mothers , or in the late stage of delivery , and the study design and screening timeline of the intestinal flora were inconsistent. A study on the antibiotic administration, including Caesarean section, has been reported . However, to the best of our knowledge, the present study is the first of its kind to evaluate the effects of antibiotic administration to mothers immediately before delivery, including the cases of Caesarean section. The study comprised of a statistically appropriate number of samples, thereby allowing sub-group analyses and the effects were evaluated until 6 months after birth using a 16S rRNA-targeting next-generation sequencer.
The delivery method (vaginal vs Caesarean section) did not affect the Bifidobacteria occupancy in 1-month-old infants, but there was a significant difference in the CEZ and non-AED groups. The delivery method did not show any difference in the effect on the Bifidobacteria population between the ABPC and CEZ groups. Thus, Bifidobacteria occupancy was significantly influenced in the Caesarean section and CEZ groups compared to non-AED infants, but this effect did not change by AED in the vaginal delivery group ( i.e., the ABPC group). These results suggest that β-lactam antibiotics may directly influence bifidobacterial occupancy. Influence of delivery method on Bifidobacteria population from immediately after birth to early infancy was shown previously, [39, 50–53] but the influence of antibiotics administered before Caesarean section [36, 37, 54] was not considered in these studies, and this effect may have been simultaneously observed. The present study is not capable of judging whether the observed effect is due to the infant not passing through the birth canal in Caesarean section or CEZ. Further studies with the use of the same antibiotics for vaginal delivery and Caesarean section are therefore warranted.
Bifidobacteria colonisation was also influenced by the presence or absence of siblings. Bifidobacteria occupancy in AED infants was significantly higher in those with siblings. The results suggested that at least until 3 months after birth, the presence of an elder sibling promoted the colonisation of Bifidobacteria, even in infants exposed to antibiotics at delivery, i.e., there may be mutual interference of the microbiome between siblings. This may explain the maintenance of high Bifidobacteria occupancy in infants, even in the AED group. In a previous pilot study,  the presence of siblings was suggested to influence IAP, and the present study confirmed this effect. Previous studies on the effects of siblings have reported that Bifidobacteria colonisation occurs more easily in infants with siblings, [19, 39, 40]. However, this effect has been scarcely studied as compared to studies on factors such as AED and the delivery method, particularly in Japanese infants. Thus, the present study is significant in showing the effect of siblings in a large cohort study. This effect was confirmed by a sub-group analysis within the AED group to allow the interpretation in the context of AED. The association of this effect with the intestinal flora of siblings requires a continuous study, including siblings living together.
The influence of Caesarean section was more substantial than that of AED in Bacteroides. This effect was firmly maintained at 3 months and persisted at least until 6 months after birth. The same tendency was observed in the sub-analysis of the AED group. This confirmed that birth via Caesarean section is an important factor in the occupancy of Bacteroides, compared to AED. The persistence of the influence of Caesarean section on Bacteroides until the weaning period and thereafter has been pointed out, [34, 39, 53, 55, 56] similar to our results. However, as described above, all Caesarean section cases received preoperative CEZ. Thus, although the Bacteroides population was not influenced by ABPC, it may have been markedly influenced by CEZ. This possibility is outstanding and was not examined in previous studies.
Bacteroides population was not affected by siblings at 1 month, but a significant effect was seen at 3 months, with no influence at 6 months. In contrast to Bifidobacteria, siblings negatively influenced the occupancy of Bacteroides. The influence of siblings on Bacteroides population has been shown before,  with the occupancy by Bacteroides at 18 months after birth being higher in infants with siblings, in contrast to the findings of this study until 6 months.  Similar to Bifidobacteria, the effect on Bacteroides until 3 months may reflect mutual interference among siblings.
Many studies have examined the clinical significance of colonisation with Bifidobacteria and Bacteroides in intestinal flora in early infancy. [17–22] The present study evaluated the influence of AED on early infants in terms of the effects on their subsequent health. The samples from this study were also used to assess the relation of allergies with changes in the intestinal flora. This study was performed as a part of a cohort study evaluating the clinical significance of changes in the intestinal flora during early infancy in healthy Japanese infants and their effect on the intestinal flora later in life. Data at 1, 3, and 6 months after birth were used in this study, but data related to longer-term time-course changes in the same individuals are needed for a complete and comprehensive investigation. The composition of intestinal flora shows substantial similarities at different time-points in the same individual,  but external factors influencing the intestinal flora increase with growth, such as the increase in baby food intake, interaction with siblings and other infants in group nursing, and further use of antibiotics for various diseases. However, this study is significant as a cohort study with a statistically significant large number of samples performed on intestinal flora in infants in the first 6 months of life, which is a crucial period for the development of the immune system since this is a time at which external factors have the least influence in the entire lifetime [24–26].
The present study is not without limitations despite the undeniable significance. This study was conducted in a single hospital, which does not allow for extrapolation of the obtained results to the population from other regions within and without Japan, with differences in ethnicity or geography. Although the protocol for the use of antimicrobial agents before the delivery was similar to that in previous studies on IAP  , multi-centre studies are needed for validation of such extrapolation. Also, while this study focused on several factors that could have a significant impact on the gut microbiota of infants, it did not account for some factors such as feeding methods (only exclusively breast-fed infants included), which could affect colonisation characteristics of some bacteria in the intestinal microbiota of infants. Inclusion of another group of infants that were milk-fed for the most part along with top feed is imperative to understand the factors influencing gut microbiota in infants.